Effects of the sulphydryl donor N-acetyl-L-cysteine on nerve conduction, perfusion, maturation and regeneration following freeze damage in diabetic rats.
Eur J Clin Invest (ENGLAND) Aug 1996, 26 (8) p698-706
Peripheral nerve conduction velocity deficits in diabetic rats depend on decreased nerve perfusion, which may be related to increased free radical activity and impaired endogenous protection by the glutathione redox cycle. We studied the effect of treatment with the glutathione precursor N-acetyl-L-cysteine on nerve conduction, blood flow, maturation and regeneration. Two months of diabetes in mature rats caused 20% and 48% deficits in sciatic motor conduction velocity and endoneurial blood flow, respectively, which were largely corrected by N-acetyl-L-cysteine treatment during the second month. In young nondiabetic rats, sciatic motor conduction velocity increased by 31% over 6 weeks. Diabetes halved the conduction velocity maturation rate, however N-acetyl-L-cysteine treatment allowed a normal pattern of development. After 1 month of treated or untreated diabetes, the sciatic nerve was lesioned by a liquid nitrogen-cooled probe. Myelinated fibre regeneration distance, determined electrophysiologically, was reduced by 12.2% with diabetes; this was prevented by N-acetyl-L-cysteine treatment. Thus, the data stress the importance of free radical-mediated changes in the aetiology of experimental diabetic neuropathy.
Mitochondria alterations and dramatic tendency to undergo apoptosis in peripheral blood lymphocytes during acute HIV syndrome
AIDS (United Kingdom), 1997, 11/1 (19-26)
Objective: To study alterations of mitochondrial membrane potential (Deltapsi) and the propensity to undergo apoptosis in peripheral blood lymphocytes (PBL) from subjects with acute HIV syndrome; and to evaluate possible modulations of these phenomena by antioxidants that can be used in therapy, such as N-acetyl-cysteine (NAC), nicotinamide (NAM), or L-acetyl-carnitine (LAC). Methods: Mitochondrial function and the tendency of PBL to undergo spontaneous apoptosis were studied on freshly collected PBL from patients with symptomatic, acute HIV-1 primary infection, which were cultured for different durations in the presence or absence of NAC, NAM or LAC. By a cytofluorimetric method allowing analysis of Deltapsi in intact cells, we studied the function of these organelles under the different conditions. PBL apoptosis was evaluated by the classic cytofluorimetric method of propidium iodide staining, capable of revealing the typical DNA hypodiploid peak. Results: Significant Deltapsi alterations and tendency to undergo apoptosis were present in PBL from the subjects we studied. Indeed, when cultured even for a few hours in the absence of any stimulus, a consistent number of cells died. However, the presence of even different levels of NAC, NAM or LAC was able to rescue most of them from apoptosis. Both a fall in Deltapsi and apoptosis were evident in PBL collected in the earliest phases of the syndrome (before seroconversion), and changed significantly after a few days. A significant correlation was found between spontaneous apoptosis and tumour necrosis factor (TNF)-alpha or p24 plasma levels, as well as between apoptosis and the percentages of circulating CD4+ or CD8+ T cells. Conclusions: PBL from patients with acute HIV syndrome are characterized by both significant mitochondrial alterations and a dramatic tendency to undergo apoptosis. The use of NAC, NAM or LAC seems to rescue cells through a protective effect on mitochondria, a well-known target for the action of TNF-alpha and for reactive oxygen species, the production of which is strongly induced by this cytokine. Thus, our data could provide the rationale for the use of such agents in addition to antiviral drugs in primary infection.
Chemoprevention of colorectal tumors: role of lactulose and of other agents.
Ponz de Leon M; Roncucci L
Dept. of Internal Medicine, University of Modena, Italy.
Scand J Gastroenterol Suppl (NORWAY) 1997, 222 p72-5
Chemoprevention can be defined as an attempt at cancer control in which the occurrence of the disease is prevented by the administration of one (or more) chemical compounds. Main problems in chemoprevention studies are the choice of a suitable drug, the choice of an appropriate intermediate or definitive end point, and the definition of the population which should be investigated. Main classes of chemopreventive agents include vitamins, non-steroid antinflammatory drugs, minerals such as calcium or selenium, and other antioxidants such as N-acetylcysteine. Chemoprevention is particularly appealing in colorectal cancer, either because these lesions develop through a multistep process, or owing to the concept of "field carcinogenesis'. Between 1985 and 1990 we carried out a controlled study in which antioxidant vitamins or lactulose were used in an attempt to prevent the recurrence of colorectal polyps after their endoscopic removal. Among the 209 patients who could be evaluated, polyps recurred in 5.7% of the individuals who were given vitamins (A, C and E), 14.7% of patients given lactulose and 35.9% of untreated controls (chi 2 = 17.1, P < 0.001). The study suggested that either antioxidant vitamins or lactulose could be effective in reducing the recurrence rate of adenomatous polyps. In a subsequent on-going study, lower doses of the same vitamins were tested versus N-acetylcysteine (60a 40% reduction of the recurrence of polyps (ver sus controls) in individuals given N-acetylcysteine, while the effect of lower doses of vitamins was less appreciable. Definitive results of the study should be available by the end of 1998.
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 (UNITED STATES) Sep 17 1996, 67 (6) p842-8
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 striynergism 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.
N-Acetylcysteine enhances T cell functions and T cell growth in culture
INT. IMMUNOL. (United Kingdom), 1993, 5/1 (97-101
N-Acetylcysteine (NAC) is highly nontoxic for peripheral blood T cells and immunostimulatory enhancing T cell functions such as mitogenesis, interleukin-2 (IL-2) production, and growth in culture. NAC has been proposed for the treatment of AIDS based on its inhibition of human immunodeficiency virus (HIV) replication in cultured cells. Therefore its effect on normal T cells from 10 young donors and one elderly donor has been investigated as a prelude to clinical consideration. T cell function was evaluated in the presence and absence of accessory cells. With concanavalin A and anti-CD3 activation, NAC enhanced mitogenesis by similar2- to 2.5-fold at 5-10 mM. Mitogenesis of purified T cells with anti-CD2 was not affected by NAC; in the presence of accessory cells, NAC enhanced mitogenesis by similar2-fold at 1-10 mM. Importantly, NAC levels above 10 mM completely inhibited activation of peripheral blood mononuclear cells by anti-CD2. IL-2 secreted by T cells was also enhanced by NAC, similar1.5-fold, but IL-2 secreted by cells from old donors was enhanced by 3-fold. In cultures of peripheral blood T cells, NAC (10 mM) stimulated growth by at least 4- to 6-fold after two passages. These results show that NAC, nontoxic even at 20 mM, is an effective enhancer of T cell function and a remarkable enhancer of growth. Results from other laboratories show that NAC, which increases glutathione levels, suppresses HIV replication presumably via suppression of the activation of transcriptional factor NF-kappa B. For normal T cells, however, this mechanism does not appear applicable because IL-2 production, regulated by several factors including NF-kappa B, is enhanced by NAC. Rather, glutathione may enhance the activity of other transcriptional factors modulating IL-2 expression. NAC did exhibit one inhibitory characteristic, however, towards T cell adhesion. Slow cluster formation, induced by PMA, was moderately inhibited (0-30%) by 5-10 mM NAC in cells from most donors studied.
N-acetylcysteine (NAC) enhances interleukin-2 but suppresses interleukin-4 secretion from normal and HIV+ CD4+ T-cells.
Cell Mol Biol (Noisy-le-grand) (FRANCE) 1995, 41 Suppl 1 pS35-40
We find that purified CD4+ T cells from 30 HIV+ individuals have a suppressed Interleukin-4 (IL-4) production compared to normal controls regardless of activator (anti-CD3 or Con A) or co-activator [phorbol ester (PMA or anti-CD28)], generally by 2-4 fold. In every case, the cells producing IL-4 respond more strongly to anti-CD28 co-activation than to PMA, ie, 1150 pg/ml compared to 2070 pg/ml for controls and 398 pg/ml compared to 1250 pg/ml for HIV+ cells, respectively. In contrast, anti-CD3 with PMA gives a more vigorous IL-2 response than with anti-CD28, ie, 37.3 ng/ml compared to 12.3 ng/ml for controls and 28.5 ng/ml versus 15.1 ng/ml for HIV+ cells, respectively. These data are not compatible with the TH1/TH2 switch hypothesis since IL-4 production is decreased, not increased for CD4+ HIV+ T-cells and while IL-2 production is decreased with PMA, it is not decreased significantly with anti-CD28. Interestingly, 5 mM N-acetylcysteine (NAC) acts as an immunoenhancer; mitogenesis was enhanced 2 fold or more in general for control and HIV+ CD4+ T-cells and IL-2 production was enhanced 2-3 fold for anti-CD3 (with PMA or anti-CD28) for both controls and HIV+ CD4+ cells. However, NAC suppressed IL-4 production induced by anti-CD3 and anti-CD28 in both control and HIV+ CD4+ T cells. In the other cases, it produced in general no significant change.
N-acetylcysteine enhances antibody-dependent cellular cytotoxicity in neutrophils and mononuclear cells from healthy adults and human immunodeficiency virus-infected patients.
J Infect Dis (UNITED STATES) Dec 1995, 172 (6) p1492-502
Patients with AIDS have decreased levels of the intracellular antioxidant, glutathione, in their circulating lymphocytes and plasma. N-acetylcysteine (NAC) increases intracellular stores of glutathione and has direct antioxidant properties. In this study, the effects of glutathione and NAC on the cytotoxicity of neutrophils and mononuclear cells were tested using cells from healthy controls and human immunodeficiency virus (HIV)-infected patients. NAC (1 and 5 mM) enhanced the antibody-dependent cellular cytotoxicity (ADCC) of neutrophils from healthy adult controls and HIV-infected adults and children. The antineoplastic drug, 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU), which depletes intracellular glutathione, inhibited the ADCC of neutrophils; the addition of NAC partially reversed this inhibition. Similar effects of BCNU and NAC were seen when the cytotoxicity of mononuclear cells was tested using CEM tumor cells bearing the HIV gp120 antigen as targets. Thus, NAC enhances various forms of cytotoxicity and may be beneficial to AIDS patients whose defects in leukocyte cytotoxicity may be due to glutathione depletion.
Glutathione precursor and antioxidant activities of N-acetylcysteine and oxothiazolidine carboxylate compared in in vitro studies of HIV replication.
AIDS Res Hum Retroviruses (UNITED STATES) Aug 1994, 10 (8) p961-7
N-Acetyl-L-cysteine (NAC) and L-2-oxothiazolidine 4-carboxylate (OTC) are pro-GSH drugs that been proposed for AIDS therapy. In this article we compare the antiviral activities of these compounds in various in vitro HIV infection models. Although both compounds blocked cytokine induction of HIV in acute and chronic infection models, and in HIV-LTR reporter cell systems, NAC was far more effective than OTC, even at suboptimal doses. To test whether this difference is due to GSH conversion efficacies of these compounds, we measured GSH restoration by NAC or OTC in GSH-depleted peripheral blood mononuclear cells (PBMCs), using flow cytometry. In isolated PBMCs, NAC fully replenishes depleted intracellular GSH whereas OTC only minimally replenishes GSH. This ability to replenish GSH in vitro and its ability to scavenge free radicals directly explain why NAC has more potent antiviral activities in vitro.
Role for oxygen radicals in self-sustained HIV-1 replication in monocyte-derived macrophages: enhanced HIV-1 replication by N-acetyl-L-cysteine.
J Leukoc Biol (UNITED STATES) Dec 1994, 56 (6) p702-7
N-acetyl-L-cysteine (NAC) has been proposed as a therapeutic agent for AIDS patients because it reduces human immunodeficiency virus type 1 (HIV-1) replication in stimulated T cells. However, NAC and glutathione enhanced acute HIV-1 replication in monocyte-derived macrophages. Buthionine sulfoximine did not affect NAC-mediated enhanced HIV-1 replication, indicating that the NAC-mediated effects are glutathione-independent. Superoxide dismutase and the hydroxyl radical scavengers dimethylthiourea and thiourea, but not urea, inhibited acute HIV-1 replication in macrophages. NAC reduced ferricytochrome c and increased dose-dependently Fe(III)-citrate and Fe(III)-EDTA-catalyzed hydroxyl radical formation in a system using glucose and glucose oxidase. Dimethylthiourea and thiourea, but not urea and superoxide dismutase, dose-dependently inhibited NAC-mediated enhancement of HIV-1 replication. These data suggest that oxygen radicals play an important role in self-sustained HIV-1 replication in macrophages and that oxygen radical scavengers other than NAC should be considered as therapeutic agents for AIDS patients.
Effects of glutathione precursors on human immunodeficiency virus replication.
Chem Biol Interact (IRELAND) Jun 1994, 91 (2-3) p217-24
Asymptomatic human immunodeficiency virus (HIV)-seropositive individuals have reduced glutathione (GSH) levels. This has led to the suggestion that elevated intracellular thiols levels may inhibit HIV replication and progression of the disease. We confirmed that N-acetyl-L-cysteine (NAC), a cysteine prodrug which maintains intracellular GSH levels during oxidative stress, inhibits in the chronically infected U1 cells, the stimulation of HIV replication induced by phorbol 12-myristate 13-acetate (PMA), interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF). However, we found no significant inhibition of PMA-mediated long terminal repeat (LTR)-directed beta-galactosidase expression in transiently transfected Jurkat T-cells. We have compared NAC effects with the effects of other GSH precursors on HIV expression. Treatment of the U1 cell line by L-2-oxo-4-thiazolidine carboxylic acid (OTC), which is converted to cysteine by 5-oxoprolinase, or by homocysteine (HC), a natural cysteine precursor, reduced the PMA-induced HIV expression, but surprisingly, markedly stimulated the expression mediated by IL-6 and GM-CSF. Several experiments to investigate the effect of OTC on LTR transactivation were carried out, but beta-galactosidase activity was never modified in a significant fashion in PMA-induced Jurkat T-cells after OTC treatment. Furthermore, HC stimulated the PMA-mediated HIV-LTR transactivation in Jurkat T-cells. GSH assays showed that treatment of U937 and Jurkat T-cells with NAC and OTC moderately increased the GSH level, while HC led to a significantly higher increase of the thiol level. In conclusion, it appeared that an increase of the GSH intracellular level did not lead solely to an inhibition of HIV replication but could also lead to an activation of viral expression. This seemed the case when HIV replication was stimulated by compounds which act mainly at a post-transcriptional level.
Effect of glutathione depletion and oral N-acetyl-cysteine treatment on CD4+ and CD8+ cells.
FASEB J (UNITED STATES) Apr 1 1994, 8 (6) p448-51
HIV-infected individuals and SIV-infected rhesus macaques have, on the average, decreased plasma cysteine and cystine concentrations and decreased intracellular glutathione levels. We show that the cysteine supply and the intracellular glutathione levels have a strong influence on the T cell system. A study of healthy human subjects revealed that persons with intracellular glutathione levels of 20-30 nmol/mg protein had significantly higher numbers of CD4+ T cells than persons with either lower or higher glutathione levels. Persons who moved during a 4-week observation period from the optimal to the suboptimal range (10-20 nmol/mg) experienced, on the average, a 30% decrease in CD4+ T cell numbers. This decrease was prevented by treatment with N-acetyl-cysteine (NAC). NAC caused this relative increase of CD4+ T cell numbers in spite of decreasing glutathione levels and not by increasing the glutathione level. Our studies suggest that the immune system may be exquisitely sensitive not only against a cysteine and glutathione deficiency but also against an excess of cysteine.
Comparative study of the anti-HIV activities of ascorbate and thiol-containing reducing agents in chronically HIV-infected cells.
Am J Clin Nutr (UNITED STATES) Dec 1991, 54 (6 Suppl) p1231S-1235S
To elucidate the action of vitamin C on pathogenic human retroviruses, we investigated and compared the effects of noncytoxic concentrations of ascorbic acid (AA), its calcium salt (Ca-ascorbate), and two thiol-based reducing agents [glutathione (GSH) and N-acetyl-L-cysteine (NAC)] against human immunodeficiency virus (HIV)-1 replication in chronically infected T lymphocytes. Ca-ascorbate reduced extracellular HIV reverse transcriptase (RT) activity by about the same magnitude as the equivalent dose of AA. Long-term experiments showed that continuous presence of ascorbate was necessary for HIV suppression. NAC (10 mmol/L) caused less than twofold inhibition of HIV RT and conferred a synergistic effect (approximately eightfold inhibition) when tested simultaneously with AA (0.426 mmol/L). In contrast, nonesterified GSH (less than or equal to 1.838 mmol/L) had no effect on RT concentrations and did not potentiate the anti-HIV effect of AA. These results further support the potent antiviral activity of ascorbate and suggest its therapeutic value in controlling HIV infection in combination with thiols.
Antioxidant status and lipid peroxidation in patients infected with HIV
CHEM.-BIOL. INTERACT. (Ireland), 1994, 91/2-3 (165-180)
Deficiency in antioxidant micronutrients have been observed in patients with AIDS. These observations concerning only some isolated nutrients demonstrate a defect in zinc, selenium, and glutathione. An increase in free radical production and lipid peroxidation has been also found in these patients, and takes a great importance with recent papers presenting an immunodeficiency and more important an increase in HIV-1 replication secondary to free radicals overproduction. We have assessed different studies, trying to obtain a global view of the antioxidant status of these patients. In adults we observe a progressive decrease for zinc, selenium, and vitamin E with the severity of disease, except that selenium remains normal at stage II. However, the main dramatic decrease concerns carotenoids whose level at stage II is only half the normal value. To understand if these decreases in antioxidant and increases in oxidative stress occur secondary to the aggravation of the disease or, conversely, are responsible for it, we undertook a longitudinal survey of asymptotic patients. The preliminary results of this evaluation are presented. Paradoxically, lipid peroxidation is higher at stage II than at stage IV. This may be consecutive to a more intense overproduction of oxygen free radicals by more viable polymorphonuclear (PMN) at the asymptomatic stage. The free radicals production and lipid peroxidation seem secondary to a direct induction by the virus of PMN stimulation and cytokines secretion. N-Acetyl cystein or ascorbate have been demonstrated in cell culture to be capable of blocking the expression of HIV-1 after oxidative stress and N-acetyl cysteine inhibits in vitro TNF-induced apoptosis of infected cells. In regard to all these experimental data, few serious and large trials of antioxidants have been conducted in HIV-infected patients, although some preliminary studies using zinc or selenium have been performed. In our opinion it is now time to evaluate in humans the beneficial effect of antioxidants. The more promising candidates for presenting synergistic effects when associated with N-acetyl cysteine seem to be beta-carotene, selenium and zinc.
N-acetylcysteine inhibits latent HIV expression in chronically infected cells
AIDS RES. HUM. RETROVIRUSES (USA), 1991, 7/6 (563-567)
The progression of the human immunodeficiency virus (HIV) infection from its early latent (asymptomatic) stage to active, late-stage acquired immunodeficiency syndrome (AIDS) apparently begins with the production of inflammatory cytokines that stimulate the expression and replication of the latent virus. We have shown that N-acetylcysteine, a cysteine precursor that is converted intracellularly into glutathione, blocks cytokine-stimulated HIV replication in an acutely infected T-cell line and in acutely infected peripheral blood mononuclear cells from normal individuals. In this report, we show that N-acetylcysteine also inhibits stimulated HIV expression in chronically infected monocyte and T-cell lines which are used as models for latent infection in AIDS. Furthermore, we show that N-acetylcysteine blocks viral production in monocyte cell lines more effectively than it blocks viral production in T cells. Since monocytes are a major reservoir for HIV in infected individuals, these results suggest that N-acetylcysteine may slow the change from latency to the later stages of AIDS in HIV-infected individuals.
The intrabronchial microbial flora in chronic bronchitis patients: a target for N-acetylcysteine therapy?
Eur Respir J (DENMARK) Jan 1994, 7 (1) p94-101
Chronic bronchitis is common among smokers, often together with recurrent infectious exacerbations. Streptococcus pneumoniae and Haemophilus influenzae are the pathogens traditionally considered most important. N-acetylcysteine (NAC) treatment has been shown to reduce the number of infectious exacerbations in patients with chronic bronchitis. The mechanism behind this is unknown. We attempted to characterize the intrabronchial bacterial flora in patients with chronic bronchitis in an infection-free interval, and to determine whether pharmacological and immunological factors effected the bacterial occurrence. Twenty two smokers with non-obstructive chronic bronchitis, 19 smokers with chronic bronchitis and chronic obstructive pulmonary disease (COPD) and 14 healthy nonsmokers underwent bronchoscopy. To obtain uncontaminated intrabronchial samples, a protected specimen brush was used. Quantitative bacterial cultures and virus isolations were performed. Significantly positive bacterial cultures (> 1,000 colony-forming units (cfu).ml-1) were found only in the patients. S. pneumoniae and H. influenzae were found in five patients, and only in the patients without NAC treatment. The most common bacterium was alpha-haemolytic streptococcus. Negative cultures were more common in the healthy controls. Of the various factors examined, only NAC medication had an influence on bacterial numbers. Significantly fewer patients with NAC medication had positive cultures (3 out of 16) than in the group of patients without NAC therapy (15 out of 21). Our results confirm that chronic bronchitis in smokers leads to increased intrabronchial bacterial colonization. We could also confirm that 1,000 cfu.ml-1 is an adequate cut-off level for significant bacterial growth when using the protected specimen brush. NAC medication was associated with low bacterial numbers.
[The influence of n-acetylcysteine on chemiluminescence of granulocytes in peripheral blood of patients with chronic bronchitis]
Pneumonol Alergol Pol (POLAND) 1993, 61 (11-12)
The effect of NAC on exacerbation of chronic obstructive pulmonary disease (COPD) may be due to its mucolytic properties due to the thiol group of NAC and to its reducing and antioxidant properties. It has been postulated that NAC may protect lung cells from inhaled oxidants or oxidants produced by inflammatory leukocytes by increasing intra and extra cellular GSH. The FMLP induced granulocyte chemiluminescence (CL) in 6 healthy and 12 patients with COPD was determined. Peripheral blood polymorphonuclear leukocytes were incubated with NAC. The results obtained show a significant decrease of CL after incubation with NAC in both groups. We also found higher CL in healthy subjects than patients with COPD. This study showed a significant increase of FVC, FEV1 and a significant decrease of granulocyte CL after treatment with oral NAC 200 mg three times daily.
Protection by N-acetylcysteine of the histopathological and cytogenetical damage produced by exposure of rats to cigarette smoke.
Cancer Lett (NETHERLANDS) Jun 15 1992, 64 (2) p123-31
Adult male Sprague-Dawley rats were exposed whole-body to mainstream cigarette smoke (CS) once daily for 40 consecutive days. Such a treatment resulted in a significant decrease of body weight growth and in intense histopathological changes of terminal airways, including a severe inflammation of bronchial and bronchiolar mucosae, with multiple hyperplastic and metaplastic lesions and foci of micropapillomatous growth as well as emphysema, with extensive disruption of alveolar walls. All histopathological changes were efficiently prevented by the daily administration of the thiol N-acetyl-L-cysteine (NAC) by gavage. Cytological and cytogenetical changes were monitored in bronchoalveolar lavage (BAL) fluid and bone marrow cells of groups of rats killed after 1, 3, 8, 28, or 40 days of treatment. From the first day of exposure, CS significantly enhanced the proportion of polymorphonucleates among BAL cells and the frequency of micronucleated (MN) bone marrow polychromatic erythrocytes. After 8 days, a reduction was observed in the polychromatic/normochromatic erythrocytes ratio and an increase in the frequency of MN pulmonary alveolar macrophages (PAM) was also recorded, followed, after 28 days, by an increase of binucleated PAM. All these alterations immediately reached a plateau and persisted unchanged until the end of the experiment. NAC administration exhibited a significant and considerable protective effect towards the CS-induced alterations of BAL cellularity, the increase of MN PAM and bone marrow cytotoxicity.
Contraction and relaxation of aortas from diabetic rats: effects of chronic anti-oxidant and aminoguanidine treatments.
Naunyn Schmiedebergs Arch Pharmacol (GERMANY) Apr 1996, 353 (5) p584-91
We examined whether chronic treatment with the free radical scavengers butylated hydroxytoluene (1 g kg-1 day-1) and N-acetyl-L-cysteine (250 mg kg-1 day-1), or the inhibitor of advanced glycosylation reactions, aminoguanidine (1 g kg-1 day-1), could prevent the development of relaxation and contraction abnormalities in aorta from 2 month streptozotocin-diabetic rats. Diabetes caused a 24% deficit in maximal endothelium-dependent relaxation to acetylcholine for phenylephrine precontracted aortas (P < 0.01). This was unaffected by tissue-bath glucose concentration (5.5 or 40 mM), or by addition of 1 mM L-arginine. Butylated hydroxytoluene, N-acetyl-L-cysteine and aminoguanidine treatments gave substantial protection, maximum relaxation remaining in the non-diabetic range. Neither diabetes nor treatment affected endothelium-independent relaxation to glyceryl trinitrate. To test the suggestion that aminoguanidine could act as an inhibitor of constitutive nitric oxide synthase, acute aminoguanidine effects on endothelium-dependent relaxation to acetylcholine were also examined. No inhibition was noted. A modest increase in phenylephrine sensitivity with diabetes (P < 0.05) was unaffected by butylated hydroxytoluene or N-acetyl-L-cysteine, but partially prevented by aminoguanidine (P < 0.05). The data, therefore, provide evidence for the involvement of reactive oxygen species and the advanced glycosylation process particularly for impaired endothelium-dependent relaxation in experimental diabetes.
Inhibition with N-acetylcysteine of enhanced production of tumor necrosis factor in streptozotocin-induced diabetic rats.
Clin Immunol Immunopathol (UNITED STATES) Jun 1994, 71 (3) p333-7
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.
Acetylcysteine: a drug with an interesting past and a fascinating future.
Respiration (SWITZERLAND) 1986, 50 Suppl 1 p26-30
N-acetylcysteine (NAC) possesses a free sulfhydryl group that can rupture disulfide bridges. Although it is considered to be a mucolytic, its mucokinetic actions include expectorant, bronchorrheic and mucoregulatory contributions. New uses include the management of acetaminophen poisoning and the scavenging of free radicals liberated by cancer chemotherapy drugs. The antioxidant effects may be of prophylactic value in lungs at risk from smoking, pollution and infection. Other uses proposed for NAC include the therapy of connective tissue diseases and its use as a component in life extension diets.
Hyperthermia, radiation carcinogenesis and the protective potential of vitamin A and N-acetylcysteine
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.
Reduction of lower motor neuron degeneration in wobbler mice by N-acetyl-L-cysteine
Journal of Neuroscience (USA), 1996, 16/23 (7574-7582)
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.
Survival in patients with amyotrophic lateral sclerosis, treated with an array of antioxidants.
J Neurol Sci (NETHERLANDS) Aug 1996, 139 Suppl p99-103
Between 1983 and 1988 we treated 36 patients with sporadic amyotrophic lateral sclerosis (ALS) by an array of antioxidants and added other drugs to the regimen whenever a patient reported deterioration. Our customary prescription sequence was N-acetylcysteine (NAC); vitamins C and E; N-acetylmethionine (NAM); and dithiothreitol (DTT) or its isomer dithioerythritol (DTE). Patients with a history of heavy exposure to metal were also given meso 2,3-dimercaptosuccinic acid (DMSA). NAC, NAM, DTT, and DTE were administered by subcutaneous injection or by mouth or by both routes, the other vitamins and DMSA by mouth alone. The hospital pharmacy supplied NAC and NAM injections fluid as 100 ml bottles of 5.0 and 5.85% solutions, respectively. DTT was delivered in special double-walled capsules of 200 mg. DTT/DTE injection fluid was added to the NAC and NAM bottles, the final DTT/DTE concentrations never exceeding 0.5%. DMSA was provided in 250 mg capsules. All of the 36 patients used NAC and DTT/DTE; 29 also used vitamins C and E; 21 also used NAM; and 7 also used DMSA, DMSA, NAM, vitamins C and E were tolerated well. In many patients, DTT, DTE, NAC and NAM induced pain, redness and swelling at the injection sites in that order of decreasing frequency. DTT and DTE did often and NAC did sometimes cause gastric pain, nausea and other abdominal discomfort. Comparison of survival in the treated group and in a cohort of untreated historical controls, disclosed a median survival of 3.4 years (95% confidence interval: 3.0-4.2) in the treated and of 2.8 (95% confidence interval 2.2-3.1) years in the control patients. This difference may be explained by self-selection of our highly motivated treated group and by its initial survival of diagnosis for an average of 8.5 months before onset of treatment. We conclude that antioxidants neither seem to harm ALS patients, nor do they seem to prolong survival.
"N-Acetylcysteine for Lung Cancer Prevention"
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.
Pearls, pitfalls, and updates in toxicology
Emergency Medicine Clinics of North America (USA), 1997, 15/2 (427-450)
Pearls, pitfalls, and updates in toxicology provide pratical information for the clinical practice of emergency medicine. Clinical pearls in toxicology include using diagnostic tests to detect end-organ toxicity, applying physiologic principles to the management of hemodynamically unstable poisoned patients, and dealing with psychologic injuries from hazardous materials incidents. Recognizing serious complications from poisoning and adverse drug effects, including the serotonin syndrome, are offered as pitfalls. New therapies for clinical toxicology and pharmaceuticals with new toxicologic challenges are rapidly developing. Therefore, updates on the evolving role of N-acetylcysteine as an antidote for acetaminophen poisoning, new psychotropic medications, and new antidotes are included.
Refining the level for anticipated hepatotoxicity in acetaminophen poisoning
Journal of Emergency Medicine (USA), 1996, 14/6 (691-695)
Treatment of an acetaminophen overdose with N-acetyl cysteine usually is based on the position of the 4-h acetaminophen (APAP) level on the Rumack- Matthew homogram; however, there is disagreement on the level at which clinically relevant hepatotoxicity occurs. A retrospective review of all acute adult formulation APAP exposures reported to our poison center between 1986 and 1993 was performed and cases corresponding to the 'possible risk or toxicity' range on the nomogram were identified. Our current poison center protocol for APAP poisoning does not recommend treatment with N- acetylcysteine (NAC) in low-risk patients if the 4-h serum APAP level or the extrapolated equivalent falls within the possible toxicity range on the nomogram. Seventeen cases met the inclusion criteria for the study and received no NAC; six additional patients met inclusion criteria but received one or two doses of NAC before therapy was discontinued. No patients in either group demonstrated clinical evidence of hepatotoxicity. This pilot study suggests that patients with no risk factors and APAP levels in the 'possible risk' range may not require NAC therapy.
Outpatient N-acetylcysteine treatment for acetaminophen poisoning: An ethical dilemma or a new financial mandate?
Veterinary and Human Toxicology (USA), 1996, 38/3 (222-224)
The mainstay of treatment for acetaminophen-induced hepatotoxicity, produced by the accumulation of the toxic metabolite N- acetylbenzoquinoneimine, is an enteral 18-dose course of N-acetylcysteine (NAC). However, absence of characteristic symptomatology is a frequent reason for premature cessation of NAC and early discharge of the toxic acetaminophen poisoned patient. We report a series of confirmed acetaminophen poisonings who were discharged early with NAC and instructions to self-administer. All cases of acute acetaminophen poisoning without concomitant drugs, reported to a certified Regional Poison Information Center for a 3-mo period of time, were reviewed. Inclusion criteria included patients who were discharged with orders to complete the course of NAC outside of a hospital, despite toxic serum acetaminophen concentrations. Data parameters evaluated included age, amount taken, symptoms, laboratory results, treatment, and medical outcome. 131 cases of confirmed toxic acetaminophen poisoning yielded 6 patients who received 4 to 6 doses of NAC during hospitalization, but were discharged to home with the remaining 11-13 doses. Patients' ages ranged from 16-28 y (mean 20.0 y). Serum acetaminophen concentrations measured at 4 h post-ingestion ranged from 171-198 mcg/ml (mean 182 mcg/ml). Follow-up by the certified Regional Poison Information Center at 1-3 w post-discharge determined dosing compliance to be 83%. All 6 patients remained asymptomatic with normal liver function testing. Since health care reform encourages practitioners to reconsider established approaches to the delivery of health care, perhaps home delivery of NAC would not only be clinically preferred to premature cessation of the antidote, but also offer cost savings. Self-administration of NAC in the home setting may be representative of a new era in America's health care delivery system.
Management of acetaminophen toxicity
American Family Physician (USA), 1996, 53/1 (185-190)
Acetaminophen poisoning is a significant medical problem in the United States and is frequently managed by family physicians. The primary clinical effect of acetaminophen poisoning is hepatotoxicity that occurs after ingestion of large single doses of acetaminophen or after ingestion of smaller doses in patients with hepatic metabolism that is altered by drugs or concurrent medical conditions. Hepatocellular damage is probably caused by accumulation of the toxic intermediate metabolite N-acetyl-p- benzoquinoneimine when hepatic glutathione stores are depleted. Treatment of acetaminophen poisoning consists of preventing gastrointestinal absorption of the drug, use of the antidote N-acetylcysteine and supportive care.
[Recommendations for treatment of paracetamol poisoning. Danish Medical Society, Study of the Liver]
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.
Factors responsible for continuing morbidity after paracetamol poisoning in Chinese patients in 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.
[Clinical-toxicological case (1). Dosage of N-acetylcysteine in acute paracetamol poisoning]
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.
Protective effect of oral acetylcysteine against the hepatorenal toxicity of carbon tetrachloride potentiated by ethyl alcohol.
Alcohol Clin Exp Res (UNITED STATES) Aug 1992
Considering the well-documented protection of acetylcysteine (AC) in hepatotoxicity related to acetaminophen, we studied the preventive potential of AC against mild hepatotoxicity of CCl4, potentiated with ethyl alcohol (ETH) and the role of tissue glutathione. Rats fed a liquid diet with 30% of energy from ETH, had-intraperitoneal CCl4 administered in three injections, at 7-day intervals. AC was ingested at the level for acetaminophen overdose. ETH markedly potentiated the injury induced by CCl4, as evidenced by higher values of serum alanine aminotransferase (ALT), urinary bile acids (BA), serum creatinine, histological score of liver cell necrosis, mortality and by lower body weights and lower liver glutathione, when compared with CCl4 alone. Protective effect of AC consisted of a lesser hepatocytic necrosis, better body weights and higher liver glutathione. We conclude, that AC favorably modifies liver damage induced by CCl4 and potentiated with ETH. There is a preventive role for AC in subjects who combine ETH overuse with exposure to hepatotoxic xenobiotics, whose toxicity is modified by tissue glutathione.
A comparison of the protective effects of N-acetyl-cysteine and S-carboxymethylcysteine against paracetamol (acetaminophen)-induced hepatotoxicity.
Toxicology (NETHERLANDS) Nov 1983
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.
Overdose of Extended-Release Acetaminophen
New England Journal of Medicine, July 20, 1995;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. The authors were concerned that the high levels of acetaminophen in their patient represented a massive overdose and elected to give higher than usual doses of acetylcysteine.
Nacystelyn, a novel lysine salt of N-acetylcysteine, to augment cellular antioxidant defence in vitro.
Respir Med (ENGLAND) Mar 1997, 91 (3) p159-68
Nacystelyn (NAL), a recently-developed lysine salt of N-acetylcysteine (NAC), and NAG, both known to have excellent mucolytic capabilities, were tested for their ability to enhance cellular antioxidant defence mechanisms. To accomplish this, both drugs were tested in vitro for their capacity: (1) to inhibit O2- and H2O2 in cell-free assay systems; (2) to reduce O2- and H2O2 released by polymorphonuclear leukocytes (PMN); and (3) for their cellular glutathione (GSH) precursor effect. In comparison with GSH, NAL and NAC inhibited H2O2, but not O2-, in cell-free, in vitro test systems in a similar manner. The anti-H2O2 effect of these drugs was as potent as that of GSH, an important antioxidant in mammalian cells. To enhance cellular GSH levels, increasing concentrations (0-2 x 10(-4) mol l-1) of both substances were added to a transformed alveolar cell line (A549 cells). After NAC administration (2 x 10(-4) mol l-1), total intracellular GSH (GSH + 2GSSG) levels reached 4.5 +/- 1.1 x 10(-6) mol per 10(6) cells, whereas NAL increased GSH to 8.3 +/- 1.6 x 10(-6) mol per 10(6) cells. NAC and NAL administration also induced extracellular GSH secretion; about two-fold (NAC), and 1.5-fold (NAL), respectively. The GSH precursor potency of cystine was about two-fold higher than that of NAL and NAC, indicating that the deacetylation process of NAL and NAC slows the ability of both drugs to induce cellular glut production and secretion. Buthionine-sulphoximine, which is an inhibitor of GSH synthetase, blocked the cellular GSH precursor effect of all substances. In addition, these data demonstrate that NAC and NAL reduce H2O2 released by freshly-isolated cultured blood PMN from smokers with chronic obstructive pulmonary disease (COPD) (n = 10) in a similar manner (about 45% reduction of H2O2 activity by NAC or NAL at 4 x 10(-6) mol l-1). In accordance with the results obtained from cell-free, in vitro assays, O2- released by PMN was not affected. Ambroxol (concentrations: 10(-9)-10(-3) mol l-1) did not reduce activity levels of H2O2 and O2- in vitro. Due to the basic effect of dissolved lysine, which separates easily in solution from NAL, the acidic function of the remaining NAC molecule is almost completely neutralized [at concentration 2 x 10(-4) M: pH 3.6 (NAC), pH 6.4 (NAL)]. Due to their function as H2O2 scavengers, and due to their ability to enhance cellular glutathione levels, NAL and NAC both have potent antioxidant capabilities in vitro. The advantage of NAL over NAC is two-fold; it enhances intracellular GSH levels twice as effectively, and it forms neutral pH solutions whereas NAC is acidic. Concluding from these in vitro results, NAL could be an interesting alternative to enhance the antioxidant capacity at the epithelial surface of the lung by aerosol administration.
Use of a microsome-mediated test system to assess efficacy and mechanisms of cancer chemopreventive agents
Carcinogenesis (United Kingdom), 1996, 17/6 (1285-1290)
There is a growing need for short-term assays which can assess the mechanisms and efficacy of cancer chemopreventive agents. In the present study we have employed a microsome-mediated test system concomitantly with DNA adduct detection to assess the efficacy of five chemopreventive agents, N-acetylcysteine, butylated hydroxytoluene (BHT), curcumin, oltipraz, and ellagic acid. 32P-Postlabeling analysis of DNA incubated with benzo(a)pyrene (BP) in the presence of Aroclor 1254-induced microsomes produced two major adducts: one derived from the interaction of benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) with deoxyguanosine (dG) and the other from further activation of 9-OH-BP (309 and 34 adducts/107 nucleotides, respectively). With the exception of N-acetylcysteine, all test agents significantly altered BP-DNA adduct levels: Intervention with ellagic acid and oltipraz substantially (64-94%) inhibited both BPDE-dG and 9-OH-BP adducts, while intervention with curcumin and BHT inhibited the BPDE-dG adduct (57% and 38%, respectively) and enhanced the 9-OH-BP adduct (230% and 650%, respectively). Furthermore, ellagic acid was the only test agent observed to inhibit the anti BPDE-dG adduct in the absence of microsomal enzymes, which is consistent with the known conjugation of ellagic acid with BPDE. These results suggest that oltipraz may be acting as an inhibitor of P4501A1, the isozyme involved in activation of BP to BPDE, or by conjugation of the electrophilic species by a metabolite of oltipraz. A plausible mechanism for inhibition of the BPDE-dG adduct and enhancement of the 9-OH-BP adduct by curcumin and BHT includes inhibition of epoxide hydrolase. Our results also indicate that N-acetylcysteine does not act as an electrophilic trapping agent of BP metabolites but may exert its protective effect in vivo by various other means, including modulation of detoxification enzymes and altering DNA repair processes. These data suggest that this cell-free system in conjunction with the sensitive 32P-postlabeling DNA adduct analysis may prove a viable test system for assessing the mechanisms and efficacy of chemopreventive agents.