Additional Aspects of the Detoxification Process
Several other mechanisms work in concert with the phase I, II, and III enzyme systems to improve their efficiency or extend their functionality. While not officially characterized as part of xenobiotic metabolism, they are nonetheless important for reducing or mitigating toxin exposure.
Bile secretion is a critical digestive process for the absorption of dietary fats and fat-soluble nutrients, but also functions as the major mechanism for moving conjugated toxins out of the liver and into the intestines, where they can be eliminated.
Antioxidation is a necessary protective measure against the harsh phase I oxidation reactions, which frequently produce free-radical byproducts. The production of antioxidant enzymes, many of which are under the same genetic regulation (by Nrf2) as the phase II enzymes, is important for minimizing this free-radical damage.
Heavy metal toxicity can lead to oxidative damage by direct generation of free radical species and depletion of antioxidant reserves.59 Mercury, arsenic, and lead, for example, effectively inactivate the glutathione molecule so it is unavailable as an antioxidant or as a substrate for xenobiotic detoxification60; lead can also reduce the activity of the enzymes of that recycle glutathione.61 One method for heavy metal removal is their chelation by the cellular proteins metallothioneins (MTs), which have a high capacity to bind various reactive metal ions, such as zinc, cadmium, mercury, copper, lead, nickel, cobalt, iron, gold, and silver.62 One molecule of MT can bind 7–9 zinc or cadmium ions (or any combination of these two), up to 12 copper ions, and up to 18 mercury ions.63 Cellular stress (particularly oxidative stress), turns up MT production, which, like the phase II enzymes, is stimulated by the activity of Nrf2.64
Prevention of absorption through trapping of potential toxins (such as surface adhesion to another molecule in the gut, like activated charcoal or kaolin clay65) is an effective means of mitigating exposure; this mechanism has the requirement of some dietary adsorbent to be taken while the toxin is in transit in the GI tract. Uptake of potential toxins and their detoxification by beneficial colonic microflora could have a similar effect.
Dietary Modification of Metabolic Detoxification
Given the sheer number of diverse enzymes and transport proteins involved in metabolic detoxification and its related pathways, it is no surprise that detoxification depends on, and is sensitive to, a large number of dietary factors.
Macronutrient and micronutrient intake influences phase I and II systems. Protein deficiency decreases CYP metabolism, while high protein diets increase it.66 The opposite effects are observed for carbohydrates; the effects of lipids on CYP metabolism are unclear. Efficient phase I reactions require sufficiency in several micronutrients; deficiencies in vitamins A, B2 and B3, folate, C, E, iron, calcium, copper, zinc, magnesium, selenium have all been shown to decrease the activities of one or more phase I enzymes, or slow the transformation of specific drugs.67
The diverse set of phase II enzymes require an equally diverse set of essential nutrients, especially B vitamins, as cofactors.
The reduced glutathione for GST conjugation depends on adequate dietary sulfur-containing amino acids (methionine or cysteine), vitamin B6 for the conversion of methionine to cysteine, as well vitamins B2 and B3 for the activity of glutathione reductase, which recycles oxidized glutathione.
The methylation reactions use SAMe as a substrate; which, in turn, is synthesized through folate- and vitamin B12-dependent enzymatic reactions.
The conjugation reactions of the NAT’s and amino acid acyltransferases use the cofactor acetyl-coenzyme A (acetyl-CoA), which is synthesized from vitamin B5, using enzymes that themselves depend on multiple B vitamins.
Several phase II reactions require the energy molecule ATP in some fashion. For example, the chemical cofactors for the phase II methylation, sulfonation, glucuronidation, and glutathione conjugation reactions are all made using ATP; these ATP mediated reactions are magnesium-dependent.
Flavonoids have been extensively studied in vitro and in animal models for their ability to lower the activity of CYPs, and increase phase II enzyme activities (except for SULTs, which they tend to inhibit.68) The inhibition of CYP activity by naringenin (the principle flavonoid in grapefruit) has been well documented in humans69; hence the recommendation to avoid grapefruit when taking prescription drugs. Other flavonoids that have demonstrated mild inhibition of multiple CYPs in animal models include genistein, diadzein, and equol from soy,70,71 and theaflavins from black tea.72
Green tea extracts and the quercetin derivatives isoquercetin and rutin are an exception to most other flavonoids; green tea tannins can increase CYP activity in vivo73, but also increase phase II activity (GST and UGT). Similarly, the quercetin derivatives were demonstrated to increase intestinal and liver CYPs in rats; quercetin had no effect on CYPs in this experiment.74
Nrf2 activators: A wide variety of dietary components have been shown in vitro or cell culture to activate Nrf2 and directly increase activity of phase II enzymes; these include epigallocatechin gallate (EGCG)75, resveratrol76, curcumin77 and its metabolite tetrahydrocurcumin, which has greater phase II activity78, cinnamaldehyde79, caffeic acid phenyethyl ester, alpha lipoic acid80, alpha tocopherol81, lycopene82, apple polyphenols (chlorogenic acid and phloridzin)83, gingko biloba84, chalcone85, capsaicin86, hydroxytyrosol from olives87, allyl sulfides from garlic88, chlorophyllin89, and xanthohumols from hops90. The beneficial effects of these phytochemicals have been demonstrated in numerous animal and human studies, particularly their chemopreventative and antioxidant abilities; these effects may be explained by their indirect stimulation of antioxidant enzyme production and phase II detoxification through Nrf2 signalling.91
Isothiocyanates derived from glucosinolates are reactive sulfur compounds with potent chemopreventive properties; the prototypical member is sulforaphane, a constituent of broccoli that is the subject of several human cancer trials.
Isothiocyanates such as sulforaphane and indoles such as indole-3-carbinol (I3C) are among the most potent natural inducers of phase II detoxification enzymes.92 Sulforaphane and a derivative of I3C both directly activate Nrf293, which increases the production of several protective enzymes, including GSTs, UGTs, glutamate-cysteine ligase (which synthesizes glutathione), and NQO1.94 I3C derivatives are also strong inducers of many phase I & II enzymes, and thus are among the most well studied phytochemicals for detoxification, as well as cancer prevention.95,96,9798,99
Compounds from the Japanese horseradish Wasabi japonica100,101, and benzyl isothiocyanate (BITC102) from cruciferous vegetables similarly stimulate phase II enzyme activity via Nrf2 activation. Both sulforaphane and HITC also lower CYP activity.103
Sulfur constituents from garlic are inhibitors of various CYPs104, and induce GST and NQO1 activity in gastrointestinal tissues in rats.105 By activating Nrf2, components in garlic were able to reverse the depletion of antioxidant enzymes caused by a toxic metal compound in the livers of laboratory rats106
D-limonene (from citrus oil) has been investigated for anticancer activity in uncontrolled human trials and animal studies with some success107; part of this chemopreventive activity is due to the induction of phase I and phase II enzymes. In rats, D-limonene has been shown to increase total CYP activity108, intestinal UGT activity109 and liver GST and UGT activity110,111.
Calcium D-glucarate is present in many fruits and vegetables, and can be produced in small amounts in humans.112 When activated in the gut, it functions as an inhibitor of beta-glucuronidase, an enzyme produced by colonic bacteria and intestinal cells. In the intestines, beta-glucuronidase removes (deconjugates) glucuronic acid from neutralized toxins -- essentially reversing the reaction catalyzed by UGTs. Deconjugation reverts the toxin to its previous dangerous form, and allows it to be reabsorbed. Elevated beta-glucuronidase activity has been associated with increased cancer risk.113
Chlorophyllin is a chlorophyll derivative114 that inhibits CYP activity115, and stimulates GST activity in cell culture and rodent models.116 The unique chemical structures of chlorophyllin and chlorophyll enable them to bind and “trap” toxins in the gut preventing their absorption. In animal models, chlorophyllin and chlorophyll lower the bioavailability and accelerate the excretion of several environmental carcinogens.117,118,119 Toxin trapping may partly explain the results of a human trial of residents of Qidong, China, an area with a high incidence of liver cancer due to exposure to aflatoxin (a toxin produced by species of the fungus Aspergillus). Among the 180 people who took 100 mg of chlorophyllin three times daily, urinary levels of DNA-aflatoxin conjugates (a marker for DNA mutation) went down 55% compared to untreated people.120
Probiotics: Certain strains of probiotic bacteria may minimize toxin exposure by trapping and metabolizing xenobiotics or heavy metals.121 Examples include the detoxification of aflatoxin and patulin (two toxins produced by Aspergillus, a type of mold)122, the metabolism of heterocyclic amines and dimethylhydrazine 123, and the binding of lead and cadmium.124 Additionally, the production of the short chain fatty acid butyrate by lactic acid bacteria (from the fermentation of dietary fiber) has been shown to stimulate GST production in intestinal cell culture; this may also contribute to some of the anticarcinogenic properties of dietary fiber.125
N-acetyl cysteine: N-acetyl cysteine can provide an alternative source of sulfur for glutathione production. It is a free radical scavenger on its own, effective at reducing oxidative stress, particularly due to heavy metal toxicity.126 Because it can directly replenish glutathione stores, NAC is more effective than methionine at preventing liver damage,127 and is the current treatment for acetaminophen toxicity. It is an effective treatment for acute liver failure due to non-acetaminophen drug toxicity as well.128
Milk Thistle (Silybum marianum), the most well-researched plant in the treatment of liver disease129, contains a mixture of several related polyphenolic compounds called silymarin. Silymarin promotes detoxification by several complementary mechanisms. The antioxidant capacity of silymarin can lower the liver oxidative stress associated with toxin metabolism, particularly lipid peroxidation130, which has the effect of conserving cellular glutathione levels.131 Like NAC, silymarin can protect against acetaminophen toxicity (possibly by the similar mechanism of preserving glutathione levels). Silymarin, however, may be a more effective antidote than NAC for acetaminophen toxicity if the treatment is delayed (in an animal model, it was effective when administered up to 24 hours after overdose).132
Phase III transporters, while important for removing toxins from healthy cells, can also decrease the effectiveness of pharmaceutical therapies by increasing their clearance. This can be especially problematic with chemotherapy drugs, to which phase III transporters enable cancer cells to become resistant. Therefore, stimulation of phase III activity may not always be desirable.
Dietary factors can have differing effects on phase III transporters. For example, apple polyphenols133, and sulforaphane (at levels equivalent to about two servings of broccoli)134 both stimulate the activity of the phase III proteins. In contrast, the curcumin metabolite tetrahydrocurcumin decreases the activity of the phase III transporters in human cervical carcinoma and breast cancer cell lines.135 Resveratrol decreases phase III protein synthesis which prevented acute myeloid leukemia cells from becoming resistant to the chemotherapy drug doxorubicin in cell culture.136 Silibinin, the chief constituent of milk thistle137, is also a phase III inhibitor, both in vitro and in vivo.138
Bile flow: As a major carrier of toxins from the body, proper bile flow is a critical final step in the metabolic detoxification process. Impairment of bile flow (cholestasis), resulting from dysfunction within the liver or blockage of the bile duct, can result in the buildup of liver toxins and liver injury. Cholestasis can also be the result of the detoxification process itself; there is increasing evidence that the detoxification and excretion of clinical drugs into the bile can produce cholestatic liver disease.139 Artichoke has been used for centuries in folk medicine as a liver protectant and to stimulate bile flow (choleresis), and is the best-studied herbal choleretic agent.
Artichoke contains several antioxidants that can protect against oxidative liver damage, as well as caffeoylquinic acids, which have been shown to stimulate bile flow in animal models.140 Caffeoylquinic acids may also be responsible for the choleretic properities of yarrow141,142, fennel143, and dandelion.144 Andrographis, garlic, cumin, ginger, ajowan (carom seed), and curry and mustard leaf have also been shown to stimulate bile flow or bile acid production in rodent models.145,146,147,148