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Abstracts

N-Acetyl-Cysteine: 214 Research Abstracts

ACETAMINOPHEN POISONING **

1. Hum Exp Toxicol. 2003 Aug;22(8):453-8. Successful treatment of acetaminophen overdose associated with hepatic failure. Pajoumand A, Jalali N, Abdollahi M, Shadnia S. Poison Centre, Loghman-Hakim Hospital, Faculty of Medicine, Shaheed-Beheshti University of Medical Science, Tehran, Iran.

Acetaminophen is the most widely used antipyretic and analgesic drug in the world. Acetaminophen poisoning and the following hepatic failure are not rare and are the most common indications of liver transplantation in the USA and Europe. In this case report, the patient was a 25-year old woman with hepatic failure who was brought to Loghman-Hakim Poison Centre 24 hours after attempted suicide with 100 tablets of acetaminophen, 325 mg. She was treated with N-acetylcysteine (NAC) and discharged from the hospital 12 days after admission and followed up for 1 month. In conclusion, acetaminophen poisoning should be considered in the differential diagnoses of hepatic failure. In acetaminophen-induced hepatic damage the administration of NAC must always be considered even after 24 hours of overdose.

2. Klin Med (Mosk). 2003;81(4):58-60. [Safety of paracetamol as a representative of nonprescription analgetics-antipyretics] [Article in Russian] Makar'iants ML.

Paracetamol is a well-known analgetic-antipiretic drug widely used in medical practice. In therapeutic doses (4 g/day for adults and 60 mg/kg/day for children) paracetamol relieves fever and non-severe pain with minimal risk of complications both in adults and children. But medical professionals should know that in extremely high doses (above 10 g for adults) paracetamol has a hepatotoxic effect. Overdose intake of paracetamol in Russia happens rarely, it occurs by chance or in attempted suicide. In such a case the antidote must be used to protect the liver. This antidote for paracetamol is N-acetylcysteine.

3. Cochrane Database Syst Rev. 2002;(3):CD003328. Interventions for paracetamol (acetaminophen) overdoses. Brok J, Buckley N, Gluud C. Centre for Clinical Intervention Research, Copenhagen University Hospital, Department 71-02, H:S Rigshospitalet, Copenhagen O, Denmark, DK 2100. jesperb@mdb.ku.dk

BACKGROUND: Self-poisoning with paracetamol (acetaminophen) is a common cause of hepatotoxicity in the Western World. Interventions for paracetamol poisoning encompass inhibition of absorption, removal from the vascular system, antidotes, and liver transplantation. OBJECTIVES: The objective was to assess the beneficial and harmful effects of interventions or combination of interventions for paracetamol overdose. SEARCH STRATEGY: The Cochrane Hepato-Biliary Group Controlled Trials Register, The Cochrane Library, MEDLINE, EMBASE, and text searches were combined (until July 2001). SELECTION CRITERIA: Randomised clinical trials (RCTs) and observational studies as well as human volunteer randomised trials were included. The studies could be unpublished or published as an article, an abstract, or a letter and no language limitations were applied. DATA COLLECTION AND ANALYSIS: All the analyses were performed according to the intention to treat. The methodological quality of the included trials was evaluated by components of methodological quality. MAIN RESULTS: Nine RCTs (all small and of low methodological quality), one quasi-randomised trials, 37 observational studies, and nine randomised trials including human volunteers were identified. It was impossible to perform meta-analyses including more than two RCTs. Activated charcoal, gastric lavage, and ipecacuanha are able to reduce the absorption of paracetamol but the clinical benefit is unclear. Of these, activated charcoal seems to have the best risk-benefit ratio. N-acetylcysteine seems preferable to placebo/supportive treatment (relative risk of mortality in patients with fulminant hepatic failure = 0.65; 95% confidence interval 0.43 to 0.99), dimercaprol, and cysteamine, but N-acetylcysteine's superiority to methionine is unproven. It is not clear which N-acetylcysteine treatment protocol offers the best efficacy. No evidence supports haemoperfusion or cimetidine for paracetamol overdose. Liver transplantation has the potential to be life saving in fulminant hepatic failure, but further refinement of selection criteria for liver transplantation and evaluation of the long-term outcome are required. REVIEWER'S CONCLUSIONS: This systematic Review has highlighted a paucity of RCTs on interventions for paracetamol overdose. Activated charcoal seems the best choice to reduce paracetamol absorption. N-acetylcysteine should be given to patients with paracetamol overdose. No N-acetylcysteine regime has been shown to be more effective than any other. It is a delicate balance when to proceed to liver transplantation, which may be life saving in patients with a poor prognosis. Interventions for paracetamol overdose need assessment in high-quality, multi-centre RCTs.

4. Hepatology. 2002 Apr;35(4):876-82. Acute versus chronic alcohol consumption in acetaminophen-induced hepatotoxicity. Schmidt LE, Dalhoff K, Poulsen HE. Departments of Hepatology and Clinical Pharmacology, Rigshospitalet, University Hospital, Copenhagen, Denmark. lars.schmidt@dadlnet.dk

The aim of this study was to determine by multivariate analysis how alcohol and other factors affect the clinical course and outcome in patients with acetaminophen (paracetamol) poisoning. A total of 645 consecutive patients admitted from 1994 to 2000 with single-dose acetaminophen poisoning were studied, giving special attention to alcohol history, time between overdose and intravenous N-acetylcysteine (NAC) treatment ("time to NAC"), and other data available at the time of admittance. Up until 72 hours after ingestion, time to NAC was the single most important independent risk factor. With a time to NAC less than 12 hours, the mortality rate was 0.42% (95% CI, 0.05-2.7). When time to NAC exceeded 12, 24, and 48 hours, the mortality rate increased to 6.1%, 13%, and 19%, respectively. Chronic alcohol abuse was an independent risk factor of mortality (odds ratio [OR], 3.52; 95% CI, 1.78-6.97). Acute alcohol ingestion was an independent protective factor regarding mortality in alcoholic patients (OR, 0.08; 95% CI, 0.01-0.66) but not in nonalcoholic patients (OR, 0.21; 95% CI, 0.03-1.67). Patient age and quantity of acetaminophen were independent risk factors. In conclusion, time to NAC was confirmed as the major risk factor in acetaminophen-induced hepatotoxicity and mortality. Chronic alcohol abuse was an independent risk factor that could be counteracted by concomitant acute alcohol ingestion. We suggest that patients with chronic alcoholism and suspected acetaminophen poisoning due to an increased risk of developing hepatotoxicity should be treated with NAC regardless of risk estimation.

5. Exp Toxicol Pathol. 2002 Feb;53(6):489-500. Acetaminophen hepatotoxicity and mechanisms of its protection by N-acetylcysteine: a study of Hep3B cells. Manov I, Hirsh M, Iancu TC. Pediatric Research and Electron Microscopy Unit, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.

Acetaminophen (AAP) hepatotoxicity, resulting in centrilobular necrosis, is frequently encountered following suicidal attempts, especially by adolescents, but also after its excessive use in infants. The subcellular and molecular sequences leading to hepatocellular cell death are not yet clear. We therefore investigated AAP hepatotoxicity by using cultured hepatoma-derived cells (Hep3B) exposed to AAP and N-acetylcysteine (NAC), used as a protective agent. Specifically, we studied the role of apoptosis and oxidative damage as putative mechanisms of AAP-associated cytotoxicity. Hep3B cells were exposed to AAP (5-25 mM) and NAC (5 mM) for different time periods. Cell viability was assessed by the Alamar Blue Reduction Test and LDH. Oxidative damage was evaluated by measuring reactive oxygen species (ROS) and glutathione. AAP-induced apoptosis was investigated by flow cytometry and transmission electron microscopy. We found that: 1. In Hep3B cells, AAP causes a time- and concentration-dependent cytotoxic effect, leading to oxidative stress, mitochondrial dysfunction, alterations of membrane permeability and apoptosis; 2. In the course of AAP cytotoxicity, the generation of ROS appears as an early event which precedes decrease of viability, LDH leakage, glutathione depletion and apoptosis; 3. NAC protects Hep3B cells from AAP-induced oxidative injury, but does not prevent apoptosis.

6. [About paracetamol again.] [Article in Hungarian] Hazai E, Monostory K, Bakos A, Zacher G, Vereczkey L. Magyar Tudomanyos Akademia, Budapest, Kemiai Kutatokozpont, Farmakobiokemiai Osztaly. Orv Hetil 2001 Feb 18;142(7):345-9

The mechanism of hepatotoxicity caused by paracetamol (acetaminophen) overdose and the treatment of patients is reviewed. Paracetamol is widely used over-the-counter drug with analgesic and antipyretic properties. Although it is considered to be safe at therapeutic doses, the incidence of hepatotoxicity caused by overdose or inadvertent application has been increasing lately. N-acetyl-p-benzoquinonimine, one of the metabolites formed from paracetamol is responsible for the hepatotoxicity. Until now there is no complete therapeutic strategy for the effective treatment of hepatotoxicity caused by paracetamol. Gut decontamination, N-acetylcysteine antidote administration and enhancement of elimination is used for the management of paracetamol overdose. Those with severe hepatotoxicity and neurological symptoms can benefit from removal of necrotic liver and undergo transplantation.

7. Prevention of acetaminophen-induced cataract by a combination of diallyl disulfide and N-acetylcysteine. Zhao C, Shichi H. Department of Ophthalmology, Kresge Eye Institute, Wayne State University School of Medicine, Detroit, Michigan, USA. J Ocul Pharmacol Ther 1998 Aug;14(4):345-55

Injection of acetaminophen (APAP) (350 mg/kg body weight) into C57BL/6 mice in which cytochrome P450 (CYP) 1A1/1A2 had been induced produced acute cataract and other ocular tissue damage. Treatment of APAP-injected mice with one of the major organosulfides in garlic oil, diallyl disulfide (DADS) (200 mg/kg body weight), prevented cataract development and prolonged survival time. N-acetyl L-cysteine (NAC) (500 mg/kg body weight), a prodrug that stimulates glutathione synthesis, also prolonged survival time but was effective only weakly to prevent cataract formation. A combination of DADS and NAC completely prevented cataractogenesis, and all of the treated animals survived APAP toxicity. Neither DADS nor NAC inhibited CYP 1A1/1A2 induction as determined by their effect on the induction of hepatic microsomal ethoxyresorufin O-dealkylase (ERD) activity. However, in the in vitro enzyme assay, DADS, but not NAC, was a potent inhibitor of ERD activity (IC50 = 3.5 mM). Treatment with DADS or NAC slowed but did not stop the decrease of hepatic glutathione (GSH) content. At 4 hours after APAP injection, hepatic GSH began to increase only when DADS and NAC were administered together. These results suggest that the protective effect of DADS is due to its inhibition of biotransformation of APAP to the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) by CYP 1A1/1A2 enzymes and that NAC provides protection by increasing cellular cysteine level and GSH synthesis, thus facilitating detoxification of NAPQI by glutathione conjugation. Assay of plasma glutamate-pyruvate transaminase activity, an indicator of liver necrosis, showed that treatment with DADS and NAC together effectively protected the liver. Therefore, the decrease of GSH as much as 30% of normal concentration, by itself, is not responsible for liver damage. The primary cause of hepatic necrosis is rapid accumulation of NAPQI.

8. Mechanism of action and value of N-acetylcysteine in the treatment of early and late acetaminophen poisoning: a critical review. Jones AL Scottish Poisons Information Bureau, Royal Infirmary of Edinburgh, Scotland. SPIB@compuserve.com J Toxicol Clin Toxicol (United States) 1998, 36 (4) p277-85

INTRODUCTION: The mechanism of action of N-acetylcysteine in early acetaminophen poisoning is well understood, but much remains to be learned of the mechanism of its possible benefit in acetaminophen poisoning presenting beyond 15 hours.

METHODS: Selective review of medical literature. N-acetylcysteine should be used in all cases of early acetaminophen poisoning where the plasma acetaminophen concentration lies "above the line;" which line is chosen depends on individual preference and whether enzyme induction is suspected. Particular care should be taken with the use of the nomogram for patients with chronic excess ingestion of acetaminophen or for those who have taken slow-release formulations.

CONCLUSIONS: While there is a trend suggesting a beneficial effect of N-acetylcysteine in some patients presenting beyond 15 hours, further research is necessary to establish just how effective N-acetylcysteine is, particularly in patients presenting with fulminant hepatic failure. Candidate mechanisms for a beneficial effect in-clude improvement of liver blood flow, glutathione replenishment, modification of cytokine production, and free radical or oxygen scavenging. Hemody-namic and oxygen delivery and utilization parameters must be monitored carefully during delayed N-acetylcysteine treatment of patients with fulminant hepatic failure, as unwanted vasodilation may be deleterious to the maintenance of mean arterial blood pressure. (75 Refs.)

9. Cimetidine enhances the hepatoprotective action of N-acetylcysteine in mice treated with toxic doses of paracetamol. Al-Mustafa ZH; Al-Ali AK; Qaw FS; Abdul-Cader Z Department of Pharmacology, College of Medicine and Medical Sciences, King Faisal University, Dammam, Saudi Arabia. Toxicology (IRELAND) Sep 5 1997, 121 (3) p223-8

Paracetamol, in toxic doses, is associated with extensive liver damage. This represents one of the common causes of morbidity and mortality in drug poisoning cases. This study was undertaken to investigate the possible potentiation of the hepatoprotective action of N-acetylcysteine (NAC) by cimetidine (CMD), an inhibitor of hepatic microsomal oxidative enzymes. The effects of NAC, cimetidine and the two in combination, administered 2 h post-paracetamol dose, on mortality, plasma glutamic oxaloacetic (GOT) and glutamic pyruvic (GPT) transaminase activities and hepatic reduced glutathione (GSH) levels were investigated in mice 24 h after treatment with a single oral dose of paracetamol (400 mg/kg). Both NAC and cimetidine caused a partial improvement of survival rate, plasma GOT and GPT activities. In addition, they prevented the depletion of hepatic GSH contents. However, concomitant administration of NAC and cimetidine produced a 100% survival rate and a marked reduction in plasma GOT and GPT activities to within the normal range, while significantly raising hepatic GSH concentrations to values close to those measured in saline-treated control animals. It is therefore concluded that cimetidine and N-acetylcysteine may have an additive hepatoprotective action in the treatment of paracetamol overdose.

10. Factors responsible for continuing morbidity after paracetamol poisoning in Chinese patients in Hong Kong. Chan TY; Chan AY; Critchley JA Department of Clinical Pharmacology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong. Singapore Med J (SINGAPORE) Jun 1996, 37 (3) p275-7,

To determine those factors responsible for continuing prevalence of liver damage after paracetamol poisoning, 222 Chinese patients presenting to the Prince of Wales Hospital, Hong Kong from 1988 to 1993 were studied. Of the 27 patients with plasma paracetamol concentrations above the recommended "treatment line", 13 developed liver damage. Time elapsed between ingestion and treatment with intravenous N-acetylcysteine (NAC) was the most important prognostic factor. Failure to give NAC appropriately (50%) and late presentation (23%) were the main reasons for the continuing morbidity. Liver damage in some of the remaining patients (30%) could have been prevented if NAC was started in the Emergency Department within 8-15 hours of ingestion. Liver damage after paracetamol poisoning remains common (5.9%) in Hong Kong because of the failure to give NAC appropriately or late presentation. We hope to improve patient management by repeatedly emphasising the importance of adherence to the standard protocols and having the toxic plasma level results phoned directly to the duty registrars.

11. Clinical-toxicological case (1). Dosage of N-acetylcysteine in acute paracetamol poisoning Kind B; Krahenbuhl S; Wyss PA; Meier-Abt PJ Schweizerisches Toxikologisches Informationszentrum (STIZ), Departement Innere Medizin, Universitatsspital Zurich. Schweiz Rundsch Med Prax (Switzerland) Aug 2 1996, 85 (31-32) p935-8

There are currently three protocols used for the administration of N-acetylcysteine in the treatment of acute paracetamol poisoning. In the USA only the oral protocol is approved, while in Europe an intravenous protocol is used. If treatment is started within 10 h. after paracetamol ingestion, all three protocols appear to be equally effective. If treatment is started 10 to 24 h. after the ingestion, the oral protocol and the Smilkstein protocol appear to be superior to the Prescott protocol. N-acetylcysteine is effective also when started more than 15 h after the ingestion. Patients who present with liver failure after paracetamol poisoning should be treated with a prolonged course of N-acetylcysteine.

12. Recommendations for treatment of paracetamol poisoning. Danish Medical Society, Study of the Liver Clemmesen J.O.; Ott P.; Dalhoff K.P.; Astrup L.B.; Tage-Jensen U.; Poulsen H.E. Medicinsk Afdeling A-2101, Rigshospitalet, DK-2100 Kobenhavn O Denmark Ugeskr Laeger (Denmark) Nov 25 1996, 158 (48) p6892-5

Based on recent reports concerning the efficacy of N-acetylcysteine (NAC) in paracetamol (acetaminophen) poisoning, guidelines for treatment and control of these patients are reviewed by a study group under the Danish Association for the Study of the Liver. It is recommended that NAC-treatment is initiated immediately after referral and continued for 36 hours in all cases. Further NAC-treatment should not be discontinued before a decrease in INR has been observed.

13. Overdose of Extended-Release Acetaminophen

Graudins A, Aaron CK, Linden CH Andis Graudins, M.B., B.S., University of Massachusetts N Engl J Med 1995 Jul 20;333(3):196

This is a case report of a healthy 13-year-old female who was seen in a hospital 19 hours after ingesting 2 handfuls of Tylenol Extended Relief (McNeil Pharmaceuticals) which is a formulation containing 650 mg of acetaminophen per tablet in a time-release manner. The patient received an oral dose of 140 mg of acetylcysteine per kg of body weight followed by 6 doses of 70 mg per kg and 11 doses of 100 mg per kg. The alanine aminotransferase level that was over 7,000 and the international normalized ratio of 4.2 peaked 59 hours after the ingestion of acetaminophen. The patient remained clinically well and was sent home on day 4 with resolving liver function values. There was a linear decline in serial acetaminophen -measurements. Tylenol Extended Relief is designed to maintain the analgesic effects for up to 8 hours. There are no published data with its overdose. Animal studies show that the dose of acetylcysteine needed to prevent hepatotoxicity is proportional to the dose of acetaminophen ingested.

14. Metabolic activation and paracetamol hepatotoxicity - An update on the management of paracetamol (acetaminophen) poisoning Chan T.Y.K.; Critchley J.A.J.H.; Chan J.C.N.; Tomlinson B. Department of Clinical Pharmacology, Chinese University of Hong Kong, Prince of Wales Hospital,Shatin Hong Kong Journal of the Hong Kong Medical Association (J. HONG KONG MED. ASSOC. ) (Hong Kong) 1994, 46/1 (87-92)

The assessment and current treatment of paracetamol (acetaminophen) poisoning are reviewed with emphasis on the significance of individual variation in metabolic activation capacity. The plasma paracetamol concentration in relation to the time since ingestion is the only reliable way to assess the risk of toxicity, and its measurement should be made in all suspected cases. Patients with levels above the traditional 'treatment line' should receive intravenous N-acetylcysteine (300 mg/kg) over 20 hours with the loading dose given over 30 to 60 min. This regimen is most effective if given within 10 hours, but treatment up to 24 hours helps prevent severe liver damage. Anti-oxidant therapy with intravenous N-acetylcysteine also improves the prognosis of patients with fulminant liver failure possibly due to direct or indirect free radical scavenging. Some chronic alcoholics and patients with hepatitis B related liver diseases may be more susceptible to liver damage due to an increased capacity for metabolic activation and a lower treatment threshold is indicated. The need to lower the threshold for patients taking hepatic enzyme inducing drugs such as anti-convulsants or rifampicin is much more equivocal. There is no convincing evidence for an increased metabolic activation capacity in these patients.

15. Intravenous N-acetylcysteine, hepatotoxicity and plasma glutathione S-transferase in patients with paracetamol overdosage. Beckett GJ; Donovan JW; Hussey AJ; Proudfoot AT; Prescott LF University Department of Clinical Chemistry, Royal Infirmary, Edinburgh, Scotland, UK. Human & experimental toxicology (ENGLAND) May 1990, 9 (3) p183-6,

The concentration of glutathione S-transferase B1 (GST B1) subunits was measured in sequential plasma samples taken at frequent intervals for 48 h from ten patients with severe paracetamol poisoning who were treated with intravenous N-acetylcysteine. No significant increase in plasma GST B1 concentration was observed over the study period and with 4 h of starting treatment with N-acetylcysteine there were significant decreases in plasma GST B1 concentrations. None of the patients subsequently developed significant liver damage. At the dose used for the treatment of paracetamol poisoning, N-acetylcysteine has no hepatotoxic effects.

16. Liver damage with non-narcotic analgesics. Prescott LF Medical toxicology (NEW ZEALAND) 1986,

Non-narcotic analgesics can produce a variety of hepatic lesions but clinically significant liver damage is uncommon with normal therapeutic use. The pattern of hepatotoxicity caused by the salicylates, non-steroidal anti-inflammatory drugs (NSAIDs), paracetamol (acetaminophen) and the pyrazolones differs but many of these drugs can cause generalised reactions which involve the liver. Depending on the drugs in question, the risks of liver injury may be conditioned by factors such as age, sex, dose and duration of treatment. Hepatotoxicity associated with the use of salicylates and most NSAIDs has been reported most often in females with collagen diseases but this may simply reflect the greater use of these drugs in such patients. Paracetamol-induced liver damage occurs almost exclusively as a result of overdosage. Except for the microvesicular fatty changes in hepatocytes in patients with Reye's syndrome attributed to salicylate, the acute centrilobular necrosis caused by paracetamol in overdosage and the marked cholestasis produced by benoxaprofen, the pathological changes in hepatic reactions to non-narcotic analgesics are rather variable and nonspecific. About 50% of patients given salicylate in full anti-inflammatory dosage develop minor abnormalities of liver function. There is usually a mild to moderate increase in plasma aminotransferase activity with patchy necrosis and degeneration of hepatocytes. These changes are related to plasma salicylate concentration and are usually rapidly reversible. In a small minority of patients, particularly the young, liver damage is more severe and may be associated with liver failure, acidosis, hypoglycaemia and encephalopathy. This picture closely resembles Reye's syndrome. In overdosage, paracetamol can cause acute hepatic necrosis. Without specific treatment, some 8% of adults suffer severe liver damage with plasma aminotransferase activity greater than 1000 U/L and about 1% die with hepatic failure and encephalopathy. The administration of sulfhydryl compounds such as N-acetylcysteine within 8 to 10 hours effectively prevents liver damage and death. Liver damage has been attributed to the therapeutic use of paracetamol. However, in most reports the dose was excessive and many patients were chronic alcoholics (who seem to be at increased risk). In these cases the features were typical of acute overdosage. A consistent and characteristic pattern of hepatotoxicity is evident with relatively few non-steroidal anti-inflammatory and pyrazolone analgesics. A rank order of relative risk cannot be established and the incidence in relation to use is not known.(ABSTRACT TRUNCATED AT 400

17. Plasma glutathione S-transferase measurements after paracetamol overdose: evidence for early hepatocellular damage. Beckett GJ; Chapman BJ; Dyson EH; Hayes JD Gut (ENGLAND) Jan 1985, 26 (1) p26-31,

Plasma glutathione S-transferase (GST) measurements have been used to study early changes in hepatocellular integrity after paracetamol overdose and treatment with N-acetylcysteine (NAC). Patients admitted within seven hours and successfully treated had raised or equivocal GST on admission and each showed a transient peak in GST approximately 12 hours after the overdose. Similar, though smaller changes in GST, were seen in untreated patients whose paracetamol level fell below the treatment line. The plasma GST concentrations in successfully treated patients were small compared with values found in patients who subsequently developed severe liver damage. The changes in GST concentration observed in patients who developed severe liver damage indicated that distinct early and late phases of paracetamol-induced hepatotoxicity occurred. Although the mechanism by which paracetamol exerts its early toxic effect is unclear, our data suggest that prompt treatment with NAC can successfully prevent both clinical and subclinical hepatotoxicity in this early period.

18. Paracetamol overdosage. Pharmacological considerations and clinical management Prescott L.F. Reg. Poison. Treatm. Cent., R. Infirm., Edinburgh EH3 GYW United Kingdom Drugs ( DRUGS ) (Australia) 1983, 25/3 (290-314)

Paracetamol (acetaminophen), normally a very safe drug, may produce acute centrilobular hepatic necrosis when taken in overdosage. There are no specific early symptoms or signs of paracetamol poisoning and consciousness is not impaired. Plasma aminotransferase (AST or ALT) activity may increase dramatically to 10,000 iu/L or more with prolongation of the prothrombin time ratio and mild jaundice. Acute renal failure is an uncommon complication. Maximum abnormalities of liver function are delayed for at least 3 days, and initially the prognosis can only be determined by estimation of the plasma paracetamol concentration in relation to the time after ingestion. Severe liver damage (plasma amino-transferase activity > 1000 iu/L) occurs in about 60% of patients with paracetamol concentrations above a 'treatment line' joining semilogarithmic plots of 200 mug/ml at 4 hours and 30 mug/ml at 15 hours after ingestion. Although recovery of liver function is usually rapid and complete, fulminant hepatic failure develops 3 to 6 days after ingestion in a small minority of severely poisoned patients and is often fatal. Contrary to popular belief, only about 15% of unselected patients taking paracetamol in overdosage are severely poisoned with plasma concentrations above the 'treatment line'. Without specific therapy, severe liver damage only occurs in about 8%, fatal hepatic failure in 1 to 2%, and renal failure in about 1%. Paracetamol damages the liver through the formation of a highly reactive metabolite which is normally trapped and inactivated by preferential conjugation with hepatic glutathione. Following an hepatotoxic dose, glutathione is depleted and the toxic metabolite binds covalently to vital proteins and enzymes causing cell damage and necrosis. Toxicity depends on the balance between the rate of formation of the reactive metabolite of paracetamol and the rate of glutathione synthesis. Glutathione precursors and other sulfhydryl compounds prevent glutathione depletion, covalent binding and liver damage, probably by facilitation of glutathione conjugation. Cysteamine, methionine and N-acetylcysteine prevent liver damage, renal failure and death following paracetamol overdosage if given within 8 to 10 hours; treatment after 15 hours is of no benefit. Cysteamine has long been abandoned because of its toxicity. N-acetylcysteine or methionine therapy is indicated in patients with plasma paracetamol concentrations above the 'treatment line'. Intravenous N-acetylcysteine appears more effective than methionine and is currently the treatment of choice. Oral therapy is unreliable and cannot be recommended.

19. A comparison of the protective effects of N-acetyl-cysteine and S-carboxymethylcysteine against paracetamol-induced hepatotoxicity. Ioannides C; Hall DE; Mulder DE; Steele CM; Spickett J; Delaforge M; Parke DV Toxicology (NETHERLANDS) Nov 1983, 28 (4) p313-21,

The protective effect of the sulphur-containing amino acids N-acetyl-cysteine and S-carboxymethylcysteine against paracetamol-induced hepatotoxicity was evaluated in the hamster by biochemical and histological methods. Of the animals receiving paracetamol alone 25% died within 24 h following administration. All surviving animals showed acute hepatocellular injury and marked loss of cytochrome P-450 and hepatic mixed-function oxidase activities. Simultaneous administration of N-acetylcysteine decreased the mortality rate, partly prevented the paracetamol-induced liver damage and partly restored enzyme activities. Simultaneous administration of S-carboxymethylcysteine with paracetamol afforded no protection. Kidneys from all animals were histologically normal. Human liver microsomes and liver microsomes from 3-methylcholanthrene-pretreated hamsters metabolished paracetamol to intermediate(s) that bind covalently to microsomal proteins. The rate of covalent binding was inhibited markedly by N-acetylcysteine and to a lesser extent by S-carboxylmethylcysteine.

20. Cimetidine protects against acetaminophen toxicity Jackson J.E. Sect. Clin. Pharmacol., Dept. Pharmacol., Univ. Arizona Health Sci. Cent., Tucson, AZ 85724 United States Life Sciences ( LIFE SCI. ) (United Kingdom) 1982, 31/1 (31-35)

Generally, acetaminophen (APAP) overdoses with elimination half-lives over 4 hr. sustain liver damage. In the following cases, cimetidine (C) seems to have protected against APAP toxicity. An 18 yr. old, 64 kg female smoker presented 6 hr. after taking 10 g APAP, 1200+ mg C, and small amounts of flurazepam and Sleepeze (methaprilene + scopolamine). Three plasma APAP levels (by HPLC) revealed an elimination half-life of 4.4 hr. C did not interfere with the APAP assay. Despite the long half-life in a patient with microsomal enzymes induced by smoking, no evidence of hepatotoxicity developed. A month later, the same patient overdosed with APAP alone. Three plasma levels revealed a 3.3 hr. half-life. Lack of toxicity in the presence of a long elimination half-life may indicate a protective action of C in APAP overdoses. To test the hypothesis that C protects against APAP heptatotoxicity, adult male Swiss mice were treated with 1200 mg/kg APAP by gavage. One hr. after APAP, the mice were treated with either 100 mg/kg C(n=19), 600 mg/kg N-acetylcysteine (standard therapy, n=17), or saline (control, n=17). Seven day survival was 24% among the controls, 68% with cimetidine treatment (v. control p <.05), and 100% with N-acetylcysteine (v. control P <.01; v. C, p>0.2). C inhibits the microsomal mono-oxygenase (cytochrome P-450) system which mediates acetaminophen hepatotoxicity. By preventing the formation of the toxic metabolites, C may prove useful in treating APAP overdosage. However, in our animal model it seems less effective than N-acetylcysteine.

21. Effects of aspirin and acetaminophen on the liver Zimmerman H.J. George Washington Univ. Med. Cent., Washington, D.C. 20037 United States Archives of Internal Medicine ( ARCH. INTERN. MED. ) (United States) 1981, 141/3 (333-342)

The mechanism for aspirin-caused liver injury is not clear. Aspirin produces hepatotoxic reactions as a cumulative phenomenon, requiring days or weeks to develop. Patients with active rheumatic or collagen disease, as well as children, are especially susceptible. Blood levels of salicylate higher than 25 mg/dL are particularly likely to lead to hepatic injury. Levels lower than 15 mg/dL rarely do. The mechanism for acetaminophen liver damage is quite clear. It produces hepatic injury as a result of a large single overdose, usually suicidal in intent. Patients with acetaminophen blood levels higher than 300 mg/dL at four hours after intake are most likely to develop hepatic damage; when N-acetylcysteine is used within the first ten hours after ingestion of an overdose, the recovery rate is reported to be virtually 100%. The conditions of patients receiving long-term full doses of either aspirin or acetaminophen should be intermittently monitored for hepatic injury.

22. Treatment of paracetamol (acetaminophen) poisoning with N-acetylcysteine Prescott L.F.; Park J.; Ballantyne A.; et al. Reg. Poisoning Treatm. Cent., Roy. Infirm., Edinburgh United Kingdom Lancet ( LANCET ) (United Kingdom) 1977, 2/8035 (432-434)

Fifteen patients with paracetamol (acetaminophen) poisoning were treated with intravenous N acetylcysteine (300 mg/kg given over 20 h). Mean admission and 4 h plasma-paracetamol concentrations were 262 and 369 mug/ml, respectively. Liver function tests remained normal or were only slightly disturbed in 11 of 12 patients treated within 10 h of paracetamol ingestion. Severe liver damage developed in the other patient and in the three in whom treatment was started more than 10 h after paracetamol ingestion. In contrast to cysteamine, N-acetylcysteine was very well tolerated and has the advantage of being available as a pharmaceutical preparation in a 20% sterile solution.

23. The disposition and kinetics of intravenous N-acetylcysteine in patients with paracetamol overdosage Prescott L.F.; Donovan J.W.; Jarvie D.R.; Proudfoot A.T. University Department of Clinical Pharmacology, Royal Infirmary, Edinburgh EH3 97W United Kingdom European Journal of Clinical Pharmacology ( EUR. J. CLIN. PHARMACOL. ) ( Germany) 1989, 37/5 (501-506)

Seventeen patients received standard treatment with intravenous N-acetylcysteine for 18 episodes of severe poisoning with paracetamol (acetaminophen). The dose of N-acetylgysteine was 150 mg/kg given in 15 min followed by 50 mg/kg in 4 h and 100 mg/kg over the next 16 h. Liver damage was absenct or mild on 13 occasions (ALT < 500 mu/l) and server on 5 (ALT > 1000 mu/l). Total plasma N-acetylcysteine was estimated by HPLC. The mean maximum plasma concentration after the initial loading dose was 554 mg/l. Concentrations then fell rapidly and after 12 h a nean steady-state level of about 36 mg/l was maintained. When the infusion was discontinued N-acetylcysteine disappeared with a half-life of 5.7 h. The mean steady-state volume of distribution, AUC, mean residence time and total clearance were 536 ml/kg, 1748 mg - h - lsup -sup 1, 2.91 h and 3.18 ml - minsup -sup 1 - kgsup -sup 1. These values are generally consistent with those previously reported with much smaller doses and the disposition of N-acetylcysteine does not appear to be dose-dependent. The elimination of N-acetylcysteine was not impaired in the patients with severe liver damage, and the pharmacokinetic variables and plasma concentrations were similar in patients with adn without hepatotoxicity. The dosage schedule for intravenous N-acetylcysteine should probably be modified since adverse reactions invariably occur early when plasma concentrations are at their highest, and liver damage was prevented just as effectively at the lowest as at the highest C(max). High initial concentrations of N-acetylcysteine can be avoided with simple alternative regimens based on the kinetic data of this study.

24. Acetaminophen hepatotoxicity and malnutrition Newman T.J.; Bargman G.J. Dept. Ped., Univ. Wisconsin Hosp., Madison, Wis. 53706 United States American Journal of Gastroenterology ( AM. J. GASTROENTEROL. ) (United States) 1979, 72/6 (647-650)

A patient with severe anorexia nervosa, who ingested 15 g of acetaminophen, was treated with oral N-acetylcysteine. Contrary to suggestions in the literature that malnutrition increases the susceptibility of patients to the hepatotoxic effects of acetaminophen this patient survived without evidence of liver damage. Changes in the metabolism of acetaminophen secondary to poor nutrition may explain the benign course in this and similar patients.

ALCOHOL **

25. Protective effect of N-acetylcysteine on rat liver cell membrane during methanol intoxication. Dobrzynska I, Skrzydlewska E, Kasacka I, Figaszewski Z. Institute of Chemistry, University in Bialystok, Poland. J Pharm Pharmacol. 2000 May;52(5):547-52

Methanol is oxidized in-vivo to formaldehyde and then to formate, and these processes are accompanied by the generation of free radicals. We have studied the effect of N-acetylcysteine on liver cell membrane from rats intoxicated with methanol (3.0 g kg(-1)). Evaluation of the effect was achieved by several methods. Lipid peroxidation and surface charge density were measured. An ultrastructural study of the liver cells was undertaken. The concentration of marker enzymes of liver damage (alanine aminotransferase and aspartate aminotransferase) in blood serum was measured. Methanol administration caused an increase in lipid peroxidation products (approximately 30%) as well as in surface charge density (approximately 60%). This might have resulted in the membrane liver cell damage visible under electron microscopy and a leak of alanine aminotransferase and aspartate aminotransferase into the blood (increase of approximately 70 and 50%, respectively). Ingestion of N-acetylcysteine with methanol partially prevented these methanol-induced changes. Compared with the control group, lipid peroxidation was increased by approximately 3% and surface charge density by approximately 30%. Alanine aminotransferase and aspartate aminotransferase activity increased by 9 and 8%, respectively, compared with the control group. The results suggested that N-acetylcysteine was an effective antioxidant in methanol intoxication. It may have efficacy in protecting free radical damage to liver cells following methanol intoxication.

ALS **

26. Neurobiol Dis. 2003 Aug;13(3):213-21. Mitochondrial dysfunction due to mutant copper/zinc superoxide dismutase associated with amyotrophic lateral sclerosis is reversed by N-acetylcysteine. Beretta S, Sala G, Mattavelli L, Ceresa C, Casciati A, Ferri A, Carri MT, Ferrarese C. Department of Neuroscience and Biomedical Technologies, University of Milano-Bicocca, San Gerardo Hospital, via Donizetti, 106, 20052, Monza (MI), Italy.

We report that the expression of mutant G93A copper/zinc superoxide dismutase (SOD1), associated with familial amyotrophic lateral sclerosis, specifically causes a decrease in MTT reduction rate and ATP levels and an increase in both cytosolic and mitochondrial reactive oxygen species (ROS) production in human neuroblastoma SH-SY5Y cells compared to cells overexpressing wild-type SOD1 and untransfected cells. Exposure to N-acetylcysteine lowers ROS production and returns mitochondrial functional assays to control levels. No large aggregates of human SOD1 are detectable under basal growth conditions in any of the investigated cell lines. After proteasome activity inhibition, SOD1 aggregates can be detected exclusively in G93A-SOD1 cells, even though they do not per se enhance cell death compared to control cell lines. Our findings indicate that mitochondrial homeostasis is affected by mutant SOD1-generated ROS independently from the formation of aggregates and that this alteration is reversed by antioxidants.

27. Neurochem Int. 2001 Aug;39(2):141-9. Glutathione elevation and its protective role in acrolein-induced protein damage in synaptosomal membranes: relevance to brain lipid peroxidation in neurodegenerative disease. Pocernich CB, Cardin AL, Racine CL, Lauderback CM, Butterfield DA. Department of Chemistry, 125 Chemistry-Physics Building, University of Kentucky, Lexington, KY 40506, USA.

Oxidative stress may be a hallmark of several neurodegenerative disorders, including Alzheimer's disease (AD) Huntington's, and Parkinson's diseases as well as amyotrophic lateral sclerosis. Acrolein is a highly reactive product of lipid peroxidation that is elevated in the brains of persons with AD. This alkenal potentially can react with proteins by Michael addition to alter their structure and function. In the present study, we used electron paramagnetic resonance in conjunction with a protein-specific spin label to monitor synaptosomal membrane protein conformational alterations induced by acrolein. A dose-dependent increased conformational alteration was observed. Consistent with this finding, protein carbonyl levels from protein-bound acrolein were significantly elevated. However, pretreatment of synaptosomes with glutathione ethyl ester (GEE) significantly ameliorated both the conformational alterations and protein carbonyls induced by acrolein. Based on this success, we tested the hypothesis that elevated levels of endogenous glutathione (GSH) would offer protection against acrolein-induced oxidative stress. In-vivo elevation of GSH (215% over control, P<0.04) was produced by i.p. injection of N-acetylcysteine (NAC), a known precursor of GSH. Synaptosomes were treated with vehicle or 2 nM acrolein, the level of this alkenal found in AD brain. In contrast to synaptosomes from control animals, which had significantly increased protein carbonyl levels following addition of 2 nM acrolein, synaptosomes that were isolated from NAC-treated rodents and treated with 2 nM acrolein showed no increased carbonyl levels compared to untreated controls. These results demonstrate protection by increased in-vivo GSH levels against acrolein-induced oxidative stress at levels found in AD brain and are consistent with the notion that methods to increase endogenous GSH levels in neurodegenerative diseases associated with oxidative stress may be promising.

28. J Neurochem. 2001 Jan;76(1):224-33. N-acetyl-L-cysteine protects SHSY5Y neuroblastoma cells from oxidative stress and cell cytotoxicity: effects on beta-amyloid secretion and tau phosphorylation. Olivieri G, Baysang G, Meier F, Muller-Spahn F, Stahelin HB, Brockhaus M, Brack C. Neurobiology Laboratory, Psychiatric University Hospital, Basel, Switzerland. gianfranco.olivieri@pukbasel.ch

Redox changes within neurones are increasingly being implicated as an important causative agent in brain ageing and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). Cells have developed a number of defensive mechanisms to maintain intracellular redox homeostasis, including the glutathione (GSH) system and antioxidant enzymes. Here we examine the effects of N-acetyl-L-cysteine (NAC) on beta-amyloid (A beta) secretion and tau phosphorylation in SHSY5Y neuroblastoma cells after exposure to oxidative stress inducing/cytotoxic compounds (H(2)O(2), UV light and toxic A beta peptides). A beta and tau protein are hallmark molecules in the pathology of AD while the stress factors are implicated in the aetiology of AD. The results show that H(2)O(2), UV light, A beta 1-42 and toxic A beta 25-35, but not the inactive A beta 35-25, produce a significant induction of oxidative stress and cell cytotoxicity. The effects are reversed when cells are pre-treated with 30 mM NAC. Cells exposed to H(2)O(2), UV light and A beta 25-35, but not A beta 35-25, secrete significantly higher amounts of A beta 1-40 and A beta 1-42 into the culture medium. NAC pre-treatment increased the release of A beta 1-40 compared with controls and potentiated the release of both A beta 1-40 and A beta 1-42 in A beta 25-35-treated cells. Tau phosphorylation was markedly reduced by H(2)O(2) and UV light but increased by A beta 25-35. NAC strongly lowered phospho-tau levels in the presence or absence of stress treatment.

29. N-acetyl-L-cysteine improves survival and preserves motor performance in an animal model of familial amyotrophic lateral sclerosis. Andreassen OA, Dedeoglu A, Klivenyi P, Beal MF, Bush AI. Neurology Service, Massachusetts General Hospital and Harvard Medical School, Boston, USA. Neuroreport 2000 Aug 3;11(11):2491-3

Increasing evidence implicates oxidative damage as a major mechanism in the pathogenesis of amyotrophic lateral sclerosis (ALS). We examined the effect of preventative treatment with N-acetyl-L-cysteine (NAC), an agent that reduces free radical damage, in transgenic mice with a superoxide dismutase (SODI) mutation (G93A), used as an animal model of familial ALS. NAC was administered at 1% concentration in the drinking water from 4-5 weeks of age. The treatment caused a significantly prolonged survival and delayed onset of motor impairment in G93A mice treated with NAC compared to control mice. These results provide further evidence for the involvement of free radical damage in the G93A mice, and support the possibility that NAC, an over-the-counter antioxidant, could be explored in clinical trials for ALS.

30. Reduction of lower motor neuron degeneration in wobbler mice by N-acetyl-L-cysteine. Henderson JT, Javaheri M, Kopko S, Roder JC. Samuel Lunenfeld Research Institute, Program in Development and Fetal Health, Mount Sinai Hospital, Toronto, Ontario, Canada. J Neurosci 1996 Dec 1;16(23):7574-82

The murine mutant wobbler is a model of lower motoneuron degeneration with associated skeletal muscle atrophy. This mutation most closely resembles Werdnig-Hofmann disease in humans and shares some of the clinical features of amyotrophic lateral sclerosis (ALS). It has been suggested that reactive oxygen species (ROS) may play a role in the pathogenesis of disorders such as ALS. To examine the relationship between ROS and neural degeneration, we have studied the effects of agents such as N-acetyl-L-cysteine (NAC), which reduce free radical damage. Litters of wobbler mice were given a 1% solution of the glutathione precursor NAC in their drinking water for a period of 9 weeks. Functional and neuroanatomical examination of these animals revealed that wobbler mice treated with NAC exhibited (1) a significant reduction in motor neuron loss and elevated glutathione peroxidase levels within the cervical spinal cord, (2) increased axon caliber in the medial facial nerve, (3) increased muscle mass and muscle fiber area in the triceps and flexor carpi ulnaris muscles, and (4) increased functional efficiency of the forelimbs, as compared with untreated wobbler littermates. These data suggest that reactive oxygen species may be involved in the degeneration of motor neurons in wobbler mice and demonstrate that oral administration of NAC effectively reduces the degree of motor degeneration in wobbler mice. This treatment thus may be applicable in the treatment of other lower motor neuropathies.

CANCER **

31. Cancer Res. 2003 Jun 15;63(12):3413-7. 2-Deoxy-D-glucose-induced cytotoxicity and radiosensitization in tumor cells is mediated via disruptions in thiol metabolism. Lin X, Zhang F, Bradbury CM, Kaushal A, Li L, Spitz DR, Aft RL, Gius D. Section of Cancer Biology, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri, USA.

Exposure to ionizing radiation is believed to cause cell injury via the production of free radicals that are thought to induce oxidative damage. It has been proposed that exposure to agents that enhance oxidative stress-induced injury by disrupting thiol metabolism may sensitize cells to the cytotoxic effects of ionizing radiation. Recently, it has been shown that glucose deprivation selectively induces cell injury in transformed human cells via metabolic oxidative stress (J. Biol. Chem., 273: 5294-5299; Ann. N.Y. Acad. Sci., 899: 349-362), resulting in profound disruptions in thiol metabolism. Because 2-deoxy-D-glucose (2DG) is a potent inhibitor of glucose metabolism thought to mimic glucose deprivation in vivo, the hypothesis that exposure to 2DG might be capable of inducing radiosensitization in transformed cells via perturbations in thiol metabolism was tested. When HeLa cells were exposed to 2DG (4-10 mM) for 4-72 h, cell survival decreased (20-90%) in a dose- and time-dependent fashion. When HeLa cells were treated with 6 mM 2DG for 16 h before ionizing radiation exposure, radiosensitization was observed with a sensitizer enhancement ratio of 1.4 at 10% isosurvival. Treatment with 2DG was also found to cause decreases in intracellular total glutathione content (50%). Simultaneous treatment with the thiol antioxidant N-acetylcysteine (NAC; 30 mM) protected HeLa cells against the cytotoxicity and radiosensitizing effects of 2DG, without altering radiosensitivity in the absence of 2DG. Furthermore, treatment with NAC partially reversed the 2DG-induced decreases in total glutathione content, as well as augmented intracellular cysteine content. Finally, the cytotoxicity and radiosensitizing effects of 2DG were more pronounced in v-Fos-transformed versus nontransformed immortalized rat cells, and this radiosensitization was also inhibited by treatment with NAC. These results support the hypothesis that exposure to 2DG causes cytotoxicity and radiosensitization via a mechanism involving perturbations in thiol metabolism and allows for the speculation that these effects may be more pronounced in transformed versus normal cells.

32. Int J Biol Markers. 2003 Jan-Mar;18(1):70-4. Antiangiogenic activity of chemopreventive drugs. Pfeffer U, Ferrari N, Morini M, Benelli R, Noonan DM, Albini A. Laboratory of Molecular Oncology, National Cancer Research Institute, Genoa, Italy. ulrich.pfeffer@istge.it

Tumors growing within the host form dynamic aberrant tissue that consists of host components, including the stroma, an expanding vasculature and often chronic inflammation, in addition to the tumor cells themselves. These host components can contribute to, rather than limit, tumor expansion, whereas deprivation of vessel formation has the potential to confine tumors in small, clinically silent foci. Therapeutic inhibition of vessel formation could be best suited to preventive strategies aimed at the suppression of angiogenesis in primary tumors in subjects at risk, or of micrometastases after surgical removal of a primary tumor. Our analysis of potential cancer chemopreventive molecules including N-acetylcysteine, green tea flavonoids and 4-hydroxyphenyl-retinamide has identified antiangiogenic activities that could account--at least in part--for the tumor prevention effects observed with these compounds. These drugs appear to target common mechanisms of tumor angiogenesis that may permit identification of critical targets for antiangiogenic therapy and antiangiogenic chemoprevention.

33. J Environ Pathol Toxicol Oncol. 2003;22(1):17-28. Reactive oxygen species, antioxidant mechanisms, and serum cytokine levels in cancer patients: impact of an antioxidant treatment. Mantovani G, Maccio A, Madeddu C, Mura L, Massa E, Gramignano G, Lusso MR, Murgia V, Camboni P, Ferreli L. Department of Medical Oncology, University of Cagliari, Cagliari, Italy. mantovan@pacs.unica.it

OBJECTIVE: It has not been well established whether the oxidative stress found in cancer patients results from an increased production of oxidants in the body or from a failure of physiological antioxidant systems. To further investigate this question, we have assessed the blood levels of reactive oxygen species as a marker of free radicals producing oxidative stress and the most relevant of the physiological body enzymes counteracting reactive oxygen species, namely glutathione peroxidase and superoxide dismutase. We also investigated serum levels of proinflammatory cytokines and IL-2. All of these parameters were studied in relation to the most important clinical index of disease progression--namely, the Eastern Cooperative Oncology Group (ECOG) Performance Status (PS). We also tested the reducing ability of different antioxidant agents on reactive oxygen species levels by measuring the increase in glutathione peroxidase activity and the reduction of serum levels of IL-6 and TNF-alpha. PATIENTS AND METHODS: We carried out an open nonrandomized study on 28 advanced stage cancer patients (stage III, 10.7% and stage IV, 89.3%) with tumors at different sites. The patients were divided into 5 groups, and a different antioxidant treatment was administered to each group. The antioxidants were alpha lipoic acid 200 mg/day orally; N-acetylcysteine 1800 mg/day i.v. or carboxycysteine-lysine salt 2.7 g/day orally; amifostine 375 mg/day i.v.; reduced glutathione 600 mg/day i.v.; and a combination of vitamin A 30,000 IU/day orally, vitamin E 70 mg/day orally, and vitamin C 500 mg/day orally. The antioxidant treatment was administered for 10 consecutive days. RESULTS: We found that all but one of the antioxidants tested were effective in reducing reactive oxygen species levels, and two of them (cysteine-containing compounds and amifostine) had the additional effect of increasing glutathione peroxidase activity. Comprehensively, the antioxidant treatment was found to have an effect on both reactive oxygen species levels and glutathione peroxidase activity. The antioxidant treatment also reduced the serum levels of IL-6 and TNF-alpha. Patients in both ECOG PS 0-1 and ECOG PS 2-3 responded to antioxidant treatment.

34. Cell Mol Life Sci. 2003 Jan;60(1):6-20. Molecular mechanisms of N-acetylcysteine actions. Zafarullah M, Li WQ, Sylvester J, Ahmad M. Departement de Medecine, Centre de Recherche du Centre Hospitalier de l'Universite de Montreal, Lab. K-5255 Mailloux, Hopital Notre-Dame du CHUM, 1560 Sherbrooke est, Montreal, Quebec H2L 4M1, Canada. Muhammad.Zafarullah@umontreal.ca

Oxidative stress generated by an imbalance between reactive oxygen species (ROS) and antioxidants contributes to the pathogenesis of arthritis, cancer, cardiovascular, liver and respiratory diseases. Proinflammatory cytokines and growth factors stimulate ROS production as signaling mediators. Antioxidants such as N-acetylcysteine (NAC) have been used as tools for investigating the role of ROS in numerous biological and pathological processes. NAC inhibits activation of c-Jun N-terminal kinase, p38 MAP kinase and redox-sensitive activating protein-1 and nuclear factor kappa B transcription factor activities regulating expression of numerous genes. NAC can also prevent apoptosis and promote cell survival by activating extracellular signal-regulated kinase pathway, a concept useful for treating certain degenerative diseases. NAC directly modifies the activity of several proteins by its reducing activity. Despite its nonspecificity, ability to modify DNA and multiple molecular modes of action, NAC has therapeutic value for reducing endothelial dysfunction, inflammation, fibrosis, invasion, cartilage erosion, acetaminophen detoxification and transplant prolongation.

35. Toxicol Pathol. 2003 Jan-Feb;31(1):39-51. Slowing tumorigenic progression in TRAMP mice and prostatic carcinoma cell lines using natural anti-oxidant from spinach, NAO--a comparative study of three anti-oxidants. Nyska A, Suttie A, Bakshi S, Lomnitski L, Grossman S, Bergman M, Ben-Shaul V, Crocket P, Haseman JK, Moser G, Goldsworthy TL, Maronpot RR. Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, North Carolina 27709, USA. nyska@niehs.nih.gov

The TRAMP model and human prostatic cancer (PCA) cell lines DU145 and PC3 are useful forchemopreventive studies. We compared the efficacy of 3 anti-oxidants [a water-soluble natural anti-oxidant. NAO (200 mg/kg). found in spinach leaves; epigallocatechin-3 gallate, EGCG (200 mg/kg), a major green tea polyphenol; and N-acetylcysteine, NAC (125 mg/kg)] plus vehicle in slowing spontaneous tumorigenic progression in TRAMP and wild-type male mice. Sacrifices occurred on weeks 5, 9, and 13. Prostatic histopathology and oxidative-stress blood markers were evaluated. Hyperplasias were ranked by a combination of severity grade and distribution (focal, multifocal, and diffuse). The effectivity of each tested compound in reducing the severity/focalness of hyperplasia varied from lobe to lobe. NAO exerted a significant effect on the dorsal and lateral lobes; NAC, on the anterior and ventral lobes, and EGCG, on the ventral lobe. When the most severe hyperplasia in all 4 lobes of TRAMPs was evaluated, only NAO reduced hyperplasia at weeks 9 and 13. Plasma peroxide levels in TRAMPs were reduced following oral administration of NAO or NAC for 13 weeks; EGCG only slightly reduced these levels. In NAO-treated DU 145 and PC3 PCA cells, inhibition of cellular proliferation occurred in a dose-dependent manner, increasing numbers of G1 cells and reducing ROS levels. The anti-oxidative and antiproliferative properties of NAO may explain its efficacy in slowing the spontaneous prostatic carcinogenic process in the TRAMP and its effects in the cell lines.

36. Toxicol Lett. 2003 Mar 3;138(3):243-51. Astroglial CYP1B1 up-regulation in inflammatory/oxidative toxic conditions: IL-1beta effect and protection by N-acetylcysteine. Malaplate-Armand C, Ferrari L, Masson C, Siest G, Batt AM. Centre du Medicament, Inserm U525, Faculte de Pharmacie, Universite Henri Poincare Nancy I, 30 rue Lionnois, 54000 Nancy, France.

The present work aims to determine the relevance of an astrocytoma cell line U373 MG, for assessing the role of some astroglial cytochrome P450 in neurotoxicity and neuroprotection. CYP1B1, CYP2C8, CYP2C9, CYP2D6, CYP2J2, CYP2E1 and CYP4A11 mRNA were detected by reverse transcriptase-polymerase chain reaction in control U373 MG cell cultures. Among them we focused on CYP1B1 expression. After 48 h treatment with a range of concentrations of interleukin-1beta (1, 5, 10 ng/ml) used to simulate stress conditions, CYP1B1 mRNA expression was enhanced in a dose-dependent way. This increased expression was followed 24 h later by an increase in protein level, determined by Western-blot. N-acetylcysteine (NAC) partially inhibited this effect both on the mRNA and protein levels. As CYP1B1 activates procarcinogenic compounds to reactive metabolites, an increase in this P450 isoform will participate to toxic consequences of an inflammatory/oxidative stress. NAC will prevent this deleterious effect. Copyright 2002 Elsevier Science Ireland Ltd.

37. J Neurooncol. 2002 Jan;56(2):109-17. Mitogen activated protein kinase activation and oxidant signaling in astrocytoma cells. Kuruganti PA, Wurster RD, Lucchesi PA. Neuroscience Program, Department of Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, IL, USA. pkuruganti@yahoo.com

Presence of increased reactive oxygen species (ROS) has been observed in most high risk factors for brain tumor development. Our past study demonstrated that ROS could induce increased brain tumor cell proliferation. Growth effects of ROS may involve modifications of cellular proteins such as mitogen-activated protein kinases (MAPKs), which regulate cell proliferation. Here, we report effects of a ROS (hydrogen peroxide, H2O2) and an antioxidant (N-acetylcysteine, NAC) on MAPK activation in astrocytoma (U373-MG) cells. MAPKs are activated by phosphorylation that can be detected by Western blot analysis. The unphosphorylated/inactivated form of MAPK exhibits slower mobility on SDS-PAGE compared to the phosphorylated/activated form. Densitometric analysis was used to measure MAPK activation. Results indicate that H2O2 caused a dose and time-dependent increase in MAPK activation in astrocytoma cells. Furthermore, ROS-induced activation was almost completely suppressed by NAC. NAC also inhibited serum-induced MAPK activation indicating there may be an oxidant-sensitive component to serum-induced growth signaling. Modifications of MAPKs by H2O2 demonstrate that ROS-induced proliferation is via biochemical pathways similar to other known growth stimuli. Understanding of processes that link a proliferation signal (ROS) to cell proliferation can aid in the selection of therapy used to suppress brain tumor growth.

38. BMC Infect Dis. 2002 Mar 28;2(1):5. Respiratory syncytial virus and TNF alpha induction of chemokine gene expression involves differential activation of Rel A and NF-kappa B1. Carpenter LR, Moy JN, Roebuck KA. Department of Immunology/Microbiology, Rush-Presbyterian-St, Luke's Medical Center Chicago, IL 60612, USA. lcarpent@rush.edu

BACKGROUND: Respiratory syncytial virus (RSV) infection of airway epithelial cells stimulates the expression and secretion of a variety of cytokines including the chemotactic cytokines interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and RANTES (regulated upon activation, normal T cell expressed and secreted). Chemokines are important chemoattractants for the recruitment of distinct sets of leukocytes to airway sites of inflammation. RESULTS: We have shown previously that chemokine expression is regulated in airway epithelial cells (A549) in a stimulus-specific manner in part through the redox-responsive transcription factors AP-1 and NF-kappaB. In this study, we examined the NF-kappaB-mediated effects of RSV and the proinflammatory cytokine TNFalpha on the induction of IL-8, MCP-1 and RANTES chemokine gene expression in A549 epithelial cells. The results demonstrate that RSV induces chemokine expression with distinct kinetics that is associated with a specific pattern of NF-kappaB binding activity. This distinction was further demonstrated by the differential effects of the NF-kappaB inhibitors dexamethasone (DEX) and N-acetyl-L-cysteine (NAC). NAC preferentially inhibited RSV induced chemokine expression, whereas DEX preferentially inhibited TNFalpha induced chemokine expression. DNA binding studies using NF-kappaB subunit specific binding ELISA demonstrated that RSV and TNFalpha induced different NF-kappaB binding complexes containing Rel A (p65) and NF-kappaB1 (p50). Both TNFalpha and RSV strongly induced Rel A the activation subunit of NF-kappaB, whereas only TNFalpha was able to substantially induce the p50 subunit. Consistent with the expression studies, RSV but not TNFalpha induction of Rel A and p50 were markedly inhibited by NAC, providing a mechanism by which TNFalpha and RSV can differentially activate chemokine gene expression via NF-kappaB. CONCLUSIONS: These data suggest that RSV induction of chemokine gene expression, in contrast to TNFalpha, involves redox-sensitive NF-kappaB complexes containing predominantly Rel A.

39. Ann N Y Acad Sci. 2002 Nov;973:555-8. Type I insulin-like growth factor receptor expression on colorectal adenocarcinoma cell lines is decreased in response to the chemopreventive agent N-acetyl-l-cysteine. Kelly RG, Nally K, Shanahan F, O'Connell J. Department of Medicine, University College Cork, Ireland.

Increased expression of the type I insulin-like growth factor receptor (IGF-1R) is associated with colon cancer, while the antioxidant N-acetyl-l-cysteine (NAC) is known to suppress colonic proliferation. We demonstrate that NAC down-regulates the expression of IGF-1R on three colorectal adenocarcinoma cell lines (HT29, SW480, and LoVo). NAC also abrogates the proliferative effect of IGF-I on HT29 cells. This indicates a novel mechanism for the therapeutic effects of NAC.

40. Nutr Cancer. 2002;43(1):59-66. Timing of supplementation with the antioxidant N-acetyl-L-cysteine reduces tumor multiplicity in novel, cancer-prone p53 haploinsufficient Tg.AC (v-Ha-ras) transgenic mice but has no impact on malignant progression. Martin KR, Saulnier MJ, Kari FW, Barrett JC, French JE. Transgenic Carcinogenesis Unit, Laboratory of Environmental Carcinogenesis and Mutagenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. krm12@psu.edu

Epidemiological studies support the protective role of dietary antioxidants in preventing cancer. However, emerging evidence suggests that antioxidant supplements may actually exacerbate carcinogenesis. We explored this paradox in a model containing two common genotypic characteristics of human cancers. We selected p53 haploinsufficient Tg.AC (v-Ha-ras) mice as a model, because it contains an activated, carcinogen-inducible ras oncogene and an inactivated p53 tumor suppressor gene. These mice develop chemically induced benign and malignant skin tumors rapidly. Mice were fed basal diet with or without 3% N-acetyl-L-cysteine (NAC) before and after topical application of the carcinogen benzo[a]pyrene (64 micrograms twice per week for 7 wk) until 50% of mice within a group displayed at least one lesion. Half each of mice fed the basal and the NAC-supplemented diet were then switched to the alternate diet. Mice fed the NAC-supplemented diet or switched from the NAC-supplemented to the basal diet displayed 38% and 26% reductions, respectively, in tumor multiplicity and a 15% reduction if switched from the basal to the NAC-supplemented diet. Although latency was unaffected, NAC induced a lag in tumor incidence, which exceeded 90% at 10 wk for all groups. The timing of NAC supplementation did not affect malignant progression. Thus dietary NAC was chemoprotective by slowing tumorigenesis but did not affect malignant conversion.

41. Acta Biol Hung. 2002;53(3):293-8. N-acetil-l-cysteine and 2-amino-2-thiiazoline N-acetyl-l-cysteinate as a possible cancer chemopreventive agents in murine models. Simkeviciene V, Straukas J, Uleckiene S. Institute of Biochemistry, Vilnius, Lithuania. vitalija@bchi.lt

The aim of this study was to investigate N-acetyl-cysteine (NAC) and its 2-amino-2-thiazoline salt (NACAT) as potential chemopreventive agents on experimentally induced lung tumours by urethane (U) in mice. Female BALB/c mice were used. U was given by intraperitoneal injections during 2 weeks (single dose - 10 mg/mouse, total - 50 mg/mouse). Mice were treated daily per os with NAC 1/10 LD50, NACAT 1/10 or 1/100 LD50 starting 2 weeks prior U administration, then during U treatment and thereafter for 2 months. The duration of experiment was 4 months. The results showed that NAC (1000 mg/kg) reduced the lung tumour incidence to 30% that of controls, P < or = 0.05. Most effective of NACAT was 100 mg/kg dose; it reduced an average of lung adenomas per mouse by 26%, P < or = 0.05, but lower dose (10 mg/kg) was less effective. In order to achieve similar chemopreventive effect (approximately 30%) on mice, it is necessary to use 0.38 mM/kg of NACAT or 6.13 mM/kg of NAC. It means that 16 times less of NACAT is required, if calculated by molar concentration. In general, NAC and NACAT have a moderate chemopreventive effect on lung tumorigenesis induced by urethane in mice.

42. Ren Fail. 2002 Jul;24(4):529-33. N-acetylcysteine as salvage therapy in cisplatin nephrotoxicity. Nisar S, Feinfeld DA. Department of Medicine, Nassau University Medical Center, East Meadow, NY 11554, USA.

N-acetylcysteine (NAC) repletes intracellular stores of reduced glutathione and may be a scavenger of oxygen free radicals. We report a 52-year-old female who developed acute renal insufficiency after administration of one dose of 150 mg of cisplatin for treatment of squamous cell cancer of the esophagus. Her blood urea nitrogen and creatinine rose from 12 and 0.7 mg/dL, respectively, to 24 and 1.8 mg/dL on day 5 after cisplatin. On that day the patient was begun on NAC, starting with a loading dose of 140-mg/kg-body weight followed by 70mg/kg every 4h for 4 days. Two days after starting NAC her renal function began to improve, and although she failed to complete a full course of the drug, by day 10 her serum creatinine had fallen to 0.8 mg/dL. A previous report showed that N-acetylcysteine might reverse cisplatin-induced renal toxicity. Our case supports this hypothesis.

43. Carcinogenesis. 2002 Sep;23(9):1455-61. Inhibition of lung tumorigenesis in A/J mice by N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine and myo-inositol, individually and in combination. Hecht SS, Upadhyaya P, Wang M, Bliss RL, McIntee EJ, Kenney PM. University of Minnesota Cancer Center, Minneapolis, MN 55455, USA. hecht002@umn.edu

Isothiocyanates, their N-acetylcysteine conjugates, and myo-inositol (MI) are inhibitors of lung tumorigenesis in A/J mice. However, chemoprevention by combinations of these compounds in different temporal sequences has not been examined. This is important for developing practical approaches to lung cancer chemoprevention in smokers and ex-smokers. We used a tumor model in which A/J mice are treated with 8 weekly doses of benzo[a]pyrene (B[a]P) plus 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and killed 19 weeks after the final treatment. In Experiment 1, isothiocyanates or their N-acetylcysteine conjugates were added to the diet (1 or 3 micro mol/g) from 1 week before until 1 week after carcinogen treatment. The compounds were 2-phenethyl isothiocyanate (PEITC), 3-phenylpropyl isothiocyanate (PPITC), N-acetyl-S-(N-benzyl-thiocarbamoyl)-L-cysteine (BITC-NAC), N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine (PEITC-NAC), and N-acetyl-S-(N-3-phenylpropylthiocarbamoyl)-L-cysteine (PPITC-NAC). Significant reductions in lung tumor multiplicity were observed in mice treated with PEITC, PEITC-NAC, PPITC and PPITC-NAC. PEITC-NAC was chosen for combination studies with MI (Experiment 2). Mice were treated with B[a]P plus NNK without or with PEITC-NAC (3 micro mol/g diet), MI (55.5 micro mol/g diet), or PEITC-NAC plus MI (3 micro mol plus 55.5 micro mol/g diet). Different temporal sequences of dietary additions were investigated: carcinogen treatment phase; post-carcinogen treatment phase; entire experiment; 50% of carcinogen treatment phase until termination; and 75% of carcinogen treatment phase until termination. All treatments reduced lung tumor multiplicity except PEITC-NAC post-carcinogen or from 75% of the carcinogen treatment phase. Reduction of lung tumor multiplicity by PEITC-NAC plus MI was greater than that in the mice treated with the agents alone in all temporal sequences. When all results were combined, PEITC-NAC plus MI was significantly more effective than the agents alone. There was a significant trend for reduction in lung tumor multiplicity with increased duration of treatment by the chemopreventive agents. These results provide a basis for further development of mixtures of PEITC-NAC and MI for chemoprevention of lung cancer.

44. J Nutr. 2002 Aug;132(8):2151-6. N-acetylcysteine, vitamin C and vitamin E diminish homocysteine thiolactone-induced apoptosis in human promyeloid HL-60 cells. Huang RF, Huang SM, Lin BS, Hung CY, Lu HT. Department of Nutrition and Food Sciences, Fu-Jen University, Hsin-Chuang, Taiwan, ROC. rweifen@mails.fju.edu.tw

We showed previously that homocysteine thiolactone (HcyT) is a potent inducer of apoptosis in HL-60 cells. In the present study, the role of some radical scavengers (N-acetylcysteine, vitamin C, vitamin E and folate) on the reduction of HcyT-induced apoptosis was investigated. Preincubation of HcyT-treated HL-60 cells with vitamin C (Vit C; 100 micro mol/L) or vitamin E (Vit E; 100 micro mol/L) for 2 h significantly reduced the proportion of apoptotic cells with hypodiploid DNA contents or with membrane phosphatidylserine exposure, and attenuated the apoptotic DNA fragmentation. Preincubation of cells with N-acetylcysteine (NAC; 5 mmol/L) for 2 h significantly reduced HcyT-promoted apoptosis measured by membrane phosphatidylserine exposure only. The reduction of HcyT-induced apoptosis by NAC, Vit C or Vit E occurred simultaneously with a significant decrease in intracellular H(2)O(2) levels and reduced caspase-3 enzymatic activity. In contrast, folate had no H(2)O(2) scavenging capacity and did not suppress caspase-3 activity 6 h after HcyT treatment, although folate exhibited antioxidant behavior toward superoxide anions, hydroxyl radicals and peroxynitrite. Preincubation of cells with folate (10 micro mol/L) for 3 d did not affect the extent of HcyT-promoted apoptotic damage. Taken together, our findings suggest that antioxidant pretreatment with NAC, Vit C or Vit E exerts more beneficial effects than folate on reducing apoptotic cell damage induced by homocysteine thiolactone.

45. J Urol. 2002 Aug;168(2):780-5. N-acetylcysteine augments the cellular redox changes and cytotoxic activity of internalized mycobacterium bovis in human bladder cancer cells. Pook SH, Esuvaranathan K, Mahendran R. Department of Surgery, National University of Singapore, Singapore.

PURPOSE: We determined whether changes in cellular reactive oxygen species correlated with mycobacteria internalization and bladder cancer cell death. MATERIALS AND METHODS: Reactive oxygen species and thiols in RT112 and MGH bladder cancer cells were determined using the fluorescence probes 5-(and 6)-carboxy-2', 7' dichlorodihydrofluorescein diacetate and monobromobimane. Superoxide and nitrite production were measured using bis-N-methylarcridinium nitrate and Griess reagents. Cytotoxicity was determined by the release of 14C-thymidine from cells with 14C labeled DNA. RESULTS: MGH cells that internalize bacillus Calmette-Guerin (BCG) had decreased cellular reactive oxygen species and thiols, although superoxide and nitric oxide production increased. RT112 cells, which do not internalize BCG, did not show a decrease in reactive oxygen species after incubation with BCG. Blocking BCG uptake in MGH cells abrogated reactive oxygen species reduction, confirming that the changes in reactive oxygen species were internalization dependent events. Treating cells with BCG and the antioxidant N-acetylcysteine caused a greater reduction in reactive oxygen species, and induced earlier and greater cytotoxicity in MGH but not in RT112 cells. CONCLUSIONS: The induction of bladder cancer cell killing by BCG parallels the ability of cells to internalize BCG, which in turn indicates that the susceptibility of tumor cells to the cytotoxic effects of BCG may be related to changes in cellular levels of reactive oxygen species and thiols. Supplementation with an antioxidant could enhance the antitumor effect of BCG.

46. Carcinogenesis. 2002 Jun;23(6):993-1001. Inhibition of chronic ulcerative colitis-associated colorectal adenocarcinoma development in a murine model by N-acetylcysteine. Seril DN, Liao J, Ho KL, Yang CS, Yang GY. Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA.

Long-term ulcerative colitis (UC) patients are at increased risk for developing colorectal cancer. In order to develop strategies for preventing UC-associated carcinogenesis, we studied the effect of the antioxidant N-acetylcysteine (NAC) on UC-associated cancer development in a mouse model. Female C57BL/6J mice were subjected to long-term administration of dextran sulfate sodium (DSS) in the drinking fluid and 2-fold iron-enriched AIN76A diet, with or without NAC. In the DSS-plus-2-fold iron positive control group, gross tumor incidence was 88.5% (23/26 mice) after 12 DSS cycles (1 DSS cycle = 7 day DSS treatment period followed by 10 day recovery period). The tumor multiplicity was 2.1 +/- 0.2 tumors/tumor-bearing mouse, and the tumor volume was 0.054 +/- 0.019 cm3. With 0.2% NAC administration, tumor incidence was significantly reduced (68%, 17/25 mice; P < 0.05), as was the tumor multiplicity (1.5 +/- 0.1 tumors/tumor-bearing mouse; P < 0.05). The tumor volume was lower (0.014 +/- 0.004 cm3), but not significantly decreased. The proliferation index was significantly decreased in non-cancerous epithelia (48.5 +/- 6.0% vs 32.0 +/- 3.7%; P < 0.05), but not in tumor cells. NAC significantly induced apoptosis in both non-cancerous epithelia and colorectal adenocarcinoma. The number of cells immunostained-positive for nitrotyrosine was markedly decreased in the non-cancerous mucosa of NAC-treated mice (102.4 +/-16.6 positive cells/mm2 mucosa vs 53.6 +/- 14.9 cells/mm2; P < 0.05). In addition, the number of inducible nitric oxide synthase (iNOS)-positive inflammatory cells in the non-cancerous mucosa of the distal colon was markedly decreased by NAC. This study indicates that the antioxidant NAC has the potential to serve as a preventive agent for UC-associated colorectal cancer, possibly via inhibition of cellular proliferation and nitrosative stress-caused cellular damage.

47. Oncol Rep. 2002 Jul-Aug;9(4):887-96. Phase II study of subcutaneously administered interleukin-2 in combination with medroxyprogesterone acetate and antioxidant agents as maintenance treatment in advanced cancer responders to previous chemotherapy. Mantovani G, Maccio A, Madeddu C, Mulas C, Massa E, Astara G, Ferreli L, Mudu MC, Gramignano G, Murgia V, Lusso MR, Mocci M, Cardia A, Mura L. Department of Medical Oncology, University of Cagliari, Italy. mantovan@pacs.unica.it

An open, non-randomized phase II study was carried out including patients with advanced solid tumors who achieved an objective response or disease stabilization as a result of previous chemotherapy, to receive a maintenance treatment with recombinant interleukin-2 (rIL-2) plus medroxyprogesterone acetate (MPA) plus antioxidant agents alpha-lipoic acid (ALA) and N-acetyl cysteine (NAC). The first study endpoints were to define clinical outcome and toxicity as well as the evaluation of quality of life. As secondary endpoints we measured the changes of lymphocyte absolute count, the serum levels of proinflammatory cytokines, IL-2, C-reactive protein (CRP) and leptin after treatment. rIL-2 was administered at a dose of 1.8 MIU subcutaneously 3 times/week on alternate days for the first two weeks of every month and MPA was given orally at a dose of 500 mg/day at alternate days without interruption. ALA 300 mg/day orally and NAC 1800 mg/day orally were also administered continuously. Twenty-eight patients were enrolled in the study. The median duration of maintenance treatment was 10 months (6-30+). The response to maintenance treatment at September 15, 2001 was: CR 11 patients (39.3%); SD 2 patients (7.1%); PD 15 patients (53.6%). The median duration of response was 11 months (6-34+). The median follow-up duration was 11 months (6-34+). The median OS was not reached. The median PFS was 21.5 months (1-40+). The 1-year survival rate was 72.2%. At September 15, 2001, 16 patients were still surviving. No grade 3/4 toxicity and one grade 2 skin toxicity were observed. We found a significant increase of the absolute lymphocyte count and serum levels of IL-2 and a significant decrease of TNF alpha after treatment. The evaluation of patient subgroups showed the following: the patients alive at the end of study had a significant increase of lymphocyte count, IL-2 and leptin, and a significant decrease of IL-1 beta, IL-6 and TNF alpha, whereas the patients who had died had only a significant increase of lymphocyte count and IL-2. Among the patients alive, those in objective clinical response (CR + PR) + those in SD had a significant increase of lymphocyte count, IL-2 and leptin and a significant decrease of IL-1 beta, IL-6 and TNFalpha, whereas those with PD had no significant changes in any of the above values. We conclude that the combination of s.c. rIL-2 with oral MPA and anti-oxidant agents ALA and NAC in an intermittent schedule, repeated for a long-term period, is feasible, has a very low toxicity and results in the improvement of biological markers which are predictive for patient outcome.

48. Int J Cancer. 2002 Apr 1;98(4):493-7. Effects of N-acetylcysteine in an esophageal carcinogenesis model in rats treated with diethylnitrosamine and diethyldithiocarbamate. Balansky RM, Ganchev G, D'Agostini F, De Flora S. National Center of Oncology, Sofia, Bulgaria.

Due to the increasing role of esophageal tumors in human cancer pathology, there is need for animal models evaluating the mechanisms of esophageal carcinogenesis and investigating protective factors toward this disease. Several N-nitrosamines have been shown to induce esophageal tumors in rats. We designed a study in BD(6) rats treated with N-diethylnitrosamine (DEN) according to a simple protocol involving weekly i.p. injections of this carcinogen for 8 consecutive weeks. This treatment resulted in a high incidence and multiplicity of liver tumors and in occurrence of preneoplastic lesions and papillomas in the esophagus. Intraperitoneal injections of diethyldithiocarbamate (DEDTC), 4 hr after each DEN injection, i.e., during the period of DEN metabolization, improved survival of rats and did not affect the liver tumor yield but doubled the incidence of esophageal tumors and enhanced 4.9x their multiplicity. Moreover, 15% of rats developed esophageal squamocellular carcinomas. The oral administration of the thiol N-acetyl-L-cysteine (NAC), a precursor and analogue of reduced glutathione, to rats treated with the DEN/DEDTC combination did not change the liver tumor yield but attenuated esophageal carcinogenesis by producing a significant shift of preneoplastic lesions to milder forms as well as a significant decrease of tumor multiplicity. Therefore, the DEN/DEDTC protocol appears to provide an interesting 2-organ model of N-nitrosamine-induced carcinogenesis in rats, in which NAC is moderately effective as an inhibitor. The mechanisms underlying enhancement of DEN-induced esophageal carcinogenesis by DEDTC and the protective effects of NAC are discussed. Copyright 2002 Wiley-Liss, Inc.

49. Free Radic Biol Med. 2002 Mar 1;32(5):431-45. Reactive oxygen species modulate Zn(2+)-induced apoptosis in cancer cells. Provinciali M, Donnini A, Argentati K, Di Stasio G, Bartozzi B, Bernardini G. Laboratory of Tumor Immunology, Immunology Centre, Gerontology Research Department, I.N.R.C.A., Ancona, Italy. m.provinciali@inrca.it

Some recent evidence has suggested a protective role of zinc against cancer. The mechanism by which zinc exerts this action has not been defined and, in particular, it has not been clarified whether zinc may directly act on cancer cells and the molecular mechanisms involved in this effect. In this study, we examined the in vitro effect of zinc on the apoptosis of mouse TS/A mammary adenocarcinoma cells, studying the zinc-dependent modulation of the intracellular levels of reactive oxygen species (ROS) and of p53 and Fas/Fas ligand pathways. We showed that zinc concentrations ranging from 33.7 to 75 muM Zn(2+) induced apoptosis in mammary cancer cells. The apoptosis was associated with an increased production of intracellular ROS, and of p53 and Fas/Fas ligand mRNA and protein. Zn(2+) induced a faint metallothionein response in TS/A cells in comparison with mouse lymphocytes. The treatment of tumor cells with the antioxidant N-acetylcysteine was able to prevent Zn(2+)-induced apoptosis, as well as the increase of p53 and Fas ligand protein induced by zinc. The data demonstrate that zinc exerts a direct action on mammary cancer cells inducing ROS-mediated apoptosis and that the effect may be mediated by the ROS-dependent induction of p53 and Fas/Fas ligand.

50. Cancer Epidemiol Biomarkers Prev. 2002 Feb;11(2):167-75. Effects of oral administration of N-acetyl-L-cysteine: a multi-biomarker study in smokers. Van Schooten FJ, Nia AB, De Flora S, D'Agostini F, Izzotti A, Camoirano A, Balm AJ, Dallinga JW, Bast A, Haenen GR, Van't Veer L, Baas P, Sakai H, Van Zandwijk N. Department of Health Risk Analysis and Toxicology, Maastricht University, The Netherlands.

N-Acetyl-L-cysteine (NAC) has been shown to exert cancer-protective mechanisms and effects in experimental models. We report here the results of a randomized, double-blind, placebo-controlled, Phase II chemoprevention trial with NAC in healthy smoking volunteers. The subjects were supplemented daily with 2 x 600 mg of oral tablets of NAC (n = 20) or placebo (n = 21) for a period of 6 months, and internal dose markers [plasma and bronchoalveolar lavage (BAL) fluid cotinine, urine mutagenicity], biologically effective dose markers [smoking-related DNA adducts and hemoglobin (Hb) adducts], and biological response markers (micronuclei frequency and antioxidants scavenging capacity) were assessed at both pre- and postsupplementation times (T(0) and T(1), respectively). Overall, the internal dose markers remained unchanged at T(1) as compared with T(0) in both NAC and placebo groups. When quantifying the biologically effective dose markers, we observed an inhibitory effect of NAC toward the formation of lipophilic-DNA adducts (5.18 +/- 0.73 versus 4.08 +/- 1.03/10(8) nucleotides; mean +/- SE; P = 0.05) as well as of 7,8-dihydro-8-oxo-2'-deoxyguanosine adducts in BAL cells (3.9 +/- 0.6 versus 2.3 +/- 0.2/10(5) nucleotides; P = 0.003). There was no effect of NAC on the formation of lipophilic-DNA adducts in peripheral blood lymphocytes or polycyclic aromatic hydrocarbon-DNA adducts in mouth floor/buccal mucosa cells or 4-aminobiphenyl-Hb adducts. Likewise, quantification of the biological response markers showed an inhibitory effect of NAC on the frequency of micronuclei in mouth floor and in soft palate cells (1.3 +/- 0.2 versus 0.9 +/- 0.2; P = 0.001) and a stimulating effect of NAC on plasma antioxidant scavenging capacity (393 +/- 14 versus 473 +/- 19 microM Trolox; P = 0.1) but not on BAL fluid antioxidant scavenging capacity. We conclude that NAC has the potential to impact upon tobacco smoke carcinogenicity in humans because it can modulate certain cancer-associated biomarkers in specific organs.

51. Cancer Res. 2002 Jan 1;62(1):2-7. Inhibition of benzo(a)pyrene-induced lung tumorigenesis in A/J mice by dietary N-acetylcysteine conjugates of benzyl and phenethyl isothiocyanates during the postinitiation phase is associated with activation of mitogen-activated protein kinases and p53 activity and induction of apoptosis. Yang YM, Conaway CC, Chiao JW, Wang CX, Amin S, Whysner J, Dai W, Reinhardt J, Chung FL. Division of Carcinogenesis and Molecular Epidemiology, American Health Foundation, Valhalla, New York 10595, USA.

Recent studies in cell culture have shown that isothiocyanates (ITCs) induce apoptosis via activation of mitogen-activated protein (MAP) kinases and p53 pathways, suggesting a potential for ITCs or their conjugates to inhibit tumorigenesis during the postinitiation phase. To evaluate whether ITC compounds administered after carcinogen treatment inhibit lung tumorigenesis, we investigated in A/J mice the effects of the N-acetylcysteine (NAC) conjugates of benzyl (BITC-NAC) and phenethyl ITC (PEITC-NAC) in the diet (15 micromol/g) administered after a single dose of 20 micromol benzo(a)pyrene [B(a)P]. The formation of lung adenomas was examined 140 days after B(a)P dosing. Both the BITC-NAC and PEITC-NAC-treated groups showed a significant reduction in lung tumor multiplicity from 6.1 +/- 3.1 tumors/mouse in the B(a)P group fed the control diet to 3.7 +/- 2.9 and 3.4 +/- 2.7 tumors/mouse (P = 0.018 and 0.006, respectively). To investigate the mechanisms of tumor inhibition, lung tissues were obtained at 21, 84, and 140 days at interim sacrifices during the bioassay. These tissues showed a significant increase in apoptosis as determined by in situ end-labeling for both ITC-NAC-treated groups. The MAP kinase pathway was activated in the ITC-NAC-treated groups. The activation of c-Jun NH(2)-terminal kinase was higher in the BITC-NAC and PEITC-NAC groups when compared with B(a)P-treated control. The phosphorylation of p38 and extracellular signal-regulated kinases (ErKs) 1 and 2 was also induced by these treatments. To determine the downstream target of MAP kinases, activator protein-1 (AP-1) and nuclear factor-kappaB activities were evaluated by gel shift assay. The AP-1 binding activity was remarkably increased in lung tissue from both the BITC-NAC and PEITC-NAC groups. No change in nuclear factor-kappaB binding activity was found, however. Phosphorylation of p53 was also higher than the constitutive levels in both ITC-NAC-treated groups, but no induction of p53 expression was detected. This study demonstrates the chemopreventive efficacy of the NAC conjugates of PEITC and BITC administered in the diet after a single dose of B(a)P for lung tumorigenesis and provides the first in vivo evidence that activation of MAP kinases, AP-1 transcription factors, p53 phosphorylation, and the induction of apoptosis may be involved in the chemopreventive activity of these compounds.

52. FASEB J. 2002 Jan;16(1):2-14. Angioprevention': angiogenesis is a common and key target for cancer chemopreventive agents. Tosetti F, Ferrari N, De Flora S, Albini A. Molecular Biology Laboratory, National Cancer Research Institute (IST), Genova, Italy.

The potential to block tumor growth by inhibition of the neoangiogenic process represents an intriguing approach to the treatment of solid tumors. The high proliferation rate in the tumor deprived of proper vascularization would be balanced by cell death due to lack of diffusion of nutrients and oxygen. Matrix metalloproteinases (MMPs), angiogenic growth factors, and their receptors are the main targets of an increasing number of clinical trials approved to test the tolerance and therapeutic efficacy of antiangiogenic agents. We observed that a series of substances proposed as possible cancer chemopreventive agents show antiangiogenic properties when tested in in vitro and in vivo angiogenesis models. We demonstrated that N-acetyl-l-cysteine is able to reduce the invasive and metastatic potential of melanoma cells, and to inhibit endothelial cell invasion by direct inhibition of MMP activity. We also showed that epigallocatechin gallate (EGCG), a flavonoid from green tea that possesses chemopreventive activity in experimental and epidemiological studies, is a potent inhibitor of MMP-2 and MMP-9. Angiogenesis has also been demonstrated to be a target for nonsteroidal anti-inflammatory drug chemopreventive activity. Based on these data, we hypothesize that other chemopreventive agents, including natural or synthetic retinoids, steroid hormone antagonists, peroxisome proliferator-activated receptor gamma ligands, vitamin D, and protease inhibitors, might have antiangiogenesis as an important mechanism of action, a novel concept we will term 'angioprevention'. We analyze the mechanisms on how and why chemopreventive agents could exert antiangiogenic effects aimed at controlling tumor growth, and their potential use in the clinic.

53. Cancer Res. 2001 Nov 15;61(22):8171-8. Inhibition of angiogenesis-driven Kaposi's sarcoma tumor growth in nude mice by oral N-acetylcysteine. Albini A, Morini M, D'Agostini F, Ferrari N, Campelli F, Arena G, Noonan DM, Pesce C, De Flora S. National Institute for Cancer Research (IST), c/o Advanced Biotechnology Center, Largo R. Benzi 10, I-16132 Genoa, Italy. albini@vega.cba.unige.it

The thiol N-acetyl-L-cysteine (NAC), an analogue and precursor of reduced glutathione, has cancer chemopreventive properties attributable to its nucleophilicity, antioxidant activity, and a variety of other mechanisms. We demonstrated recently that NAC has anti-invasive, antimetastatic, and antiangiogenic effects in in vitro and in vivo test systems. In the present study, s.c. transplantation of KS-Imm cells in (CD-1)BR nude mice resulted in the local growth of Kaposi's sarcoma, a highly vascularized human tumor. The daily administration of NAC with drinking water, initiated after the tumor mass had become established and detectable, produced a sharp inhibition of tumor growth, with regression of tumors in half of the treated mice along with a markedly prolonged median survival time. The production of vascular endothelial growth factor (VEGF) and certain proliferation markers (proliferating cell nuclear antigen and Ki-67) were significantly lower in Kaposi's sarcomas from NAC-treated mice than from control mice. Treatment of KS-Imm cells with NAC in vitro resulted in a dose-dependent inhibition of chemotaxis and invasion through inhibition of gelatinase-A (matrix metalloproteinase-2, MMP-2) activity without altering MMP-2 or MMP-9 mRNA levels. NAC also significantly inhibited VEGF production but did not affect proliferation markers in vitro. Reverse transcription-PCR analysis indicated that total VEGF mRNAs were reduced by 10 mM NAC. Taken together, these findings provide evidence that NAC, the safety of which even at high doses has been established in almost 40 years of clinical use, in addition to its chemopreventive action, has a strong antiangiogenic potential that could be exploited for preventing cancer progression as well as used in cancer adjuvant therapy.

54. Biogerontology. 2001;2(1):55-60. N-Acetyl-L-Cystein downregulates beta-amyloid precursor protein gene transcription in human neuroblastoma cells. Studer R, Baysang G, Brack C. Laboratory of Molecular Gerontology, Basel University, Psychiatric University Clinic, Switzerland. Rolf.Studer@Actelion.Com

The causes for the sporadic form of Alzheimer's disease (AD) are still poorly understood, except from the fact that age is an important risk factor. The main component of the characteristic amyloid plaques in brains of AD patients are Abeta peptides, derivatives of the amyloid precursor protein APP. Oxidative stress may contribute to the aetiology of AD by dysregulation of APP metabolism. Overexpression of the APP gene could result in an increased secretion of neurotoxic Abeta peptides, while preventing the overexpression might be protective. We here report that the antioxidant N-Acetyl-L-Cystein (NAC) downregulates APP gene transcription in human neuroblastoma cells. The effect is reversible when cells are returned to NAC free medium. These results open up new possibilities for the development of therapeutic agents that intervene at the transcriptional level.

55. Cancer Res. 2001 Nov 1;61(21):7868-74. Therapeutic efficacy of aortic administration of N-acetylcysteine as a chemoprotectant against bone marrow toxicity after intracarotid administration of alkylators, with or without glutathione depletion in a rat model. Neuwelt EA, Pagel MA, Hasler BP, Deloughery TG, Muldoon LL. Department of Neurology, Oregon Health Sciences University, Portland, Oregon 97201, USA. neuwelte@ohsu.edu

Modulation of thiol levels may alter both the efficacy and toxicity of chemotherapeutic agents. We investigated cytoenhancement, using L-buthionine-[S,R]-sulfoximine (BSO) to reduce cellular glutathione levels prior to intracarotid alkylator administration. We also evaluated chemoprotection against chemotherapy-induced systemic toxicity when the thiol agents N-acetylcysteine (NAC) and sodium thiosulfate were administered into the descending aorta to limit brain delivery. BSO treatment reduced rat brain and intracerebral tumor glutathione levels by 50-65%, equivalent to the reduction in liver and s.c. tumor. BSO treatment significantly enhanced the toxicity of chemotherapy with carboplatin, melphalan, and etoposide phosphate against granulocytes, total white cells, and platelets. Intracarotid administration of NAC resulted in high delivery to the brain, whereas infusion via the descending aorta minimized brain delivery. When NAC, with or without sodium thiosulfate, was administered via aortic infusion prior to chemotherapy, the magnitude of the bone marrow toxicity nadir was minimized, even with BSO-enhanced myelosuppression. Thus, BSO depleted brain and brain tumor glutathione but thereby increased chemotherapy-induced myelosuppression. Surprisingly, although NAC was found to readily cross the blood-brain barrier when given into the carotid artery, aortic infusion of NAC resulted in minimal exposure to the central nervous system (CNS) vasculature because of rapid clearance. As a result, aortic infusion of NAC to perfuse bone marrow and minimize myelosuppression and toxicity to visceral organs could be performed without interfering with the CNS cytotoxicity of intracarotid alkylators, even after BSO depletion of CNS glutathione.

56. Carcinogenesis. 2001 Sep;22(9):1373-8. Dietary N-acetyl-L-cysteine modulates benzo[a]pyrene-induced skin tumors in cancer-prone p53 haploinsufficient Tg.AC (v-Ha-ras) mice. Martin KR, Trempus C, Saulnier M, Kari FW, Barrett JC, French JE. Transgenic Carcinogenesis Unit, Laboratory of Environmental Carcinogenesis and Mutagenesis and Laboratory of Molecular Carcinogenesis, NIEHS, NIH, Research Triangle Park, NC, USA.

Epidemiologic studies support the protective role of dietary antioxidants in preventing cancer. However, emerging evidence from clinical trials and laboratory data suggest that in some cases individual antioxidant supplements may actually exacerbate carcinogenesis. Our goal was to explore these paradoxical activities in a rodent model that possesses genotypic characteristics of human cancers. We selected the p53 haploinsufficient Tg.AC (v-Ha-ras) mouse as a model, because it contains an activated, carcinogeninducible ras oncogene and an inactivated p53 tumor suppressor gene, which are frequent genetic alterations in human cancers. These mice develop chemically induced benign and malignant skin tumors rapidly which can easily be quantified. Mice were fed basal diets with or without 3% N-acetyl-L-cysteine (NAC), a well-recognized antioxidant, prior to, during and after topical application of the carcinogen benzo[a]pyrene (64 microg/mouse) applied twice per week for 7 weeks. Tumor incidence exceeded 90% for both groups, and NAC did not reduce tumor latency. Mice fed NAC displayed a 43% reduction (P < 0.05) in tumor multiplicity and delayed the appearance of lesions (P < 0.05). Dietary NAC also significantly (P < 0.05) improved group survival by 5 weeks. Total tumor yields were reduced in both dietary groups but malignant spindle cell tumors (SCT) increased by 25% in NAC-fed mice. The v-Ha-ras oncogene and p53 protein products were clearly co-expressed in both benign and malignant lesions from both dietary groups. In summary, dietary supplementation with NAC was chemopreventive, but the marginal increase in SCT suggests a paradoxical effect.

57. Carcinogenesis. 2001 Jul;22(7):999-1013. Mechanisms of N-acetylcysteine in the prevention of DNA damage and cancer, with special reference to smoking-related end-points. De Flora S, Izzotti A, D'Agostini F, Balansky RM. Department of Health Sciences, Section of Hygiene and Preventive Medicine, University of Genoa, Via A. Pastore 1, I-16132 Genoa, Italy. sdf@unige.it

Although smoking cessation is the primary goal for the control of cancer and other smoking-related diseases, chemoprevention provides a complementary approach applicable to high risk individuals such as current smokers and ex-smokers. The thiol N-acetylcysteine (NAC) works per se in the extracellular environment, and is a precursor of intracellular cysteine and glutathione (GSH). Almost 40 years of experience in the prophylaxis and therapy of a variety of clinical conditions, mostly involving GSH depletion and alterations of the redox status, have established the safety of this drug, even at very high doses and for long-term treatments. A number of studies performed since 1984 have indicated that NAC has the potential to prevent cancer and other mutation-related diseases. N-acetylcysteine has an impressive array of mechanisms and protective effects towards DNA damage and carcinogenesis, which are related to its nucleophilicity, antioxidant activity, modulation of metabolism, effects in mitochondria, decrease of the biologically effective dose of carcinogens, modulation of DNA repair, inhibition of genotoxicity and cell transformation, modulation of gene expression and signal transduction pathways, regulation of cell survival and apoptosis, anti-inflammatory activity, anti-angiogenetic activity, immunological effects, inhibition of progression to malignancy, influence on cell cycle progression, inhibition of pre-neoplastic and neoplastic lesions, inhibition of invasion and metastasis, and protection towards adverse effects of other chemopreventive agents or chemotherapeutical agents. These mechanisms are herein reviewed and commented on with special reference to smoking-related end-points, as evaluated in in vitro test systems, experimental animals and clinical trials. It is important that all protective effects of NAC were observed under a range of conditions produced by a variety of treatments or imbalances of homeostasis. However, our recent data show that, at least in mouse lung, under physiological conditions NAC does not alter per se the expression of multiple genes detected by cDNA array technology. On the whole, there is overwhelming evidence that NAC has the ability to modulate a variety of DNA damage- and cancer-related end-points.

58. Cancer Res. 2001 Mar 15;61(6):2472-9. Modulation of biomarkers by chemopreventive agents in smoke-exposed rats. Izzotti A, Balansky RM, Dagostini F, Bennicelli C, Myers SR, Grubbs CJ, Lubet RA, Kelloff GJ, De Flora S. Department of Health Sciences, University of Genoa, Italy.

Chemoprevention opens new perspectives in the prevention of cancer and other chronic degenerative diseases associated with tobacco smoking, exploitable in current smokers and, even more, in exsmokers and passive smokers. Evaluation of biomarkers in animal models is an essential step for the preclinical assessment of efficacy and safety of potential chemopreventive agents. Groups of Sprague Dawley rats were exposed whole body to a mixture of mainstream and sidestream cigarette smoke for 28 consecutive days. Five chemopreventive agents were given either with drinking water (N-acetyl-L-cysteine, 1 g/kg body weight/day) or with the diet (1,2-dithiole-3-thione, 400 mg; Oltipraz, 400 mg; phenethyl isothiocyanate, 500 mg; and 5,6-benzoflavone, 500 mg/kg diet). The monitored biomarkers included: DNA adducts in bronchoalveolar lavage cells, tracheal epithelium, lung and heart; oxidative damage to pulmonary DNA; hemoglobin adducts of 4-aminobiphenyl and benzo(a)pyrene-7,8-diol-9,10-epoxide; micronucleated and polynucleated alveolar macrophages and micronucleated polychromatic erythrocytes in bone marrow. Exposure of rats to smoke resulted in dramatic alterations of all investigated parameters. N-Acetyl-L-cysteine, phenylethyl isothiocyanate, and 5,6-benzoflavone exerted a significant protective effect on all alterations. 1,2-Dithiole-3-thione was a less effective inhibitor and exhibited both a systemic toxicity and genotoxicity in alveolar macrophages, whereas its substituted analogue Oltipraz showed limited protective effects in this model. Interestingly, combination of N-acetyl-L-cysteine with Oltipraz was the most potent treatment, resulting in an additive or more than additive inhibition of smoke-related DNA adducts in the lung and hemoglobin adducts. These results provide evidence for the differential ability of test agents to modulate smoke-related biomarkers in the respiratory tract and other body compartments and highlight the potential advantages in combining chemopreventive agents working with distinctive mechanisms.

59. Carcinogenesis. 2001 Mar;22(3):375-80. Modulation of apoptosis by cigarette smoke and cancer chemopreventive agents in the respiratory tract of rats. D'Agostini F, Balansky RM, Izzotti A, Lubet RA, Kelloff GJ, De Flora S. Department of Health Sciences, University of Genoa, Via A. Pastore 1, I-16132 Genoa, Italy.

Preclinical studies may elucidate the meaning of biomarkers applicable to epidemiologic studies and to clinical trials for cancer prevention. No study has explored so far the effect of cigarette smoke on apoptosis in vivo. We evaluated modulation of apoptosis in cells of the respiratory tract of smoke-exposed Sprague-Dawley rats both by morphological analysis and TUNEL method. In a first study, exposure of rats to mainstream cigarette smoke for either 18 or 100 consecutive days produced a significant and time-dependent increase in the proportion of apoptotic cells in the bronchial and bronchiolar epithelium. Oral N:-acetylcysteine did not affect the background frequency of apoptosis but significantly and sharply decreased smoke-induced apoptosis. In a second study, exposure of rats to a mixture of sidestream and mainstream smoke for 28 consecutive days resulted in a >10-fold increase in the frequency of pulmonary alveolar macrophages undergoing apoptosis. Dietary administration of either 5,6-benzoflavone, 1,2-dithiole-3-thione or oltipraz did not affect the frequency of smoke-induced apoptosis, whereas phenethyl isothiocyanate produced a further significant enhancement. Again, N-acetylcysteine and its combination with oltipraz significantly decreased smoke-induced apoptosis. In both studies exposure to smoke resulted in a sharp increase of cells positive for proliferating cell nuclear antigen (PCNA), which was unaffected by the examined chemopreventive agents. These findings highlight the concept that modulation of apoptosis has diversified meanings. Different meanings (as explained in the following lines). First, the apoptotic process is triggered as a defense system against genotoxic agents, such as the components of cigarette smoke. The further induction produced by phenethyl isothiocyanate, favoring removal of damaged cells, represents an example of a detoxification mechanism. Inhibition of smoke-induced apoptosis by N:-acetylcysteine should be interpreted as an epiphenomenon of antigenotoxic mechanisms, as shown in parallel studies evaluating modulation of DNA alterations in the respiratory tract of the same animals. Thus, it is important to discriminate between whether the opposite modulation of apoptosis is per se a protective mechanism or the beneficial outcome of other mechanisms inhibiting genotoxicity.

60. J Gastrointest Surg. 2001 Jan-Feb;5(1):91-7. The antioxidant N-acetylcysteine increases 5-fluorouracil activity against colorectal cancer xenografts in nude mice. Bach SP, Williamson SE, Marshman E, Kumar S, O'Dwyer ST, Potten CS, Watson AJ. Cancer Research Campaign, Department of Epithelial Biology, The Paterson Institute, Christie Hospital, Withington, Manchester M20 4BX, UK. Sbach@picr.man.ac.uk

The antioxidant pyrrolidinedithiocarbamate improves the therapeutic efficacy of 5-fluorouracil (5-FU) against HCT-15 colorectal cancer cell line xenografts in nude mice without increasing toxicity to normal intestinal or hematopoietic tissues. In the current study we have shown that a similar clinically licensed antioxidant, N-acetylcysteine (200 mg/kg), can modulate the activity of 5-FU (120 mg/kg) against HCT-15 tumor xenografts in nude mice. We demonstrate that this effect is accompanied by a sustained elevation in p53-independent apoptosis without accompanying alterations in cell cycle kinetics. Extensive tumor necrosis is also a prominent feature of treatment; however, no significant impairment of neovascularization as assessed by intratumor microvessel density occurred. We believe that the clinical efficacy of N-acetylcysteine as an adjunct to 5-FU in advanced colorectal cancer should be investigated further.

61. Se-methylselenocysteine induces apoptosis mediated by reactive oxygen species in HL-60 cells. Jung U, Zheng X, Yoon SO, Chung AS. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusung-gu, Taejon, Republic of Korea. Free Radic Biol Med 2001 Aug 15;31(4):479-89

Recent studies have implicated apoptosis as one of the most plausible mechanisms of the chemopreventive effects of selenium compounds, and reactive oxygen species (ROS) as important mediators in apoptosis induced by various stimuli. In the present study, we demonstrate that Se-methylselenocysteine (MSC), one of the most effective selenium compounds at chemoprevention, induced apoptosis in HL-60 cells and that ROS plays a crucial role in MSC-induced apoptosis. The uptake of MSC by HL-60 cells occurred quite early, reaching the maximum within 1 h. The dose-dependent decrease in cell viability was observed by MSC treatment and was coincident with increased DNA fragmentation and sub-G(1) population. 50 microM of MSC was able to induce apoptosis in 48% of cell population at a 24 h time point. Moreover, the release of cytochrome c from mitochondria and the activation of caspase-3 and caspase-9 were also observed. The measurement of ROS by dichlorofluorescein fluorescence revealed that dose- and time-dependent increase in ROS was induced by MSC. N-acetylcysteine, glutathione, and deferoxamine blocked cell death, DNA fragmentation, and ROS generation induced by MSC. Moreover, N-acetylcysteine effectively blocked caspase-3 activation and the increase of the sub-G(1) population induced by MSC. These results imply that ROS is a critical mediator of the MSC-induced apoptosis in HL-60 cells.

62. Anticancer Res. 2001 Jan-Feb;21(1A):213-9. Inhibitory effect of N-acetylcysteine on invasion and MMP-9 production of T24 human bladder cancer cells. Kawakami S, Kageyama Y, Fujii Y, Kihara K, Oshima H. Department of Urology and Reproductive Medicine, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-Ku, Tokyo 113-8519, Japan. s-kawakami@med.tmd.ac.jp

BACKGROUND: MMPs play a crucial role in the process of cancer invasion and metastasis. METHODS: The influence of NAC on invasion and MMP-9 production of human bladder cancer cell line T24 was investigated using an in vitro invasion assay, gelatin zymography, Western and Northern blot analyses and RT-PCR assays. RESULTS: TPA increased the number of invading T24 cells through reconstituted basement membrane more than 10-fold compared to basal condition. NAC inhibited TPA-enhanced invasion dose-dependently. TPA increased the MMP-9 production by T24 cells without altering expression of TIMP-1 gene, while NAC suppressed TPA-enhanced production of MMP-9. Neither TPA nor NAC altered TIMP-1 mRNA level in T24 cells. In vitro experiments demonstrated that MMP-9 was directly inhibited by NAC but was not influenced by TPA. CONCLUSION: NAC limits invasion of T24 human bladder cancer cells by inhibiting the MMP-9 production in addition to a direct inhibition of MMP-9 activity.

63. J Neurochem. 2001 Jan;76(1):224-33. N-acetyl-L-cysteine protects SHSY5Y neuroblastoma cells from oxidative stress and cell cytotoxicity: effects on beta-amyloid secretion and tau phosphorylation. Olivieri G, Baysang G, Meier F, Muller-Spahn F, Stahelin HB, Brockhaus M, Brack C. Neurobiology Laboratory, Psychiatric University Hospital, Basel, Switzerland. gianfranco.olivieri@pukbasel.ch

Redox changes within neurones are increasingly being implicated as an important causative agent in brain ageing and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). Cells have developed a number of defensive mechanisms to maintain intracellular redox homeostasis, including the glutathione (GSH) system and antioxidant enzymes. Here we examine the effects of N-acetyl-L-cysteine (NAC) on beta-amyloid (A beta) secretion and tau phosphorylation in SHSY5Y neuroblastoma cells after exposure to oxidative stress inducing/cytotoxic compounds (H(2)O(2), UV light and toxic A beta peptides). A beta and tau protein are hallmark molecules in the pathology of AD while the stress factors are implicated in the aetiology of AD. The results show that H(2)O(2), UV light, A beta 1-42 and toxic A beta 25-35, but not the inactive A beta 35-25, produce a significant induction of oxidative stress and cell cytotoxicity. The effects are reversed when cells are pre-treated with 30 mM NAC. Cells exposed to H(2)O(2), UV light and A beta 25-35, but not A beta 35-25, secrete significantly higher amounts of A beta 1-40 and A beta 1-42 into the culture medium. NAC pre-treatment increased the release of A beta 1-40 compared with controls and potentiated the release of both A beta 1-40 and A beta 1-42 in A beta 25-35-treated cells. Tau phosphorylation was markedly reduced by H(2)O(2) and UV light but increased by A beta 25-35. NAC strongly lowered phospho-tau levels in the presence or absence of stress treatment.

64. Int J Cancer. 2000 Dec 1;88(5):702-7. Interactions between N-acetylcysteine and ascorbic acid in modulating mutagenesis and carcinogenesis. D'Agostini F, Balansky RM, Camoirano A, de Flora S. Department of Health Sciences, University of Genoa, Genoa, Italy.

Both ascorbic acid (AsA, vitamin C) and N-acetylcysteine (NAC), a precursor and analogue of glutathione, possess a broad array of biological properties underlying their protective role in a variety of pathophysiological conditions. However, under certain circumstances, AsA behaves as a pro-oxidant rather than an anti-oxidant and produces adverse effects. This prompted us to evaluate whether NAC could interact with AsA in preventing mutation and cancer. AsA significantly increased spontaneous revertants in the Salmonella typhimurium strains TA102 and TA104, which are sensitive to oxidative mutagens. In contrast, NAC lowered the spontaneous background in TA104 and neutralized the negative effects of AsA. Moreover, NAC and AsA showed additive effects in reducing chromium(VI) and in reverting its mutagenicity. A single i.p. injection of urethane (1 g/kg body weight) to 120 A/J mice resulted, after 4 months, in the formation of a total of 1,532 lung tumors, 425 in the 30 mice treated with the carcinogen only, 404 in those treated with urethane plus AsA, 365 in those treated with urethane plus NAC and 338 in those treated with urethane plus the combination of AsA and NAC (both given daily with drinking water at the dose of 1 g/kg body weight). Compared to positive controls, tumor multiplicity was poorly affected by AsA, whereas it was significantly decreased by NAC and even more so by its combination with AsA. The overall volumes of lung tumors in the 4 groups were 107.5, 89.3, 61.3 and 49.7 mm(3), respectively. Tumor sizes were slightly but significantly decreased in mice treated with AsA and more so in those treated with NAC and NAC plus AsA, their combination being significantly more effective than each individually. All protective effects elicited by combining the 2 drugs were additive. Therefore, NAC prevents the adverse effects of AsA on spontaneous mutagenicity; at the same time, this thiol behaves in an additive fashion with AsA, inhibiting the mutagenicity of chromium(VI) and the lung tumorigenicity of urethane in mice. These findings suggest that NAC and AsA could conveniently be combined in cancer chemoprevention and other pharmacological interventions. Copyright 2000 Wiley-Liss, Inc.

65. Int J Cancer. 2000 Jun 15;86(6):842-7. Restoration of functional defects in peripheral blood mononuclear cells isolated from cancer patients by thiol antioxidants alpha-lipoic acid and N-acetyl cysteine. Mantovani G, Maccio A, Melis G, Mura L, Massa E, Mudu MC. Department of Medical Oncology and Internal Medical Sciences, University of Cagliari, Cagliari, Italy. mantovan@pacs.unica.it

The ability of Alpha-Lipoic Acid (ALA) and N-acetyl cysteine (NAC), two active antioxidant agents, to correct in vitro the most significant functional defects of peripheral blood mononuclear cells (PBMC) isolated from advanced stage cancer patients was studied. The proliferative response of PBMC isolated from cancer patients to anti-CD3 monoclonal antibody (MAb) and the expression of CD25 (IL-2R) and CD95 (Fas) on unstimulated and anti-CD3 MAb-stimulated PBMC were studied, and the serum levels of proinflammatory cytokines IL-1, IL-6, TNFalpha as markers of pro-cachectic activity in cancer patients, and the serum levels of IL-2 and sIL-2R were assessed. Twenty patients (mean age 64.6 years) with cancer of lung, ovary, endometrium, and head and neck, all in advanced (III, IV) stage of disease, were studied. The serum levels of IL-1beta, IL-2, IL-6, TNFalpha, and sIL-2R were significantly higher in cancer patients than in normal subjects. The response of PBMC isolated from cancer patients to anti-CD3 MAb was significantly lower than that of controls. The addition of either ALA 0.001 mM or NAC 0.004 mM in the PBMC cultures stimulated with anti-CD3 MAb significantly increased the response of PBMC isolated from cancer patients and normal subjects. After 24 and 72 hr of culture with anti-CD3 MAb, the expression of CD25 and CD95 on PBMC isolated from cancer patients was significantly lower than that of PBMC isolated from normal subjects. The addition of either ALA or NAC into cultures of PBMC isolated from cancer patients significantly increased the percentage of cells expressing CD25 as well as those expressing CD95. The results of the present study show a favorable effect of antioxidant agents ALA and NAC on several important T-cell functions in vitro in advanced-stage cancer patients. Copyright 2000 Wiley-Liss, Inc.

66. Altern Med Rev. 2000 Oct;5(5):467-71. N-acetylcysteine. [No authors listed]

N-acetylcysteine (NAC) is the acetylated precursor of both the amino acid L-cysteine and reduced glutathione (GSH). Historically it has been used as a mucolytic agent in chronic respiratory illnesses as well as an antidote for hepatotoxicity due to acetaminophen overdose. More recently, animal and human studies of NAC have shown it to be a powerful antioxidant and a potential therapeutic agent in the treatment of cancer, heart disease, HIV infection, heavy metal toxicity, and other diseases characterized by free radical oxidant damage. NAC has also been shown to be of some value in treating Sjogren's syndrome, smoking cessation, influenza, hepatitis C, and myoclonus epilepsy.

Cytokine. 2000 Apr;12(4):374-8. Vascular endothelial growth factor (VEGF) and melanoma. N-acetylcysteine downregulates VEGF production in vitro. Redondo P, Bandres E, Solano T, Okroujnov I, Garcia-Foncillas J. Department of Dermatology, University Clinic of Navarra, School of Medicine, Pamplona, Spain.

Vascular endothelial growth factor (VEGF), the most potent angiogenic factor identified to date, is associated with growth and metastasis of solid tumours, including melanoma. It has been shown in vitro that melanoma cells produce raised concentrations of VEGF. We examined the VEGF concentrations in plasma of 20 patients with primary melanoma, local recurrence and metastatic melanoma. We also studied the inhibiting effect of one antioxidant, N-acetylcysteine, on VEGF production in three human melanoma cell lines. We found elevated levels of VEGF (median 205 pg ml; 95 percent confidence interval, 80-414) in metastatic melanoma, with respect to primary and locally recurrent melanoma (75 pg/ml; 95 percent confidence interval, 35-130). The health control patients had levels of 25 pg/ml (95 percent confidence interval, 10-35). Human melanoma cell lines secreted VEGF in basal conditions (550-963 +/- 125 pg/ml) and N-acetylcysteine (0.5-20 mM) significantly decreased the VEGF production in a dose-dependent manner. VEGF concentrations were found to be raised in patients with primary melanoma, local recurrence, and above all, metastatic melanoma (P=0.008). N-acetylcysteine inhibits VEGF production in three human melanoma cell lines. This antioxidant might have therapeutic applications in metastatic melanoma in combination with other cytotoxic drugs.

67. Int J Oncol. 2000 Jun;16(6):1215-9. Modulation of growth of human prostate cancer cells by the N-acetylcysteine conjugate of phenethyl isothiocyanate. Chiao JW, Chung F, Krzeminski J, Amin S, Arshad R, Ahmed T, Conaway CC. Department of Medicine, New York Medical College, Valhalla, NY 10595, USA.

There is growing evidence that thiol conjugates of isothiocyanates present in cruciferous vegetables are effective cancer chemopreventive and potentially active therapeutic agents. The effects of the N-acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC) on tumor cell growth were analyzed in human prostate cancer cell lines LNCaP, androgen-dependent, and DU-145, androgen-independent. Exposure of the cells to PEITC-NAC at high concentrations caused cytolysis, while at lower concentrations PEITC-NAC mediated a dose-dependent growth modulation, with reduction of DNA synthesis and growth rate, inhibition of clonogenicity and induction of apoptosis in both types of prostate cancer cells. PEITC-NAC decreased cells in S and G2M phases of cell cycle, blocking cells entering replicating phases. In parallel, a significant enhancement of cells expressing the cell cycle regulator p21 as well as its intensity was determined using a fluorescent antibody technique. The action of PEITC-NAC was time-dependent, with the magnitude of inhibition increasing to 50-65% after PEITC-NAC exposure for several days. Interaction of tumor cells with dissociation products of PEITC-NAC, PEITC and NAC, are proposed as the mechanism of growth regulation.

68. Cancer Res. 2000 Jan 15;60(2):350-8. Effective elimination of lung metastases induced by tumor cells treated with hydrostatic pressure and N-acetyl-L-cysteine. Goldman Y, Peled A, Shinitzky M. Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.

In previous studies, we have demonstrated that application of high hydrostatic pressure (P) to tumor cells in the presence of a slow-reacting membrane-impermeable cross-linker (CL), 2'-3'-adenosine dialdehyde, can rearrange cell surface proteins into immunogenic clusters. Here, we present evidence indicating that subsequent reduction of surface protein disulfides with N-acetyl-L-cysteine (NAC) further augments the immunogenic potential of PCL-modified tumor cells both in vitro and in vivo. Immunotherapy with PCL+NAC-modified 3LL-D122 Lewis lung carcinoma cells plus i.v. delivery of NAC in mice bearing established lung metastases provoked an antitumor response capable of eradicating the metastatic nodules as demonstrated by restoration of normal lung weight and histology. In addition, immunization with PCL+NAC-modified tumor cells gave rise to a strong delayed-type hypersensitivity recall response against parental D122 cells. We propose that this novel two-prong strategy, based on local immunization with autologous PCL+NAC-modified tumor cells and systemic boosting with NAC, could provide a practical, effective immunotherapeutic regimen for the treatment of human cancer.

69. Cancer Lett. 1999 Dec 1;147(1-2):109-14. N-acetylcysteine suppression of the proliferative index in the colon of patients with previous adenomatous colonic polyps. Estensen RD, Levy M, Klopp SJ, Galbraith AR, Mandel JS, Blomquist JA, Wattenberg LW. Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455, USA.

This investigation is part of an effort to develop chemoprevention for carcinogenesis of the large bowel. The agent investigated is N-acetylcysteine (NAC). We used as a predictive biomarker, the proliferative index (PI), in a short-term human study. Patients with previous adenomatous colonic polyps are a cohort with increased risk for colon cancer and an increased PI of colonic crypts. They were randomly assigned to an experimental group given 800 mg/day of NAC for 12 weeks or a placebo group. Using proliferative cell nuclear antigen immunostaining, the PI of colonic crypts was measured prior to and after the treatments. The PI of the NAC group was decreased significantly (P < 0.02) while the placebo group showed no difference (P > 0.45). Since this decrease in PI may be an indicator of decreased risk of colon cancer, more extensive studies of the potential of NAC as a chemopreventive agent for colon cancer appear warranted.

70. Lab Invest. 1999 Sep;79(9):1151-9. N-acetylcysteine inhibits endothelial cell invasion and angiogenesis. Cai T, Fassina G, Morini M, Aluigi MG, Masiello L, Fontanini G, D'Agostini F, De Flora S, Noonan DM, Albini A. National Institute for Cancer Research, Genoa, Italy.

The thiol N-acetylcysteine (NAC) is a chemopreventive agent that acts through a variety of mechanisms and can prevent in vivo carcinogenesis. We have previously shown that NAC inhibits invasion and metastasis of malignant cells as well as tumor take. Neovascularization is critical for tumor mass expansion and metastasis formation. We investigated whether a target of the anti-cancer activity of NAC could be the inhibition of the tumor angiogenesis-associated phenotype in vitro and in vivo using the potent angiogenic mixture of Kaposi's sarcoma cell products as a stimulus. Two endothelial (EAhy926 and human umbilical vein endothelial [HUVE]) cell lines were utilized in a panel of assays to test NAC ability in inhibiting chemotaxis, invasion, and gelatinolytic activity in vitro. NAC treatment of EAhy926 and HUVE cells in vitro dose-dependently reduced their ability to invade a reconstituted basement membrane, an indicator of endothelial cell activation. Invasion of HUVE cells was inhibited with an ID50 of 0.24 mM NAC, whereas inhibition of chemotaxis required a 10 fold higher doses, indicating that invasion is a preferential target. NAC inhibited the enzymatic activity and conversion to active forms of the gelatinase produced by endothelial cells. The matrigel in vivo assay was used for the evaluation of angiogenesis; NAC strongly inhibited neovascularization of the matrigel sponges in response to Kaposi's sarcoma cell products. NAC prevented angiogenesis while preserving endothelial cells, implying that it could be safely used as an anti-angiogenic treatment.

71. Carcinogenesis. 1999 Sep;20(9):1869-72. Induction of cyclin-dependent kinase inhibitors and G(1) prolongation by the chemopreventive agent N-acetylcysteine. Liu M, Wikonkal NM, Brash DE. Department of Therapeutic Radiology and Department of Genetics, Yale University School of Medicine, New Haven, CT 06520-8040, USA.

Cyclin-dependent kinase (cdk) inhibitors, such as p16(INK4a) and p21(WAF1/CIP1), often inhibit G(1) cyclin kinases and result in G(1) arrest. It has been suggested that p21(WAF1/CIP1) may also play a role in other chemopreventive activities such as DNA repair, slowdown of DNA replication and induction of cellular differentiation. In this report we demonstrate that the antioxidant N-acetylcysteine (NAC), a well-known chemopreventive agent, induces p16(INK4a) and p21(WAF1/CIP1) gene expression and prolongs cell-cycle transition through G(1) phase. A portion of the G(1) arrest by NAC is governed by p16(INK4a); it is independent of p53. NAC's usual mechanism of increasing intracellular glutathione level is not required for the G(1) arrest. An antioxidant whose action is limited to scavenging radicals, Trolox, does not induce G(1) arrest. Taken together, these results suggest a potential novel molecular basis for chemoprevention by NAC.

72. Jpn J Cancer Res. 1999 May;90(5):565-70. N-acetylcysteine modifies cis-dichlorodiammineplatinum-induced effects in bladder cancer cells. Miyajima A, Nakashima J, Tachibana M, Nakamura K, Hayakawa M, Murai M. Department of Urology, National Defense Medical College, Tokorozawa, Saitama. aandt@ix.netcom.com

We previously demonstrated a role of reactive oxygen species (ROS) in cytotoxicity induced by cis-dichlorodiammineplatinum (CDDP) in combination with glutathione (GSH) depletors in bladder cancer cells. However, the relationship between CDDP and ROS is still unclear, although many mechanisms of drug resistance have been well characterized. The present study was undertaken to investigate the effects of N-acetylcysteine (NAC), a GSH precursor, on the CDDP-induced effects in bladder cancer cells (KU1). The cytotoxic effects of CDDP were significantly blunted by NAC (1 mM) in KU1 cells. The IC50 of CDDP only (10.2+/-1.2 microM) is significantly lower than that of CDDP with NAC (IC50: 20.3+/-1.6 microM) in KU1 cells. NAC also significantly increased the intracellular concentration of GSH in KU1 cells (37.2+/-1.6 nmol/10(6) cells), compared to controls (15.9+/-7.6 nmol/10(6) cells). While CDDP produced a significant increase in ROS as measured in terms of dichlorofluorescein (DCF) production in KU1 cells in a time-dependent manner, pretreatment with NAC significantly reduced CDDP-induced intracellular DCF in KU1 cells. Moreover, TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay showed that CDDP-induced apoptosis (31.1+/-3.8%) was significantly inhibited by pretreatment with NAC in KU1 cells (11.2+/-2.6%). These results demonstrated that NAC scavenges CDDP-induced ROS and inhibits CDDP-induced cytotoxicity, suggesting that ROS mediate the CDDP-induced cytotoxicity in bladder cancer cells.

73. Int J Biol Markers. 1999 Oct-Dec;14(4):268-71. The role of the thiol N-acetylcysteine in the prevention of tumor invasion and angiogenesis. Morini M, Cai T, Aluigi MG, Noonan DM, Masiello L, De Flora S, D'Agostini F, Albini A, Fassina G. National Institute for Cancer Research, Genova, Italy.

We have extensively studied the effects of N-acetylcysteine (NAC), a cytoprotective drug that can prevent in vivo carcinogenesis. Here we review our findings NAC completely inhibits gelatinolytic activity of metalloproteases and chemotactic and invasive activities of tumor cells. In addition, NAC reduces the number of lung metastases when malignant murine melanoma cells are injected into nude mice. NAC treatment decreases the weight of primary tumors and produces a dose-related increase in tumor latency. Moreover, oral administration of NAC reduces the formation of spontaneous metastases. In experimental metastasis assays, we have found a synergistic reduction in the number of lung metastases after treatment with doxorubicin (DOX) and NAC in nude mice. In tumorigenicity and spontaneous metastasis assays, the combined administration of DOX and oral NAC again has shown synergistic effects on the frequency and weight of primary tumors and local recurrences and completely prevented the formation of lung metastases. The addition of NAC to endothelial cells strongly reduces their invasive activity in response to angiogenic stimuli. NAC inhibited the degradation and release of radiolabeled type IV collagen by activated endothelial cells, indicating that NAC blocks gelatinase activity. Oral administration of NAC reduces the angiogenic response induced by KS tumor cell products, confirming the ability of NAC to inhibit the invasive activity of endothelial cells in vivo and thereby blocking angiogenesis.

74. Hum Cell. 2000 Dec;13(4):213-20. N-acetyl cysteine inhibits cell cycle progression in pancreatic carcinoma cells. Kusano C, Takao S, Noma H, Yoh H, Aikou T, Okumura H, Akiyama S, Kawamura M, Makino M, Baba M. First Department of Surgery, Faculty of Medicine, Kagoshima University.

The antioxidant N-acetyl cysteine (NAC) is a precursor of intracellular glutathione (GSH) and is also a well known as one of the chemopreventive agents which act through a variety of cellular mechanisms. We examined the effects of NAC on cell cycle progression in the pancreatic carcinoma cell lines, SW1990 and JHP1. Cells were incubated with or without NAC. Cell cycle distribution was analyzed by flow cytometry and immunoblotting. NAC suppressed cell proliferation in a concentration-dependent manner, whereas NAC increased intracellular glutathione content significantly in a dose-dependent manner. The percentage of cells in the G1 phase after treatment with NAC was significantly higher than the percentage seen for control cells. Cyclin D1 expression of carcinoma cells treated with NAC decreased remarkably compared with cells without NAC treatment. Thus, the antiproliferative effect of NAC by prolongation of the G1 phase in human pancreatic carcinoma cells shows its possible utility as an antitumor agent.

75. In Vitr Mol Toxicol. 2000 Winter;13(4):237-48. N-acetyl-L-cysteine simultaneously increases mitogenesis and suppresses apoptosis in mitogen-stimulated B-lymphocytes from p53 haploinsufficient Tg.AC (v-Ha-ras) mice. Martin KR, Kari FW, Barrett JC, French JE. Transgenic Carcinogenesis Unit, Laboratory of Environmental Carcinogenesis and Mutagenesis, National Institute of Environmental Health Services, NIH, Research Triangle Park, North Carolina 27709, USA. martin9@niehs.nih.gov

Recent epidemiological evidence suggests that antioxidants may enhance carcinogenesis by promoting cellular proliferation and/or impeding programmed cell death. We examined the effect of N-acetyl-L-cysteine (NAC) on mitogenesis and apoptosis in splenocytes from p53 haploinsufficient Tg.AC (v-Ha-ras) mice. This model contains genetic lesions found frequently in human cancer and is predisposed to develop carcinogen-induced cancer. Splenocytes were incubated with NAC alone or with the B- and T-cell-specific mitogens Concanavalin A (Con A) and E. coli lipopolysaccharide (LPS), respectively. Mitogenesis increased 17-fold in mitogen-stimulated cultures and 10-fold in cultures incubated with NAC alone. Co-incubation with both NAC (1000 microg/mL) and mitogen increased mitogenesis by 33-fold without changing apoptosis rates. Strikingly, incubation with NAC and LPS attenuated LPS-induced apoptosis. Mitogen alone did not affect GSH levels but NAC-induced increases were significantly depleted by co-incubation with mitogen. Furthermore, NAC increased the number of CD45R+ B cells, but decreased CD3+ T cells showing enhanced survival of B cells under these conditions. These results demonstrate concurrent reduced apoptosis and increased mitogenesis in B lymphocytes that may favor clonal selection of preneoplastic cells.

76. Antiapoptotic and antigenotoxic effects of N-acetylcysteine in human cells of endothelial origin. Aluigi MG, De Flora S, D'Agostini F, Albini A, Fassina G. Advanced Biotechnology Center, Genoa, Italy. Anticancer Res 2000 Sep-Oct;20(5A):3183-7

N-Acetylcysteine (NAC) is a drug bearing multiple preventive properties that can inhibit genotoxicity and carcinogenicity. NAC also inhibits invasion and metastasis of malignant cells, as well as tumor take. We recently demonstrated the effects of NAC on Kaposi's sarcoma cells supernatant-induced invasion in vitro and angiogenesis in vivo. Many anticancer agents act through cytotoxicity of rapidly proliferating cells and several antineoplastic drugs induce apoptosis of cancer cells. Since endothelial cells are the target for the inhibition of angiogenesis, we wanted to verify that NAC, while inhibiting tumor vascularization and endothelial cell invasion would not induce endothelial cell apoptosis. We tested the ability of NAC to modulate apoptosis and cytogenetic damage in vitro and to promote differentiation on a reconstituted basement membrane (matrigel) in two endothelial cell lines (EAhy926 and HUVE). Treatment with NAC protected endothelial cells from TGF-beta-induced apoptosis and paraquat-induced cytogenetic damage. Therefore, NAC acts as an antiangiogenic agent and, at the same time, appears to prevent apoptosis and oxygen-related genotoxicity in endothelial cells.

77. Modulation of growth of human prostate cancer cells by the N-acetylcysteine conjugate of phenethyl isothiocyanate. Chiao JW, Chung F, Krzeminski J, Amin S, Arshad R, Ahmed T, Conaway CC. Department of Medicine, New York Medical College, Valhalla, NY 10595, USA. Int J Oncol 2000 Jun;16(6):1215-9

There is growing evidence that thiol conjugates of isothiocyanates present in cruciferous vegetables are effective cancer chemopreventive and potentially active therapeutic agents. The effects of the N-acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC) on tumor cell growth were analyzed in human prostate cancer cell lines LNCaP, androgen-dependent, and DU-145, androgen-independent. Exposure of the cells to PEITC-NAC at high concentrations caused cytolysis, while at lower concentrations PEITC-NAC mediated a dose-dependent growth modulation, with reduction of DNA synthesis and growth rate, inhibition of clonogenicity and induction of apoptosis in both types of prostate cancer cells. PEITC-NAC decreased cells in S and G2M phases of cell cycle, blocking cells entering replicating phases. In parallel, a significant enhancement of cells expressing the cell cycle regulator p21 as well as its intensity was determined using a fluorescent antibody technique. The action of PEITC-NAC was time-dependent, with the magnitude of inhibition increasing to 50-65% after PEITC-NAC exposure for several days. Interaction of tumor cells with dissociation products of PEITC-NAC, PEITC and NAC, are proposed as the mechanism of growth regulation.

78. N-acetylcysteine inhibits endothelial cell invasion and angiogenesis. Cai T, Fassina G, Morini M, Aluigi MG, Masiello L, Fontanini G, D'Agostini F, De Flora S, Noonan DM, Albini A. National Institute for Cancer Research, Genoa, Italy. Lab Invest 1999 Sep;79(9):1151-9

The thiol N-acetylcysteine (NAC) is a chemopreventive agent that acts through a variety of mechanisms and can prevent in vivo carcinogenesis. We have previously shown that NAC inhibits invasion and metastasis of malignant cells as well as tumor take. Neovascularization is critical for tumor mass expansion and metastasis formation. We investigated whether a target of the anti-cancer activity of NAC could be the inhibition of the tumor angiogenesis-associated phenotype in vitro and in vivo using the potent angiogenic mixture of Kaposi's sarcoma cell products as a stimulus. Two endothelial (EAhy926 and human umbilical vein endothelial [HUVE]) cell lines were utilized in a panel of assays to test NAC ability in inhibiting chemotaxis, invasion, and gelatinolytic activity in vitro. NAC treatment of EAhy926 and HUVE cells in vitro dose-dependently reduced their ability to invade a reconstituted basement membrane, an indicator of endothelial cell activation. Invasion of HUVE cells was inhibited with an ID50 of 0.24 mM NAC, whereas inhibition of chemotaxis required a 10 fold higher doses, indicating that invasion is a preferential target. NAC inhibited the enzymatic activity and conversion to active forms of the gelatinase produced by endothelial cells. The matrigel in vivo assay was used for the evaluation of angiogenesis; NAC strongly inhibited neovascularization of the matrigel sponges in response to Kaposi's sarcoma cell products. NAC prevented angiogenesis while preserving endothelial cells, implying that it could be safely used as an anti-angiogenic treatment.

79. Inhibition by oral N-acetylcysteine of doxorubicin-induced clastogenicity and alopecia, and prevention of primary tumors and lung micrometastases in mice.

D'Agostini F, Bagnasco M, Giunciuglio D, Albini A, De Flora S. Institute of Hygiene and Preventive Medicine, University of Genoa, I-16132 Genoa, Italy.

Int J Oncol 1998 Aug;13(2):217-24

The thiol N-acetylcysteine (NAC), an analog and precursor of glutathione, displays cancer preventive properties not only in early stages of the carcinogenesis process but also in its advanced stages. NAC inhibited type-IV collagenase activity as well as invasion, tumor take, and metastasis of malignant cells in murine models. Previously, we provided evidence for synergistic effects of oral NAC with intravenously injected doxorubicin (DOX). In the present study B16-BL6 melanoma cells were injected s.c. into the footpad of C57BL/6 mice. The animals were divided into 5 groups: i) untreated mice; ii) mice receiving daily NAC with drinking water (12.25 mmol/kg body weight) starting 16 h after injection of cancer cells; iii) mice receiving a single i.v. injection of DOX (2 micromol/kg body weight) 24 h after injection of cancer cells; iv) mice receiving a combination of NAC and DOX, with NAC treatment starting 72 h before injection of cancer cells; and v) mice treated as in iv) but with NAC treatment starting 16 h after injection of cancer cells. Both NAC and DOX, either individually or in combination, significantly enhanced the survival time as compared to controls. The weight of local primary tumors was significantly decreased by either drug, and was further decreased to a significant extent, compared to the individual treatments, in the two groups of mice receiving combinations of NAC and DOX. No lung micrometastases, evaluated by immunohistochemistry as S-100-positive foci of melanocytic cells, were detectable in the two groups of mice receiving the combined treatments. NAC significantly, attenuated the time-related increase of micronucleated polychromatic erythrocytes in the peripheral blood of DOX-treated mice. All mice individually treated with DOX developed a partial but well evident alopecia, diffusely affecting their back hair, which was totally prevented by NAC, irrespective of the combination schedule. Thus, besides preventing DOX cardiotoxicity, as extensively documented in the literature, oral NAC protects mice from DOX-induced myelogenotoxicity and alopecia, and at the same time interacts with this cytotoxic agent in inhibiting cancer cell invasion and metastasis.

80. Hyperthermia, radiation carcinogenesis and the protective potential of vitamin A and N-acetylcysteine Sminia P.; Van der Kracht A.H.W.; Frederiks W.M.; Jansen W. Academic Medical Centre, Department of Radiotherapy, Meibergdreef 9, 1105 AZ Amsterdam Netherlands Journal of Cancer Research and Clinical Oncology (Germany), 1996, 122/6 (343-350)

The in vivo carcinogenic risk of hyperthermia, alone or in combination with irradiation, and the anti-carcinogenic potential of vitamin A and N-acetylcysteine (AcCys) were investigated. Starting 1 month before treatment, 160 rats were divided into four diet groups: no additives, vitamin A-enriched diet, AcCys and the combination vitamin A+ AcCys. In 10 animals per diet group, the hind leg was treated with either X-irradiation alone (16 Gy), hyperthermia alone (60 min at 43degreeC), hyperthermia 5 h prior to irradiation or hyperthermia 5 h after irradiation. Animals were observed for 2 years after treatment with regard to the development of tumours either inside or outside the treated volume. After 16 Gy alone 12 plus or minus 5% of the animals developed a tumour. Tumour incidence increased to 37 plus or minus 9% (borderline significance P = 0.07 versus treatment with X-rays alone) when hyperthermia was applied prior to X-rays, and to 24 plus or minus 8% (NS) with hyperthermia after irradiation. The relative risk ratio (RRR) for tumour induction was increased to 2.4 by hyperthermia if combined with X-irradiation. Pathological characterization of induced tumours showed that these were of the fibrosarcoma, osteosarcoma and carcinoma type. Vitamin A alone or in combination with AcCys slightly protected against the induction of tumours by X-rays without or with hyperthermia (RRR of 0.4). However, morphological changes such as lipid accumulation in hepatocytes and damage to the parenchyma were noticed in livers from all animals that were given a vitamin-A-enriched diet (P < 0.0001). Data from the present and past reports show that hyperthermia alone is not carcinogenic, but that it may increase radiation carcinogenesis. Treatment temperature and time of exposure to heat in addition to the radiation dose applied are important factors in the carcinogenic process. The enhancement of radiation carcinogenesis seems to occur independently of the sequence and time interval between irradiation and hyperthermia. However, not all data are consistent with this interpretation.

Pyrrolidine dithiocarbamate inhibits the production of interleukin-6, interleukin-8, and granulocyte-macrophage colony-stimulating factor by human endothelial cells in response to inflammatory mediators: modulation of NF-kappa B and AP-1 transcription factors activity. Munoz C, Pascual-Salcedo D, Castellanos MC, Alfranca A, Aragones J, Vara A, Redondo MJ, de Landazuri MO. Servicio de Inmunologia, Hospital de la Princesa, Universidad Autonoma de Madrid, Spain.

81, Blood 1996 Nov 1;88(9):3482-90

Endothelial cells (EC) play a key role in the inflammatory response, both by the production of proinflammatory cytokines and by their interaction with leukocytes. Molecular genetic analysis has demonstrated that functional NF-kappa B sites are involved in the transcription of interleukin-6 (IL-6), IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes in response to inflammatory mediators. Thus, we have explored the effect of two inhibitors of the NF-kappa B activation, pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC), on the production of these cytokines by EC. Both PDTC and NAC inhibited, in a dose-dependent manner, the synthesis of IL-6, IL-8, and GM-CSF induced by tumor necrosis factor (TNF)-alpha or bacterial lipopolysaccharides (LPS) in human umbilical vein endothelial cells (HUVEC). PDTC appeared to prevent IL-6, IL-8, and GM-CSF gene transcription, as it blocked the induction of specific mRNA by TNF-alpha or LPS. The TNF-alpha mediated transcriptional activation of a chloramphenicol acetyltransferase (CAT) plasmid containing three copies of the -72 kappa B binding site from the IL-6 promoter was abrogated by PDTC. According to transfection experiments, electrophoretic mobility shift assays (EMSA) demonstrated that the antioxidant prevented the induction of NF-kappa B DNA-binding activity by TNF-alpha. Under the same conditions, PDTC by itself or in combination with TNF-alpha, enhanced the DNA-binding activity of AP-1, as well as c-fos and c-jun mRNA levels. Altogether, these results indicate that the antioxidant PDTC specifically inhibits the transcription of IL-6, IL-8, and GM-CSF genes through the inhibition of the NF-kappa B activation, while increasing the expression of AP-1. Our data make evident the antiinflammatory and immunoregulatory potential of the pharmacological inhibition of the NF-kappa B activation. In addition, PDTC and related molecules may be a useful tool to explore the expression of genes involved in the inflammatory response.

82. Inhibition of invasion, gelatinase activity, tumor take and metastasis of malignant cells by N-acetylcysteine. Albini A, D'Agostini F, Giunciuglio D, Paglieri I, Balansky R, De Flora S. Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy. Int J Cancer 1995 Mar 29;61(1):121-9

The thiol N-acetylcysteine (NAC) is currently considered one of the most promising cancer chemopreventive agents by virtue of its multiple and coordinated mechanisms affecting the process of chemical carcinogenesis. Recent studies have shown that an unpaired cysteine residue in the propeptide plays a key role in inactivation of latent metastasis-associated metalloproteinases: the present study was designed to assess whether NAC could also affect tumor take, invasion and metastasis of malignant cells. As assessed by zymographic analysis, NAC completely inhibited the gelatinolytic activity of type-IV collagenases in the cells tested (gelatinases A and B). Moreover, NAC was efficient in inhibiting the chemotactic and invasive activities of tumor cells of human (A2058 melanoma) and murine origin (K1735 and B16-F10 melanoma cells as well as C87 Lewis lung carcinoma cells) in Boyden-chamber assays, which are predictive of the invasive and metastatic properties. Reduced glutathione (GSH) had a similar, although less effective activity. The number of lung metastases decreased sharply when B16-F10 murine melanoma cells, injected i.v. into nude mice, were pre-treated with NAC and resuspended in medium supplemented with 10 mM NAC. In other experiments NAC was given in drinking water, starting 48-72 hr before subcutaneous inoculation of either B16-F10 cells or of their highly metastatic variant B16-BL6, or intramuscular injection of LLC cells. In all experiments NAC treatment decreased the weight of the locally formed primary tumor and produced a dose-related delay in tumor formation. Spontaneous metastasis formation by B16-F10 and B16-BL6 tumors was slightly yet significantly reduced by oral administration of NAC. However, this was not observed for Lewis lung tumors. These data indicate that NAC affects the process of tumor-cell invasion and metastasis, probably due to inhibition of gelatinases by its sulfhydryl group, with the possible contribution of other mechanisms, including the potent antioxidant activity of this thiol.

83. "N-Acetylcysteine for Lung Cancer Prevention" van Zandwijk N Department of Chest Oncology, The Netherlands, Cancer Institute/Antoni van Leeuwenhoek Huis, Amsterdam. Nico Chest May 1995;107(5):1437-1441

In 1981 it was estimated by Doll and Peto that of all cancer deaths in the United States 30% were due to tobacco, 3% to alcohol and 35% to diet and other causes. Twelve percent of lung cancers were not attributable to tobacco and dietary factors were implicated in the causation of cancer in tissues other than the gastrointestinal tract. Damage to cellular DNA not only occurs from environmental mutagens but also from the endogenous production of oxidants which damage DNA and other mechanisms related to the conversion of food, in particular fats to energy. Inflammation and the healing process can also result in damage. Dietary antioxidants have also been shown to prevent this oxidative cellular DNA damage; these include vitamin A, the carotene family, vitamin C, E and selenium. In reviewing approximately 200 published studies there was overwhelming evidence that the consumption of fruit and vegetables is associated with reduced cancer incidence. Cigarette smoke contains oxidants as well as several precarcinogens. Metabolism of carcinogens and the steps of carcinogenesis are a balance between forces such as metabolic activation and detoxification, formation and scavenging of radicals and DNA damage and repair. This suggests that carcinogenic compounds can initiate tumor growth only when they saturate detoxification pathways. Glutathione plays a role in the detoxification of xenobiotics. N-acetylcysteine which is an amino thiol and precursor of intracellular cysteine and glutathione has been shown not only to be an efficient antidote in acetaminophen poisoning but also has important chemopreventive properties. N-acetylcysteine appears to exert its chemopreventive effects by multiple mechanisms and may provide protection against different mutagens and carcinogens in different stages of carcinogenesis. N-acetylcysteine has reached the Phase III trial stage in chemoprevention in Europe and has been used in clinical practice for more than 30 years. In large groups of patients with chronic obstructive lung disease N- acetylcysteine has turned out to be a safe agent with minor effects even when prescribed for a prolonged period of time. N- acetylcysteine is well tolerated when taken continuously in a dose of 600 mg per day. Dyspepsia has been reported as a mild side effect. N-acetylcysteine holds promise and it may turn out to be effective in preventing secondary tumors. It may have a wider use in chemopreventive purposes.

84. Metabolic, desmutagenic and anticarcinogenic effects of N-acetylcysteine. De Flora S, Rossi GA, De Flora A. Respiration 1986;50 Suppl 1:43-9

N-acetylcysteine (NAC) is often administered to respiratory patients with histories of exposure to noxious agents (e.g. cigarette smoke and atmospheric pollutants), which are known to act as glutathione (GSH) depletors and as cancer initiators and/or promoters. Since NAC is a precursor of intracellular GSH, we investigated its effects on GSH metabolism and on the biotransformation of carcinogenic and/or mutagenic compounds. In vitro, NAC induced a significant increase in oxidized glutathione (GSSG) reductase activity in rat liver preparations and counteracted the mutagenicity of direct-acting compounds (such as epichlorohydrin, hydrogen peroxide, 4-nitroquinoline-N-oxide and dichromate), as a result of its reducing and scavenging properties. At high concentrations, the drug completely inhibited the mutagenicity of procarcinogens (cigarette smoke condensate, tryptophan pyrolysate, cyclophosphamide, 2-aminofluorene, benzo(a)pyrene and aflatoxin B1) by binding their electrophilic metabolites. In contrast, their metabolic activation was stimulated by decreasing NAC concentrations, especially when liver preparations from enzyme-induced rats were used. Lung and liver subcellular preparations of rats treated in vivo with NAC, in various combinations with enzyme inducers and/or GSH depletors, also affected the mutagenicity of a number of compounds. NAC generally increased intracellular GSH and restored its levels following depletion. It did not affect the levels nor the spectral properties of cytochromes P-450 in pulmonary and hepatic microsomes, whereas it stimulated, especially in Aroclor-pretreated animals, cytosolic enzyme activities involved in NADP or GSSG reduction (G6PD, 6PGD and GSSG reductase) and in the reductive detoxification of xenobiotics (DT diaphorase). When administered with the diet, at a nontoxic posology (120 mg/kg b.w.), NAC markedly inhibited the induction of lung tumors in mice by a potent carcinogen (urethane).

85. N-acetylcysteine as antimutagen and anticarcinogen. De Flora, S. et al. Toxicol. Lett. 1992; 53: W4/L2 (abstract). No abstract available.

Synergism between N-acetylcysteine and doxorubicin in the prevention of tumorigenicity and metastasis in murine models. De Flora S, D'Agostini F, Masiello L, Giunciuglio D, Albini A. Institute of Hygiene and Preventive Medicine, University of Genoa, Italy. Int J Cancer 1996 Sep 17;67(6):842-8

The thiol N-acetylcysteine (NAC) is a promising cancer chemopreventive agent which acts through a variety of mechanisms, including its nucleophilic and antioxidant properties. We have recently shown that NAC inhibits type-IV collagenase activity as well as invasion, tumor take and metastasis of malignant cells in mice. NAC is also known to attenuate the cardiotoxicity of the cytostatic drug doxorubicin (DOX, Adriamycin). The present study was designed to evaluate whether the combination of NAC and DOX treatments in mice injected with cancer cells could affect their tumorigenic and metastatic properties. Six separate experiments were carried out, using a total of 291 adult female mice. In experimental metastasis assays, in which B16-F10 melanoma cells were injected i.v. into (CD-1)BR nude mice, DOX significantly reduced the number of lung metastases when administered i.v. at a dose of 10 mg/kg body weight, 3 days after the i.v. injection of cancer cells. NAC inhibited lung metastases when added to the medium of cancer cells before their i.v. injection. The combined treatment with DOX and NAC, under various experimental conditions, was highly effective, showing a synergistic reduction in the number of mestastases. In tumorigenicity and spontaneous metastasis assays, in which B16-BL6 melanoma cells were injected s.c. into the footpad of C57BL/6 mice, DOX decreased the number of lung metastases when given i.p. at 2 mg/kg body weight. Oral NAC exerted significant protective effects, and considerably prolonged survival of mice. The combined treatment with DOX and NAC again showed synergistic effects on the frequency and weight of primary tumors and local recurrences, and completely prevented the formation of lung metastases in the experiment in which these end-points were evaluated at fixed times. While injection of DOX 7 days after implantation of cancer cells failed to improve the cancer-protective effects of NAC, its injection after I day resulted in a striking inhibition of lung metastases. These findings demonstrate an evident synergism between DOX (given parenterally) and NAC (given with drinking water) in preventing tumorigenicity and metastases. The indications of these animal studies warrant further evaluation in clinical trials.

CARDIOVASCULAR w/kidney **

86. J Surg Res. 2003 May 15;111(2):236-9. Attenuation of ischemia/reperfusion injury by N-acetylcysteine in a rat hind limb model. Koksal C, Bozkurt AK, Cangel U, Ustundag N, Konukoglu D, Musellim B, Sayin AG. Sureyyapasa Thoracic and Cardiovascular Disease Hospital, Departmemnt of Cardiovascular Surgery, Istanbul, Turkey. cengizkoksal@hotmail.com

BACKGROUND: Ischemia/reperfusion is a complex set of events with severe pathologic consequences. Reperfusion initiates both the local and systemic damage in part through rapid oxygen generation. N-acetylcysteine (NAC) is a scavenger of free radical species, inhibits neutrophil accumulation, acts as a vasodilator and also improves microcirculation. In present study, we examined the protective effect of NAC in a rat hind limb ischemia/ reperfusion model. Dimethyl-sulfoxide (DMSO), a well-known antioxidant was also tested for comparison. MATERIALS AND METHODS: Ischemia was induced for 4 h by vascular clamping and followed by 1 h of reperfusion. Muscle injury was evaluated in 3 groups as a saline group (control), DMSO group, and NAC group. Plasma levels of creatine kinase, lactate dehydrogenase, thiobarbituric acid reactive substances (TBARS), and blood HCO(3), as well as muscle tissue TBARS, were measured at the end of reperfusion. Muscle tissue samples were taken for histological evaluation. RESULTS: DMSO and NAC group showed significant amelioration of plasma CPK (P < 0.05, P < 0.05), plasma TBARS (P < 0.05, P < 0.05), and muscle tissue TBARS (P < 0.05, P < 0.05) compared with the control group. Similarly, neutrophil infiltration in DMSO and NAC groups were significantly less prominent than the control group (P < 0.01, P < 0.01). CONCLUSIONS: These results show that NAC improved effectively ischemia reperfusion injury in a rat hind limb model.

87. J Invasive Cardiol. 2003 Jun;15(6):311-4. Prevention of radiocontrast-induced nephropathy with N-acetylcysteine in patients undergoing coronary angiography. Tadros GM, Mouhayar EN, Akinwande AO, Campbell B, Wood C, Blankenship JA. Department of Internal Medicine, Geisinger Medical Center, Danville, PA 17822, USA. gmtadros@geisinger.edu

OBJECTIVES: Acetylcysteine in patients undergoing computerized tomography with intravenous contrast reduces the incidence of acute renal dysfunction. We examined the effect of N-acetylcysteine in patients undergoing coronary angiography. METHODS: Fifty-five consecutive patients receiving 3 doses of N-acetylcysteine prior to cardiac catheterization were compared to 55 historical controls. All patients in both groups had baseline serum creatinine > 1.2 mg/dl and received intravenous hydration before and after the procedure. Serum creatinine levels at baseline and 48 hours after the procedure were compared. RESULTS: Univariate analysis of clinical variables revealed no significant differences between the groups except for a higher baseline creatinine in the treatment group (2.0 0.7 vs. 1.8 0.4 mg/dl; p = 0.04). There was no difference in the amount or type of contrast used. The mean change in creatinine after 48 hours was -0.4 0.3 versus +0.1 0.3 mg/dl for treatment and control groups (p < 0.001). In patients with baseline creatinine > 2 mg/dl, the benefit was larger (-0.4 0.4 vs. +0.5 0.3 mg/dl; p < 0.001). Multivariate analysis confirmed pre-treatment with N-acetylcysteine as an independent predictor of renal protection (p < 0.001). CONCLUSIONS: Prophylactic use of acetylcysteine prevented reduction of renal function after coronary angiography. The benefit was greater in patients with baseline serum creatinine > 2 mg/dl.

88. Neurosci Res. 2003 Jun;46(2):191-7. Antioxidant NAC and AMPA/KA receptor antagonist DNQX inhibited JNK3 activation following global ischemia in rat hippocampus. Tian H, Zhang G, Li H, Zhang Q. Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, Jiangsu 221002, PR China.

c-Jun N-terminal kinase-3 (JNK3), the only neural-specific isoform, may play an important role in excitotoxicity and neuronal injury. To analyze the variation of JNK3 activation, levels of phospho-JNK3 were measured at various time points of ischemia and selected time points of reperfusion, respectively. Our study illustrated that JNK3 was rapidly activated and translocated from cytosol to nucleus during ischemia. During reperfusion, two peaks of JNK3 activation occurred at 30 min and 3 days, respectively. To further define the mechanism of JNK3 activation, antioxidant N-acetylcysteine (NAC), alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate (KA) receptor antagonist 6,7-dinitro-quinoxaline-2,3(1H,4H)-dione (DNQX), N-methyl-D-aspartate (NMDA) receptor antagonist ketamine and L-type voltage-gated Ca(2+) channel (L-VGCC) antagonist nifedipine were given to the rats 20 min prior to ischemia. The results showed that NAC obviously inhibited JNK3 activation during the early reperfusion, whereas DNQX preferably attenuated JNK3 activation during the latter reperfusion. Ketamine and nifedipine had no significant effects on JNK3 activation during reperfusion. Consequently, reactive oxygen species (ROS) and AMPA/KA receptor were closely associated with JNK3 activation following global ischemia.

89. Anesthesiology. 2003 Jun;98(6):1384-90. N-acetylcysteine restores isoflurane-induced preconditioning against myocardial infarction during hyperglycemia. Kehl F, Krolikowski JG, Weihrauch D, Pagel PS, Warltier DC, Kersten JR. Department of Anesthesiology, Medical College of Wisconsin and the Zablocki VA Medical Center, Milwaukee, Wisconsin 53226, USA.

BACKGROUND: Hyperglycemia generates reactive oxygen species and prevents isoflurane-induced preconditioning. The authors tested the hypothesis that scavenging reactive oxygen species with N-acetylcysteine will restore protection against myocardial infarction produced by isoflurane in vivo. METHODS: Barbiturate-anesthetized dogs (n = 45) were instrumented for measurement of systemic hemodynamics. Myocardial infarct size and coronary collateral blood flow were measured with triphenyltetrazolium staining and radioactive microspheres, respectively. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3 h of reperfusion. Dogs were randomly assigned to receive an infusion of 0.9% saline or 15% dextrose in water to increase blood glucose concentrations to 600 mg/dl (hyperglycemia) in the absence or presence of isoflurane (1.0 minimum alveolar concentration) with or without pretreatment with N-acetylcysteine (150 mg/kg i.v.) in six experimental groups. Isoflurane was discontinued, and blood glucose concentrations were allowed to return to baseline values before left anterior descending coronary artery occlusion. RESULTS: Myocardial infarct size was 27 +/- 2% (n = 8) of the left ventricular area at risk in control experiments. Isoflurane significantly (P < 0.05) decreased infarct size (13 +/- 2%; n = 7). Hyperglycemia alone did not alter infarct size (29 +/- 3%; n = 7) but abolished the protective effect of isoflurane (25 +/- 2%; n = 8). N-acetylcysteine alone did not affect infarct size (28 +/- 2%; n = 8) but restored isoflurane-induced cardioprotection during hyperglycemia (10 +/- 1%; n = 7). CONCLUSIONS: Acute hyperglycemia abolishes reductions in myocardial infarct size produced by isoflurane, but N-acetylcysteine restores these beneficial effects. The results suggest that excessive quantities of reactive oxygen species generated during hyperglycemia impair isoflurane-induced preconditioning in dogs.

90. Am J Hematol. 2003 May;73(1):26-32. Effects of N-acetylcysteine on dense cell formation in sickle cell disease. Pace BS, Shartava A, Pack-Mabien A, Mulekar M, Ardia A, Goodman SR. Department of Molecular and Cell Biology, University of Texas at Dallas, 2601 Floyd Road, Mail Station FO 3.1, Richardson, TX 75083, USA. bpace@utdallas.edu

The extent to which dense and irreversible sickle cells (ISCs) contribute to vaso-occlusive episodes in sickle cell disease remains unclear. N-Acetylcysteine (NAC) inhibits dense cell and ISC formation in sickle erythrocytes in vitro and restores glutathione levels toward normal. A phase II double-blind randomized clinical trial was completed to determine the efficacy of NAC in decreasing dense cell and ISC formation, and vaso-occlusive episodes in sickle cell disease. Twenty-one subjects with a history of at least two vaso-occlusive episodes per year and 6% dense cells were enrolled. Four treatment groups were analyzed; NAC at a dose of 2,400 mg per day decreased the percent dense cells from 20.1 +/- 2.9 to 12.6 +/- 2.1 (P < 0.05) and increased red cell glutathione levels from 292.8 +/- 74.5 to 576.7 +/- 155.1 (P < 0.05). In addition, we observed a decrease in vaso-occlusive episodes from 0.03 to 0.006 episodes per person-days and a decreased in relative risk to R = 0.39. Although NAC did not significantly decrease the number of ISCs, there was a downward trend at all doses tested. In summary, NAC inhibited dense cell formation, restored glutathione levels toward normal, and decreased vaso-occlusive episodes at a well-tolerated dose of 2,400 mg per day. To determine the long-term efficacy and safety of NAC, a multicenter phase III clinical trial is required. Copyright 2003 Wiley-Liss, Inc.

91. Brain Res. 2003 May 2;971(1):1-8. N-Acetyl cysteine protects against injury in a rat model of focal cerebral ischemia. Sekhon B, Sekhon C, Khan M, Patel SJ, Singh I, Singh AK. Department of Pediatrics, Division of Developmental Neurogenetics, Medical University of South Carolina, 29425, Charleston, SC, USA

Ischemic cerebrovascular disease (stroke) is one of the leading causes of death and long-time disability. Ischemia/reperfusion to any organ triggers a complex series of biochemical events, which affect the structure and function of every organelle and subcellular system of the affected cells. The purpose of this study was to investigate the therapeutic efficacy of N-Acetyl cysteine (NAC), a precursor of glutathione and a potent antioxidant, to attenuate ischemia/reperfusion injury to brain tissue caused by a focal cerebral ischemia model in rats. A total of 27 male Sprague-Dawley rats weighing 250-300 g were used in this study. Focal cerebral ischemia (45 min) was induced in anesthetized rats by occluding the middle cerebral artery (MCA) with an intra-luminal suture through the internal carotid artery. The rats were scored post-reperfusion for neurological deficits. They were then sacrificed after 24 h of reperfusion and infarct volume in the brain was assessed by 2,3,5-triphenyl tetrazolium chloride (TTC). Brain sections were immunostained for tumor necrosis factor (TNF-alpha) and inducible nitric oxide synthase (iNOS). Animals treated with NAC showed a 49.7% (S.E.M.=1.25) reduction in brain infarct volume and 50% (S.E.M.=0.48) reduction in the neurological evaluation score as compared to the untreated animals. NAC treatment also blocked the ischemia/reperfusion-induced expression of tumor necrosis factor and inducible nitric oxide synthase. The data suggest that pre-administration of NAC attenuates cerebral ischemia and reperfusion injury in this brain ischemia model. This protective effect may be as a result of suppression of TNF-alpha and iNOS.

92. J Nephrol. 2003 Jan-Feb;16(1):75-80. Protective effect of N-acetylcysteine on renal ischemia/reperfusion injury in the rat. Sehirli AO, Sener G, Satiroglu H, Ayanoglu-Dulger G. Marmara University, School of Pharmacy, Department of Pharmacology, Istanbul, Turkey.

BACKGROUND: Oxygen free radicals are important components involved in the pathophysiological tissue alterations observed during ischemia/reperfusion (I/R). METHODS: The protective effect of N-acetylcysteine (NAC) against the damage inflicted by reactive oxygen species during renal I/R was investigated in Wistar Albino rats using biochemical parameters. Animals were unilaterally nephrectomized, and subjected to 45 min of renal pedicle occlusion followed by lh of reperfusion. N-acetylcysteine (150 mg/kg, i.p.) or vehicle was administered twice, 15 min prior to ischemia and immediately before the reperfusion period. At the end of the reperfusion period, rats were killed by decapitation. For biochemical analysis, the lipid peroxidation product malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and protein oxidation (PO) were tested. Serum creatinine and BUN concentrations were measured for the evaluation of renal function. RESULTS: I/R induced nephrotoxicity, as evidenced by increases in BUN and creatinine, was reversed by NAC. The decrease in GSH and increases in MDA, MPO and PO induced by I/R indicated that renal injury involves free radical formation. CONCLUSIONS: Since NAC reversed these oxidant responses, and protected rat renal proximal tubules from in vitro simulated reperfusion injury, it seems that NAC protects kidney tissue against oxidative damage.

93. Dig Liver Dis. 2002 Aug;34(8):560-9. Gut barrier permeability, reticuloendothelial system function and protease inhibitor levels following intestinal ischaemia and reperfusion--effects of pretreatment with N-acetyl-L-cysteine and indomethacin. Sun Z, Lasson A, Olanders K, Deng X, Andersson R. Department of Surgery, Lund University Hospital, Lund, Sweden.

BACKGROUND: Pathophysiological mechanisms and ways to intervene on intestinal barrier dysfunction following small intestinal ischaemia and prolonged reperfusion are still not fully clarified. AIMS: To evaluate the effect of oxygen free radical and prostaglandin inhibition on intestinal barrier injury following intestinal ischaemia/reperfusion. METHODS: Endothelial and epithelial barrier permeability was evaluated by clearance of radiolabelled albumin. Parameters included 125I-Escherichia coli uptake rate index, host reticuloendothelial system function and organ distribution, as well as protease inhibitor and proenzyme activities in rats subjected to small intestinal ischaemia for 40 minutes followed by 12 hours reperfusion (ischaemia/reperfusion), pretreated with N-acetyl-L-cysteine or indomethacin. RESULTS: Following ischaemia/reperfusion, endothelial and epithelial permeability increased, reticuloendothelial system activation occurred and plasma protease inhibitors were consumed. N-acetyl-L-cysteine pretreatment resulted in improved endothelial and epithelial barrier integrity, a decrease in protease inhibitor consumption and less pronounced reticuloendothelial system activation. Pretreatment with indomethacin was not effective. CONCLUSION: Oxygen free radicals seem to play an important role in the development of intestinal barrier impairment following ischaemia/reperfusion. N-acetyl-L-cystine may be a potential agent for preventing ischaemia/reperfusion damage.

94. Mol Cell Biochem. 2002 Nov;240(1-2):19-29. Attenuation of ischemia/reperfusion induced MAP kinases by N-acetyl cysteine, sodium nitroprusside and phosphoramidon. Mehta A, Sekhon CP, Giri S, Orak JK, Singh AK. Department of Pediatrics, Medical University of South Carolina, Charleston, SC 29425, USA.

Ischemia followed by reperfusion has a number of clinically significant consequences. A number of pathophysiological processes appear to be involved in ischemia/reperfusion (I/R) injury. The mitogen activated protein kinases (MAPK) are integral components of the parallel MAP kinase cascades activated in response to a variety of cellular stress inducing ischemia/ATP depletion and inflammatory cytokines. Many studies suggest that members of the MAP kinase family in particular Jun N-terminal kinase (JNK) are activated in kidney following ischemia/reperfusion of this tissue. The present study underlines the therapeutic potential of the combination of N-Acetyl cysteine (NAC), a potent antioxidant, sodium nitroprusside (SNP), a nitric oxide donor and phosphoramidon (P), an endothelin-1 converting enzyme inhibitor in ameliorating the MAPK induced damage during renal ischemia/reperfusion injury. Our previous results showed that 90 min of ischemia followed by reperfusion caused very severe injury and that the untreated animals had 100% mortality after the 3rd day whereas there was improved renal function and 100% survival of animals in the three drug combination treatment group. The present study, mainly on tissue sections, further supports the protection provided by the triple drug therapy. A higher degree of expression of all the three classes of MAPK, i.e. JNK, P38 MAP kinases and P-extracellular signal regulated kinases (ERKs) can be seen in kidneys subjected to ischemia/reperfusion insult. Pretreatment with a combination of N-acetyl cysteine, sodium nitroprusside, and phosphoramidon completely inhibits all three classes of MAPK and ameliorates AP-1 whereas individual or a combination of any two drugs is not as effective.

95. Mol Cell Biochem. 2002 Nov;240(1-2):9-17. Combination therapy of N-acetylcysteine, sodium nitroprusside and phosphoramidon attenuates ischemia-reperfusion injury in rat kidney. Dobashi K, Singh I, Orak JK, Asayama K, Singh AK. Department of Pediatrics and Pathology, Medical University of South Carolina, Charleston, SC 29425, USA.

Renal ischemia is of clinical interest because of its role in renal failure and also renal graft rejection. To evaluate the effect of the combination of N-acetylcysteine (NAC), a potent antioxidant, sodium nitroprusside (SNP), a nitric oxide donor, and phosphoramidon (P), an endothelin converting enzyme inhibitor, on tissue protection against ischemia-reperfusion injury, we studied the biochemical and morphological changes due to 90 min of renal ischemia-reperfusion in the rat model. Ninety min of ischemia caused very severe injury and the animals could not survive after 4 days without any treatment. Whereas, animals in the treated groups survived i.e. the NAC group (25%), NAC + SNP group (43%) and in the NAC + SNP + P group (100%), 2 weeks after 90 min of ischemia. A significant increase in the serum levels of creatinine and urea nitrogen was shown in the untreated group and to a much lesser extent in the treated group, especially in the NAC + SNP + P group. The protective effect was also supported by light microscopic studies on renal tissue sections. We also measured the activities of antioxidant enzymes in tissue homogenates. With the exception of Mn-superoxide dismutase, the activities of antioxidant enzymes (catalase, glutathione peroxidase, CuZn-superoxide dismutase) were decreased in the untreated kidney. The administration of NAC alone and NAC + SNP protected against the loss of activities. Treatment with a combination of NAC, SNP and P showed a synergistic effect as evidenced by the best protection. These results suggest that pre-administration of a combination of antioxidant (NAC) with endothelin derived vasodilators (sodium nitroprusside and Phosphoramidon) attenuates renal ischemia-reperfusion injury, e.g. in donor kidney for transplantation, by protecting cells against free radical damage.

96. Effect of ascorbate or N-acetylcysteine treatment in a patient with hereditary glutathione synthetase deficiency. Jain A, Buist NR, Kennaway NG, Powell BR, Auld PA, Martensson J. Department of Internal Medicine, Greenwich Hospital/Yale University School of Medicine, CT 06830-4697. J Pediatr 1994 Feb;124(2):229-33

A 45-month-old girl with 5-oxoprolinuria (pyroglutamic aciduria), hemolysis, and marked glutathione depletion caused by deficiency of glutathione synthetase was followed before and during treatment with ascorbate or N-acetylcysteine. High doses of ascorbate (0.7 mmol/kg per day) or N-acetylcysteine (6 mmol/kg per day) were given for 1 to 2 weeks without any obvious deleterious side effects. Ascorbate markedly increased lymphocyte (4-fold) and plasma (8-fold) levels of glutathione. N-Acetylcysteine also increased lymphocyte (3.5-fold) and plasma (6-fold) levels of glutathione. After these treatments were discontinued, lymphocyte and plasma glutathione levels decreased rapidly to pretreatment levels. Ascorbate treatment was extended for 1 year, and lymphocyte (4-fold) and plasma (2- to 5-fold) glutathione levels remained elevated above baseline. In parallel, the hematocrit increased from 25.4% to 32.6%, and the reticulocyte count decreased from 11% to 4%. The results demonstrate that ascorbate and N-acetylcysteine can decrease erythrocyte turnover in patients with hereditary glutathione deficiency by increasing glutathione levels.

DIABETES **

97.. Fertil Steril. 2002 Jun;77(6):1128-35. N-acetyl-cysteine treatment improves insulin sensitivity in women with polycystic ovary syndrome. Fulghesu AM, Ciampelli M, Muzj G, Belosi C, Selvaggi L, Ayala GF, Lanzone A. Department of Obstetrics and Gynecology, Universita Cattolica del Sacro Cuore, Roma, Italy.

OBJECTIVE: To evaluate the effect of N-acetyl-cysteine (NAC) on insulin secretion and peripheral insulin resistance in subjects with polycystic ovary syndrome (PCOS). DESIGN: Prospective data analysis. SETTING: Volunteer women in an academic research environment. PATIENT(S): Six lean and 31 obese subjects, aged 19-33 years. INTERVENTION(S): Patients were treated for 5-6 weeks with NAC at a dose of 1.8 g/day orally. A dose of 3 g/day was arbitrarily chosen for massively obese subjects. Six of 31 obese patients with PCOS were treated with placebo and served as controls. MAIN OUTCOME MEASURE(S): Before and after the treatment period, the hormonal and lipid blood profile and insulin sensitivity, assessed by an hyperinsulinemic euglycemic clamp, were evaluated and an oral glucose tolerance test (OGTT) was performed. RESULT(S): Fasting glucose, fasting insulin, and glucose area under curve (AUC) were unchanged after treatment. Insulin AUC after OGTT was significantly reduced, and the peripheral insulin sensitivity increased after NAC administration, whereas the hepatic insulin extraction was unaffected. The NAC treatment induced a significant fall in T levels and in free androgen index values (P<.05). In analyzing patients according to their insulinemic response to OGTT, normoinsulinemic subjects and placebo-treated patients did not show any modification of the above parameters, whereas a significant improvement was observed in hyperinsulinemic subjects. CONCLUSION(S): NAC may be a new treatment for the improvement of insulin circulating levels and insulin sensitivity in hyperinsulinemic patients with polycystic ovary syndrome.

98. Free Radic Biol Med. 2002 Dec 15;33(12):1615-21. Effect of high-glucose levels on protein oxidation in cultured lens cells, and in crystalline and albumin solution and its inhibition by vitamin B(6) and N-acetylcysteine: its possible relevance to cataract formation in diabetes. Jain AK, Lim G, Langford M, Jain SK. Caddo Magnet High School, Shreveport, LA, USA

Diabetic patients have elevated levels of glucose in their blood and other body fluids. This project studied the effect of high-glucose concentrations (HG) on the protein oxidation in cultured lens cells and in crystalline protein solution. In addition, we also examined the effect of HG on the oxidation and turbidity (aggregation) of albumin protein solution. This study also examined whether vitamin B(6) [pyridoxine (P), pyridoxamine (PM)] or n-acetylcysteine (NAC) is capable of preventing protein oxidation similar to that seen in cataracts. For cell culture studies, rabbit lens cells were cultured in control or HG medium at 37 degrees C for 2 d. For studies with protein solution, a buffered solution of serum albumin or crystalline protein was incubated with normal glucose (5 mM) or HG (50-100 mM) in a water bath at 37 degrees C for 4 d. All treatments were carried out with and without the addition of P, PM, or NAC. We found significantly higher levels of carbonyl protein (an index of protein oxidation) in HG-treated compared with normal glucose-treated lens cells and in crystalline protein solution. P, PM, and NAC significantly decreased the protein oxidation in lens cells and crystalline protein solution. We also found significantly higher levels of protein oxidation and turbidity (an index of protein aggregation) and its inhibition by P, PM, and NAC in HG-treated compared with normal glucose-treated albumin solution. This suggests that HG can cause the oxidation and modification of proteins in the lens, and that vitamin B(6) and NAC supplementation may be helpful in slowing the oxidation of lens proteins. This study explains the cause of early cataract development and the potential benefit of supplementation with vitamin B(6) and NAC in the prevention of the development of cataract among the diabetic population.

99. Diabetologia. 1996 Mar;39(3):263-9. Inhibition of development of peripheral neuropathy in streptozotocin-induced diabetic rats with N-acetylcysteine. Sagara M, Satoh J, Wada R, Yagihashi S, Takahashi K, Fukuzawa M, Muto G, Muto Y, Toyota T. Third Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan.

N-acetylcysteine (NAC) is a precursor of glutathione (GSH) synthesis, a free radical scavenger and an inhibitor of tumour necrosis factor alpha (TNF). Because these functions might be beneficial in diabetic complications, in this study we examined whether NAC inhibits peripheral neuropathy. Motor nerve conduction velocity (MNCV) was significantly decreased in streptozotocin-induced-diabetic Wistar rats compared to control rats. Oral administration of NAC reduced the decline of MNCV in diabetic rats. Structural analysis of the sural nerve disclosed significant reduction of fibres undergoing myelin wrinkling and inhibition of myelinated fibre atrophy in NAC-treated diabetic rats. NAC treatment had no effect on blood glucose levels or on the nerve glucose, sorbitol and cAMP contents, whereas it corrected the decreased GSH levels in erythrocytes, the increased lipid peroxide levels in plasma and the increased lipopolysaccharide-induced TNF activity in sera of diabetic rats. Thus, NAC inhibited the development of functional and structural abnormalities of the peripheral nerve in streptozotocin-induced diabetic rats.

100. Diabetologia. 1996 Apr;39(4):401-11. High glucose concentration inhibits migration of rat cranial neural crest cells in vitro. Suzuki N, Svensson K, Eriksson UJ. Department of Medical Cell Biology, University of Uppsala, Sweden.

Cranial neural crest cells give rise to a large part of the facial structures, and disturbed development of these cells may therefore cause congenital malformations affecting the head and face. We studied the effects of increased glucose concentration on the migration and development of cranial neural crest cells, maintained in vitro for 48 h. Pre-migratory cranial neural crest cells were removed from embryos of normal and diabetic rats on gestational day 9. After 24 h in 10 mmol/l glucose the cells were exposed to glucose concentrations of 10, 30, or 50 mmol/l for another 24 h. The cultures were photographed at 24 h and 48 h in a phase-contrast microscope to evaluate cell morphology, cell number, and cell migration. Exposure to 50 mmol/l glucose reduced the total number of neural crest cells, their mean migratory distance and migratory area expansion compared to cells cultured in 10 mmol/l glucose. To investigate the effect of antioxidant agents, high glucose cultures were studied after addition of N-acetylcysteine (NAC), or superoxide dismutase (SOD). Addition of NAC diminished the inhibitory effect of high glucose, whereas SOD did not offer any improvement in cell development. Neural crest cell culture from embryos of diabetic rats showed reduced cell migration in vitro at all glucose concentrations compared to normal cells. In addition, the cells from embryos of diabetic rats showed reduced migratory area expansion after culture in the basal 10 mmol/l glucose concentration, indicating that maternal diabetes permanently influences the future development of premigratory cranial neural crest cells. These findings indicate that high glucose concentration inhibits cranial neural crest development in vitro, and that antioxidant therapy may diminish this inhibition. Free radical oxygen species may be involved in the induction of malformations and antioxidants may therefore have a role in future attempts to block the teratogenic effects of diabetic pregnancy.

101. J Clin Invest. 1995 Sep;96(3):1395-403. Advanced glycation endproducts interacting with their endothelial receptor induce expression of vascular cell adhesion molecule-1 (VCAM-1) in cultured human endothelial cells and in mice. A potential mechanism for the accelerated vasculopathy of diabetes. Schmidt AM, Hori O, Chen JX, Li JF, Crandall J, Zhang J, Cao R, Yan SD, Brett J, Stern D. Department of Medicine, Columbia University-College of Physicians and Surgeons, New York, New York 10032, USA.

Vascular cell adhesion molecule-1 (VCAM-1), an inducible cell-cell recognition protein on the endothelial cell surface (EC), has been associated with early stages of atherosclerosis. In view of the accelerated vascular disease observed in patients with diabetes, and the enhanced expression of VCAM-1 in diabetic rabbits, we examined whether irreversible advanced glycation endproducts (AGEs), could mediate VCAM-1 expression by interacting with their endothelial cell receptor (receptor for AGE, RAGE). Exposure of cultured human ECs to AGEs induced expression of VCAM-1, increased adhesivity of the monolayer for Molt-4 cells, and was associated with increased levels of VCAM-1 transcripts. The inhibitory effect of anti-RAGE IgG, a truncated form of the receptor (soluble RAGE) or N-acetylcysteine on VCAM-1 expression indicated that AGE-RAGE-induced oxidant stress was central to VCAM-1 induction. Electrophoretic mobility shift assays on nuclear extracts from AGE-treated ECs showed induction of specific DNA binding activity for NF-kB in the VCAM-1 promoter, which was blocked by anti-RAGE IgG or N-acetylcysteine. Soluble VCAM-1 antigen was elevated in human diabetic plasma. These data are consistent with the hypothesis that AGE-RAGE interaction induces expression of VCAM-1 which can prime diabetic vasculature for enhanced interaction with circulating monocytes.

102. Clin Immunol Immunopathol. 1994 Jun;71(3):333-7. Inhibition with N-acetylcysteine of enhanced production of tumor necrosis factor in streptozotocin-induced diabetic rats. Sagara M, Satoh J, Zhu XP, Takahashi K, Fukuzawa M, Muto G, Muto Y, Toyota T. Third Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan.

We previously reported that the in vivo production of the tumor necrosis factor alpha (TNF) was significantly enhanced after the onset of diabetes in spontaneous type 1 and 2 diabetic animals. In this report we confirmed the enhanced production of TNF in streptozotocin (STZ)-induced diabetes and then attempted to suppress the enhanced TNF production with N-acetylcysteine (NAC), a precursor of glutathione synthesis. The lipopolysaccharide-induced serum TNF activities were significantly enhanced in STZ-induced diabetic rats (6-18 weeks of age) compared with those of nondiabetic rats throughout the 12-week experiment. A single, oral administration of NAC (200 or 1000 mg/kg body wt) significantly suppressed the enhanced TNF production in the diabetic rats compared with that in untreated rats in a dose-dependent manner. On the other hand, in the long-term (6 or 12 weeks) administrations, smaller doses of NAC (50 or 200 mg/kg/day) also significantly inhibited the enhanced production of TNF regardless of the dose of NAC. NAC administration, however, did not suppress the TNF production of nondiabetic rats. The long-term NAC administration affected neither body weight nor levels of serum glucose, fructosamine, albumin, and triglyceride. These results show that NAC administration significantly suppressed the enhanced TNF production in diabetic rats and indicate that NAC might be useful in preventing TNF-mediated pathological conditions in diabetes.

103. Biochim Biophys Acta. 1995 Jan 20;1254(2):187-92. The effect of reduction of lipoprotein (a) on cellular cholesterol synthesis in non-diabetic and type 2 diabetic subjects. Gilligan S, Owens D, Stinson J, Collins P, Johnson A, Tomkin GH. Department of Biochemistry, Royal College of Surgeons in Ireland, Dublin.

This study investigates the effect of Lipoprotein (a) (Lp(a)) on cellular cholesterol synthesis in non-diabetic (n = 7) and Type 2 (non-insulin-dependent) diabetic subjects (n = 7) with elevated levels of Lp(a) (> 20 mg/dl). N-acetylcysteine was used to lower Lp(a) in the control subjects and their lipoproteins were re-examined after 7 days of treatment. Low-density lipoprotein (LDL) was isolated and separated from Lp(a) by sequential ultracentrifugation. Regulation of cellular cholesterol synthesis was assessed by measuring incorporation of [14C]acetate into mononuclear leucocytes in the presence of LDL and Lp(a). Cellular cholesterol content was determined by a fluorometric assay. Delivery of cholesterol to the cell was examined using [3H]cholesteryl oleate-labelled LDL or Lp(a). LDL (5 micrograms/ml) from non-diabetic subjects suppressed cellular cholesterol synthesis by 66.2%, while Lp(a) at a similar concentration only suppressed cholesterol synthesis by 5.8% (P < 0.001). At a concentration of 20 micrograms/ml, Lp(a) suppressed cholesterol synthesis by 31.7%. The situation was similar in the diabetic subjects. Serum LDL cholesterol in non-diabetic subjects was 4.2 +/- 0.5 mmol/l and the LDL esterified/free cholesterol ratio was 2.6 +/- 0.2. Following treatment with N-acetylcysteine, LDL cholesterol did not change, while Lp(a) decreased significantly by 24% (P < 0.05). The LDL esterified/free cholesterol ratio decreased to 2.2 +/- 0.2 (P < 0.05) and there was a significant increase in the ability of the subjects LDL to inhibit cellular cholesterol synthesis (P < 0.05). There was a significant negative correlation between plasma Lp(a) and the ability of the patients' LDL to inhibit cellular cholesterol synthesis (r = -0.68, P < 0.01). [3H]Cholesteryl-oleate-LDL (5 micrograms/ml) delivered 266 +/- 13 ng cholesteryl oleate/mg cell protein, while it took 20 micrograms of [3H]cholesteryl oleate-labelled-Lp(a) to deliver a similar concentration (315 +/- 21 ng cholesteryl oleate/mg cell protein). In conclusion it appears possible that the atherogenicity of Lp(a) may be associated with its effect on the LDL receptor which alters LDL receptor uptake, LDL composition and cellular cholesterol synthesis.

HEAVY METALS **

104. Toxicol Appl Pharmacol. 2003 Jul 1;190(1):17-24. Cadmium induces apoptosis in anterior pituitary cells that can be reversed by treatment with antioxidants. Poliandri AH, Cabilla JP, Velardez MO, Bodo CC, Duvilanski BH. Centro de Investigaciones en Reproduccion, Facultad de Medicina, Universidad de Buenos Aires C1121ABG, Argentina.

Cadmium (Cd(2+)) is an ubiquitous toxic metal that is involved in a variety of pathological conditions. Several reports indicate that Cd(2+) alters normal pituitary hormone secretion; however, little is known about the mechanisms that induce this misregulation. This paper reports the effect of Cd(2+) on anterior pituitary cell viability and its relation to prolactin secretion. Cd(2+) concentrations above 10 microM were found to be cytotoxic for pituitary cells. Morphological studies as well as DNA ladder fragmentation and caspase activation showed that Cd(2+)-treated cells undergo apoptosis. Even though several hours were needed to detect Cd(2+)-induced cytotoxicity, the effect of the metal became irreversible very quickly, requiring only 3 h of treatment. Prolactin release (measured at 48 h) was inhibited when the cells were exposed to Cd(2+) for 1 h, before any change in cell viability was observed. The antioxidants N-acetyl-cysteine and Trolox (a hydrosoluble derivative of vitamin E), but not ascorbic acid, reversed both Cd(2+)-mediated cytotoxicity and the inhibition of prolactin release, supporting the involvement of oxidative stress in the mechanism of Cd(2+) action. In summary, the present work demonstrates that Cd(2+) is cytotoxic for anterior pituitary cells, that this effect is due to an induction of apoptosis, and that it can be reversed by antioxidants.

105. Altern Med Rev. 2003 Apr;8(2):106-28. Toxic metals and antioxidants: Part II. The role of antioxidants in arsenic and cadmium toxicity. Patrick L. 1984 graduate, Bastyr University; associate editor, Alternative Medicine Review; private practice, Tucson, Arizona, 1984-2002. Correspondence address: 21415 Hwy 140, Hesperus, CO 81326 Email: lpatrick@frontier.net

Exposure to toxic metals has become an increasingly recognized source of illness worldwide. Both cadmium and arsenic are ubiquitous in the environment, and exposure through food and water as well as occupational sources can contribute to a well-defined spectrum of disease. The symptom picture of arsenic toxicity is characterized by dermal lesions, anemia, and an increased risk for cardiovascular disease, diabetes, and liver damage. Cadmium has a significant effect on renal function, and as a result alters bone metabolism, leading to osteoporosis and osteomalacia. Cadmium-induced genotoxicity also increases risk for several cancers. The mechanisms of arsenic- and cadmium-induced damage include the production of free radicals that alter mitochondrial activity and genetic information. The metabolism and excretion of these heavy metals depend on the presence of antioxidants and thiols that aid arsenic methylation and both arsenic and cadmium metallothionein-binding. S-adenosylmethionine, lipoic acid, glutathione, selenium, zinc, N-acetylcysteine (NAC), methionine, cysteine, alpha-tocopherol, and ascorbic acid have specific roles in the mitigation of heavy metal toxicity. Several antioxidants including NAC, zinc, methionine, and cysteine, when used in conjunction with standard chelating agents, can improve the mobilization and excretion of arsenic and cadmium.

106. Environ Res. 2002 Sep;90(1):61-6. Reversal of lead-induced oxidative stress by chelating agent, antioxidant, or their combination in the rat. Tandon SK, Singh S, Prasad S, Srivastava S, Siddiqui MK. Chemical Toxicology, Industrial Toxicology Research Center, 80, Lucknow 226001, India. sktandon19@yahoo.com

The influence of N-acetyl cysteine (NAC), an antioxidant, on the therapeutic efficacy of meso-2,3-dimercaptosuccinic acid (DMSA), a hydrophilic, and its ester, monoisoamyl 2,3-dimercaptosuccinate (MiADMS), a lipophilic, both soft tissue lead mobilizers, was investigated in lead-preexposed rats. The subsequent treatment of lead-exposed animals with DMSA, MiADMS, or NAC reversed the lead-induced alterations in blood delta-aminolevulinic acid dehydratase, catalase, malondialdehyde (MDA), reduced glutathione, oxidized glutathione, and brain MDA levels. The combined treatment with DMSA and NAC was more effective than that with MiADMS and NAC in enhancing the restoration of all these parameters indicative of lead-induced oxidative stress. These reversals were consistent with the lead-removing ability of DMSA and MiADMS but not that of NAC. As the reversal of these parameters by NAC was independent of its lead-mobilizing capability, this ought to be mainly due to its strong antioxidant property. The increase in blood and brain zinc levels upon lead exposure appears to be the result of the redistribution of endogenous zinc due to lead. Subsequent treatment with DMSA, MiADMS, NAC, or their combination decreased the brain zinc as its excretable complexes with a transient increase in blood zinc level. The ideal treatment of lead poisoning seems to be a combination of a lead chelator and an antioxidant.

107. J Appl Toxicol. 2002 Jan-Feb;22(1):67-71. Chelation in metal intoxication: influence of cysteine or N-acetyl cysteine on the efficacy of 2,3-dimercaptopropane-1-sulphonate in the treatment of cadmium toxicity. Tandon SK, Prasad S, Singh S. Chemical Toxicology, Industrial Toxicology Research Centre, Lucknow, India. sktandon19@yahoo.com

The influence of cysteine or N-acetyl cysteine administration on the efficacy of 2,3-dimercaptopropane-1-sulphonate (DMPS) in the treatment of cadmium intoxication was investigated in cadmium-pre-exposed rats. Cysteine, N-acetyl cysteine, DMPS, DMPS + cysteine or DMPS + N-acetyl cysteine were about equal in effectiveness in mobilizing hepatic cadmium mainly from its supernatant cytosolic fraction (SCF) and both of the combinations were more effective than either of them alone in mobilizing cadmium from its nuclear mitochondrial fraction (NMF). The DMPS was apparently more effective than cysteine or N-acetyl cysteine in mobilizing renal cadmium from its SCF or NMF and it was more effective than even their combinations in mobilizing cadmium from renal SCF. The treatment with cysteine or N-acetyl cysteine reduced cadmium-induced hepatic and renal metallothionein (MT) and the treatment with DMPS reduced renal MT only, probably due to removal of hepatic and renal SCF cadmium by these agents. However, MT levels were high in animals treated with DMPS + cysteine or DMPS + N-acetyl cysteine, despite lowering of cadmium in these tissues, suggesting a contribution of MT induced by cysteine or N-acetyl cysteine itself. The cadmium exposure increased hepatic and renal zinc and renal copper levels, probably as a result of cadmium-induced MT, and some of the levels were normalized considerably by the subsequent treatment with cysteine, DMPS or to a lesser extent N-acetyl cysteine and their combinations, showing their protective effects against cadmium toxicity. The increase in blood cadmium and the decrease in blood zinc and copper levels due to cadmium exposure also were reversed appreciably by some of these treatments. The results have shown a limited benefit of cysteine or N-acetyl cysteine administration on the efficacy of DMPS in the treatment of cadmium intoxication. Copyright 2002 John Wiley & Sons, Ltd.