Modulation of Metabolic Detoxification Pathways Using Foods and Food-Derived Components: A Scientific Review with Clinical Application

Research into human biotransformation and elimination systems continues to evolve. Various clinical and in vivo studies have been undertaken to evaluate the effects of foods and food-derived components on the activity of detoxification pathways, including phase I cytochrome P450 enzymes, phase II conjugation enzymes, Nrf2 signaling, and metallothionein. This review summarizes the research in this area to date, highlighting the potential for foods and nutrients to support and/or modulate detoxification functions. Clinical applications to alter detoxification pathway activity and improve patient outcomes are considered, drawing on the growing understanding of the relationship between detoxification functions and different disease states, genetic polymorphisms, and drug-nutrient interactions. Some caution is recommended, however, due to the limitations of current research as well as indications that many nutrients exert biphasic, dose-dependent effects and that genetic polymorphisms may alter outcomes. A whole-foods approach may, therefore, be prudent.


Introduction
Food-based nutrients have been and continue to be investigated for their role in the modulation of metabolic pathways involved in detoxification processes. Several publications to date have leveraged cell, animal, and clinical studies to demonstrate that food-derived components and nutrients can modulate processes of conversion and eventual excretion of toxins from the body [1]. In general, the nature of these findings indicates that specific foods may upregulate or favorably balance metabolic pathways to assist with toxin biotransformation and subsequent elimination [2,3]. Various whole foods such as cruciferous vegetables [2,4,5], berries [6], soy [7], garlic [8,9], and even spices like turmeric [10,11] have been suggested to be beneficial and commonly prescribed as part of naturopathic-oriented and functional medicine-based therapies [12,13].
While these foods are important to note, the science in this active area of inquiry continues to evolve to reveal new findings about food-based nutrients and their effect on health. Thus, the purpose of this review article is to summarize the science to date on the influence of whole foods, with a special focus directed towards phytonutrients and other food-based components, on influencing specific metabolic detoxification pathways, including phase I cytochrome enzymes, phase II conjugation enzymes, antioxidant support systems, and metallothionein upregulation for heavy metal metabolism. Based on this current science, the paper will conclude with clinical recommendations that may be applied in a personalized manner for patients via the discretion of a qualified health professional.

The Metabolic Pathways of Detoxification
Discussion of physiological pathways for detoxification has been mainly centered around phase I and phase II enzyme systems. This review will cover phase I cytochrome P450 It is well-known for its role in the carcinogenic bioactivation of polycyclic aromatic hydrocarbons (PAHs), heterocyclic aromatic amines/amides, polychlorinated biphenyls (PCBs), and other environmental toxins [18,19]. Low CYP1A2 activity, for example, has been linked to higher risk of testicular cancer [20]. However, due to their rapid conversion to highly reactive intermediates, excessive activity of CYP1A enzymes without adequate phase II support may enhance the destructive effects of environmental procarcinogens [21]. Indeed, genetic polymorphisms in this cytochrome family have been suggested as useful markers for predisposition to certain cancers [15]. CYP1 enzymes are also involved in the formation of clinically relevant estrogen metabolites: CYP1A1/1A2 and CYP1B1 catalyze the 2-hydroxylation and 4-hydroxylation of estrogens, respectively [22]. The potential role of 4-hydroxyestradiol in estrogen-related carcinogenesis, via the production of free radicals and related cellular damage [22], has prompted investigation into factors that modulate CYP1 enzymes.
Various foods and phytonutrients alter CYP1 activity (Tables 1(a) and 1(b)). Cruciferous vegetables have been shown, in humans, to act as inducers of CYP1A1 and 1A2, and animal studies also suggest an upregulation of CYP1B1 [4,[23][24][25][26][27]. The inductory effect of crucifers on CYP1A2 seems especially well established. Clinical studies also indicate that resveratrol and resveratrol-containing foods are CYP1A1 enhancers [28]. Conversely, berries and their constituent polyphenol, ellagic acid, may reduce CYP1A1 overactivity [6], and apiaceous vegetables and quercetin may attenuate excessive CYP1A2 action [24,29]. Cruciferous vegetables and berries have been suggested as possible modulators of estrogen metabolites: berries for their reducing effect on CYP1A1 [6] and cruciferous vegetables for their stronger induction of CYP1A versus 1B1 enzymes [25][26][27]30]. Chrysoeriol, present in rooibos tea and celery, acts selectively to inhibit CYP1B1 in vitro [31] and may be especially relevant to patients with CYP1B1 overactivity. However, further research is needed to confirm this finding.
Many foods appear to act as both inducers and inhibitors of CYP1 enzymes, an effect which may be dose dependent or altered by the isolation of bioactive compounds derived from food. Curcumin at 0.1% of the diet has been shown, in animals, to induce CYP1A1, for example, [35], yet a diet of 1% turmeric was inhibitory [46]. Black tea at 54 mL/d induced both CYP1A1 and 1A2 [33], yet 20 mg/kg of theaflavins was inhibitory to CYP1A1 [45]. Soybean intake at 100 mg/kg upregulated CYP1A1 activity [7], yet at 1 g/kg black soybean extract [44] and 200 mg daidzein twice daily [49], its effect was inhibitory. Further research is needed to confirm different dose effects and impact in humans.
Varied effects may also occur from different members of the same food group. Seemingly contradictory to research showing that cruciferous vegetables activate CYP1 enzymes, kale (another member of the cruciferous family) appears to inhibit CYP1A2 (as well as 2C19, 2D6, and 3A4) in animals [51]. The dose used, at 2 g/kg per day, is 15-fold higher than the typical level of human consumption [51], and more research would be required to determine whether lower intake levels would also have a similar effect. The same authors also tested In vivo Diet of 1% turmeric [46] the effects of an equivalent volume of cabbage consumption and found no such inhibitory effect, pointing to the possibility that different cruciferous vegetables may have distinct effects on cytochrome activity.

CYP2A-E Enzymes.
The large CYP2 family of enzymes is involved in the metabolism of drugs, xenobiotics, hormones, and other endogenous compounds such as ketones, glycerol, and fatty acids [15,54]. Some notable polymorphisms occur in the CYP2C and CYP2D subgroups, leading to the classification of patients as "poor metabolizers" of various pharmaceuticals: warfarin and CYP2C9, antiarrhythmia agents, metoprolol and propafenone, and CYP2D6, phenytoin, cyclobarbital, omeprazole, and CYP2C19, for example, [15,17]. CYP2D polymorphisms may be associated with Parkinson's disease and lung cancer [15]. Clinical evidence exists for the induction of CYP2A6 by quercetin and broccoli [4,29] (Table 2(a)). In animals, chicory appears to induce CYP2A enzymes [41] and rosemary and garlic may upregulate CYP2B activity [9,37]. Clinical studies using resveratrol and garden cress indicate CYP2D6 inhibition [28,55] (Table 2(b)). Ellagic acid, green tea, black tea, and cruciferous vegetables also appear to inhibit various CYP2 enzymes. CYP2E1 enzymes have also attracted particular interest for their role in various diseases. 2E1 metabolizes nervous system agents such as halothane, isoflurane, chlorzoxazone, and ethanol and bioactivates procarcinogenic nitrosamines and aflatoxin B1 [15,65]. It produces free radicals regardless of substrate [15], and CYP2E1 polymorphisms have been associated with altered risk for coronary artery disease [66] and gastric cancer [67]. CYP2E1-induced oxidative stress has also been shown to lead to impaired insulin action via the suppression of GLUT4 expression [68]. Attenuation of 2E1 overactivity may therefore be an important consideration in high-risk patients.

CYP3A Enzymes.
The occurrence of the different CYP3A isoforms is tissue-specific [15]. Rooibos tea, garlic, and fish oil appear to induce the activity of CYP3A, 3A1, and 3A2 [8,36,69,70] (Table 3(a)). Possible inhibitory foods include green tea, black tea, and quercetin [33,56,71,72] ( Table 3(b)). The most clinically relevant of the enzymes is CYP3A4, which is expressed mainly in the liver and to a lesser extent in the kidney [13]. Caffeine, testosterone, progesterone, and androstenedione are substrates of the CYP3A4 enzyme system, as are various procarcinogens including PAHs and aflatoxin B1 [15]. To date, however, the principal driver for research on CYP3A4 has been due to its role in the metabolism of over 50 percent of all pharmaceuticals [73]. The potential for drug interaction with this single enzyme, coupled with the wide interindividual differences in enzymatic activity, generates some level of risk in administration of high doses and multiple drugs as well as food-drug In vivo 30 mg/kg/d ellagic acid [43] CYP2C6 Ellagic acid Berries, pomegranate, grapes, walnuts, and blackcurrants [42] In vivo 30 mg/kg/d ellagic acid [43] CYP2C9 Resveratrol Grapes, wine, peanuts, soy, and itadori tea [32] Clinical 1 g/d resveratrol [28]: note high dose used Myricetin Onions, berries, grapes, and red wine [58] In vivo 2 and 8 mg/kg myricetin [58] CYP2C19 Kale In vivo 2 g/kg/d kale, as freeze-dried kale drink [51] Resveratrol Grapes, wine, peanuts, soy, and itadori tea [32] Clinical 1 g/d resveratrol [28]: note high dose used

CYP2D6
Garden cress Clinical 7.5 g twice daily intake of garden cress seed powder [55] Kale In vivo 2 g/kg/d kale, as freeze-dried kale drink [51] [52] 200 mg/kg diallyl sulfide [8] 30 to 200 mg/kg garlic oil [36] Diet of 2% and 5% garlic powder [61] N-acetyl cysteine Allium vegetables [54] In vivo 25 mg/kg and 50 mg/kg N-acetyl cysteine [54] CYP2E1 Ellagic acid Berries, pomegranate, grapes, walnuts, and blackcurrants [42] In vivo 10 and 30 mg/kg/d ellagic acid [43] Green tea In vivo 45 mL/d/rat (avg. 150 g animal weight) green tea [33] Black tea In vivo 54 mL/d/rat (avg. 150 g animal weight) black tea [33] Dandelion In vivo 0.5 and 2 g/kg dandelion leaf water extract [62] Chrysin Honey, honeycomb [63] In vivo 20 and 40 mg/kg/d chrysin [63] Medium-chain triglycerides (MCTs) Coconut and coconut oil In vivo 32% calories as MCTs [64] and herb-drug interactions. Grapefruit juice is perhaps the most well-known food inhibitor of this enzyme [74], though resveratrol and garden cress, a member of the cruciferous vegetable family, appear to have similar effects in humans, albeit at intakes above what would be expected without highdose supplementation [28,55]. Curcumin may upregulate 3A4 activity [11]. Once again, there are indications that a biphasic effect may be seen from dietary bioactive compounds; Davenport and Wargovich (2005) found that shorter-term or lower dosing with garlic organosulfur compounds produced potentially anticarcinogenic effects but that longer-term higher doses (200 mg/kg) of allyl sulfides led to minor hepatic toxicity [8]. One garlic clove contains only 2,500-4,500 g of the allyl sulfide precursor, allicin [76], so the higher dose is much more than would be consumed in a typical human diet. In another example, two components of cruciferous vegetables, sulforaphanes and indole-3-carbinol, inhibited and increased activity, respectively [57,75], highlighting the potential for human studies using whole foods to clarify the outcome of consumption.

CYP4 Enzymes.
Less is known about this family of enzymes, since it is thought to play a smaller role in drug metabolism. It is, however, understood to be a primarily extrahepatic family of cytochromes, inducible by clofibrate and ciprofibrate (hypolipidemic drugs), NSAIDs, prostaglandins, and toxicants such as phthalate esters [15,77]. The CYP4B1 isoform is involved in the metabolism of MCTs (medium chain triglycerides), as well as the bioactivation of pneumotoxic and carcinogenic compounds [78].
Polymorphisms and overexpression of this subgroup may be associated with bladder cancer [15] and colitis [79]. A report by Ye et al. (2009) which examined the link between colitis and CYP4B1 activity found that the promotion of CYP4B1 activity by caffeic acid (found in caffeine-containing foods) (Table 4) correlated with reduced inflammation and disease activity [79]. Green tea may act to induce CYP4A1, as suggested by animal studies [40]. More research is needed to clearly identify food influences on this enzyme family.

Phase II Conjugation Enzymes.
After a xenobiotic has gone through the process of becoming hydrophilic through reactions overseen by CYP450 enzymes, its reactive site can be conjugated with an endogenous hydrophilic substance. This reaction is often referred to as "phase II detoxification." Conjugation involves the transfer of a number of hydrophilic compounds (via their corresponding enzymes), including glucuronic acid (glucuronyl transferases), sulfate (sulfotransferases), glutathione (glutathione transferases), amino acids (amino acid transferases), an acetyl group (N-acetyl transferases), and a methyl group (N-and O-methyltransferases) [81]. The result of the collective activity of these enzymes is an increase in the hydrophilicity of the metabolite, theoretically leading to enhanced excretion in the bile and/or urine [81]. Similar to the CYP450 enzymes, genetic polymorphisms can have profound influence on the function of these conjugating  [58] In vivo 0.4, 2, and 8 mg/kg myricetin [58]  In vivo 179 mg/kg caffeic acid [79] enzymes [82], with potential implication in the development of several forms of cancer [83]. It is conceivable that modulation of phase II enzymes by food-based bioactive compounds may be advantageous in patients who have altered enzyme activity due to genetic polymorphisms or who have a high toxic burden due to chronic exposure to environmental pollutants, overactive phase I activity, or hormonal imbalance. For example, James et al. (2008) suggest that upregulation of glucuronidation and sulfonation by certain bioactive compounds may be 8 Journal of Nutrition and Metabolism a useful consideration for the elimination of environmental PCBs [19].

UDP-Glucuronosyltransferases.
This class of enzymes, comprising multiple proteins and even subfamilies, plays an essential role in enhancing the elimination of biotransformed toxins in urine and feces, as well as metabolizing steroid hormones and bilirubin [84,85]. Their function is to catalyze the covalent linkage of glucuronic acid from UDP-glucuronic acid to an accepting functional group on the molecule, a process referred to as glucuronidation [86]. Glucuronidation occurs primarily in the liver but can occur in other tissues, such as the small intestine [86,87]. Bilirubin, specifically, is principally conjugated by UGT1A1 in hepatocytes [88] and then excreted with bile into the intestinal tract. It has been estimated that 40-70% of all medications are subject to glucuronidation reactions in humans, thereby suggesting the significance of this conjugation enzyme family [88]. Since UDP-glucuronosyltransferases (UGTs) also metabolize phytochemicals, alterations in their effects may be seen with genetically downregulated enzyme activity; flavonoids are conjugated with glucuronide and sulfate; therefore, UGT or sulfotransferase (SULT) polymorphisms may produce variability in phytochemical clearance and efficacy [89].
Meaningful interpretations of these studies may still be elusive, however: in one combined dietary trial, the consumption of 10 servings per day of a combination of cruciferous vegetables, soy foods, and citrus fruits did not have a significant effect on UGT enzyme activity compared with a diet devoid of fruits and vegetables [85]. The authors hypothesize that these results may be due to their choice of specific foods within those groups or due to Nrf2 activation (discussed in subsequent sections) when fruits and vegetables were avoided.
The effects of UGT activity may also be enhanced by Dglucaric acid by theoretical inhibition of beta-glucuronidase enzymes [100]. Beta-glucuronidase enzymes act to reverse UGT conjugation reactions. D-glucaric acid is found in many fruits, vegetables and legumes (Table 5(b)). When tested in humans, however, a diet supplemented with cruciferous vegetables (2/3 cup broccoli, 1/2 cup cabbage, and 1/2 cup radish sprouts), citrus fruits (1 cup grapefruit juice, 1/2 cup orange juice, 1 cup orange/grapefruit segments, and 1 orange peel), and soy foods was found to have no effect on betaglucuronidase activity [101] (amounts standardized for 55 kg body weight), indicating that the clinical effects of D-glucaric acid consumption still need further clarification.
In vivo research suggests that polyphenol extracts of certain berries, specifically strawberries and blackcurrant, may inhibit beta-glucuronidase activity in the intestinal lumen; Kosmala et al. (2014) observed this effect using both strawberry pomace water extract and water-alcohol extract containing 5.1% and 17.1% ellagic acid, and 0.2% and 10.9% proanthocyanidins, respectively [100]. Jurgoński et al. (2014) found a similar inhibitory effect using a diet of 1.5% blackcurrant extract (total polyphenolic content 66.8 g/100 g extract) [102]. Interestingly, the highest levels of beta-glucuronidase activity were seen in rabbits fed a high fat diet (32% calories from fat, including 10% from lard), without blackcurrant extract supplementation, suggesting that dietary fat may also alter enzyme activity [102].
Inhibition of UGT enzymatic activity may be a consideration for modulation of hormone levels and the risk of certain cancers, such as prostate cancer [84]. In vitro studies suggest that various foods and food-based components may inhibit UGT activity, including green and black tea, quercetin, rutin, naringenin, allspice, peppermint oil, cacao, and silymarin [84], although further research is needed to evaluate their in vivo and clinical effects.

Sulfotransferases.
As the name of this superfamily of enzymes might suggest, SULTs are responsible for the transfer of a sulfuryl group donated by 3 -phosphoadenosine-5 -phosphosulfate (PAPS) to hydroxyl or amine groups, particularly in the areas of liver, intestine, adrenal gland, brain, and skin tissues [103]. This process is often referred to as sulfation but is more accurately termed sulfonation or sulfurylation. Decreased function of these enzymes, through genetic variability or presence of environmental chemicals, can lead to eventual interference with thyroid hormone, estrogen, and androgen levels [104,105], as well as variable polyphenol effects [106], since the active forms of these compounds can be degraded via sulfonation. Typically, once compounds have been conjugated with sulfate, there is less reactivity and toxicity incurred from the precursor molecule [105].

Legumes
Mung bean seeds, adzuki bean sprouts [97] Vegetables and fruits Oranges, spinach, apples, carrots, alfalfa sprouts, cabbage, Brussel sprouts, cauliflower, broccoli, grapefruit, grapes, peaches, plums, lemons, apricots, sweet cherries, corn, cucumber, lettuce, celery, green pepper, tomato, and potatoes [97][98][99] may be vastly different. Of note, caffeic acid demonstrates in vitro SULT-inhibitory properties [105]. This finding conflicts with its in vivo ability to induce SULT enzymes, as described by Zhou et al. (2012) [107], highlighting the difficulty of extrapolating meaningful conclusions from in vitro data. SULT enzyme activity is dependent on a depletable reserve of inorganic sulfate [112]. Dietary sources of sulfurcontaining compounds may therefore play an essential role in SULT function, by providing the substrate for enzyme action (Table 6(b)).

Glutathione S-Transferases.
Similar to the aforementioned categories of conjugating enzymes, glutathione Stransferases (GSTs) include a complex of enzymes, whose main function is to attach a glutathione group to a biotransformed metabolite. The production of these enzymes can be induced through the production of reactive oxygen species and via gene transcription involving the antioxidantresponsive element (ARE) and the xenobiotic-responsive element (XRE), which will be subsequently discussed in this paper [113].
Cruciferous and allium vegetables and resveratrol demonstrate ability to induce GSTs in humans [28,[114][115][116][117] (Table 7(a)). Observational research also associates citrus consumption with increased GST activity [115]. In vivo data also suggest many foods and food constituents to be upregulators of these enzymes, including garlic, fish oil, black soybean, purple sweet potato, curcumin, green tea, rooibos tea, honeybush tea, ellagic acid, rosemary, ghee, and genistein [36,43,44,70,93,[118][119][120][121][122][123]. Conjugated linoleic acid has been shown to be at least partly responsible for the effect of ghee [122]. It is possible that the effects of at least some of these foods and bioactive compounds may be due to their upregulation of the Nrf2 signaling pathway.  [111] In vivo 2, 10, and 50 mg/kg caffeine [107] Retinoic acid (bioactive form of vitamin A) Meat (especially liver), fish, egg, and dairy products contain retinol; apple, apricot, artichokes, arugula, asparagus, and other plant foods contain provitamin A carotenes [111] In vivo 2, 10, and 50 mg/kg/d retinoic acid suspension in corn oil [108] (b) Genetic variances, gender, and even possibly body weight appear to play a role in the effects of dietary factors on GST enzymes [114][115][116]. Clinical investigation of cruciferous and allium vegetables by Lampe et al. (2000) found that an upregulated effect was most marked in women, indicating gender variability, and that the effect was also genotypedependent, occurring only in GSTM1-null individuals [116]. The same investigators also found that apiaceous vegetables inhibited GST activity, but only in GSTM1+ men [116] (Table 7(b)). High doses of quercetin and genistein have also shown inhibitory effects [123,126].

Animal products
There is evidence that at least some of these foods and phytonutrients may exert modulatory rather than absolute inductive/inhibitory effects; Chow et al. (2010) found that resveratrol increased GST only in those with low baseline enzyme levels or activity [28]. It is also noteworthy that bioactive components of crucifers, including isothiocyanates, are substrates for GST enzymes and that GST genotype may therefore alter the response to cruciferous vegetables consumption on other mechanisms such as glutathione peroxidase and superoxide dismutase [134,135]. GSTM1null genotype is associated with a more rapid excretion of isothiocyanates, leading some researchers to conclude that the benefits of cruciferous vegetable consumption may be lessened in individuals with this genetic variation [89]. Support for glutathione conjugation also involves enhancing reduced glutathione (GSH) status. Glutathione is a low-molecular weight tripeptide containing residues of cysteine, glutamate, and glycine [136]. Most glutathione from foods and supplements is poorly absorbed, so liposomal delivery has been used [137]. The sulfur-containing amino acids methionine and cystine are important precursors to glutathione formation; their depletion leads to depressed GSH levels [138]. N-acetyl cysteine has also been used to restore depleted GSH levels in a clinical setting [139].
Various nutrients may also enhance endogenous glutathione synthesis, including vitamin B6, magnesium, and selenium [140,141]. Curcuminoids (from turmeric), silymarin (from milk thistle), folic acid, and alpha-lipoic acid have been shown, in humans, to restore depleted GSH [129,130,142,143]. In animal studies, cruciferous vegetables and artichoke have also demonstrated a GSH-protective effect [131][132][133]. There is therefore the potential to improve glutathione status via diet or supplementation (Table 7(c)).

Amino Acid Transferases.
Amino acids of various types (e.g., taurine, glycine), whether endogenous or exogenous (from dietary sources) in origin, can be utilized for attaching to molecules for their excretion. For the benefit of providing a substrate to these enzymes, it is generally thought that dietary protein is required for an effective detoxification protocol. Table 8 lists amino acids used in phase II conjugation reactions and selected food sources.

N-Acetyl Transferases (NAT)
. This class of enzymes is responsible for the transfer of an acetyl group to convert aromatic amines or hydrazines to aromatic amides and hydrazides, which is significant for those taking pharmaceuticals such as isoniazid, hydralazine, and sulphonamides [83]. Polymorphisms in genes for this category of enzymes, leading to slow metabolism, have been shown to be associated with hepatoxicity during drug treatment [146]. One small human study found that 500 mg quercetin daily enhanced NAT activity [29]. However, more research is needed to understand the relationship between dietary nutrients and NAT function.

Glycine
Turkey, pork, chicken, soybean, seaweed, eggs, amaranth, beef, mollusks, peanuts, pumpkin seeds, almonds, duck, goose, mung beans, sunflower seeds, lentils, lamb, bison, lobster, and fish [111] Taurine Many cooked meats and fish supply taurine. Taurine is also synthesized in the body from cystine (requiring niacin and vitamin B6) and homocysteine (requiring additionally betaine and serine) [144] Glutamine Plant and animal proteins such as beef, pork, chicken, dairy products, spinach, parsley, and cabbage [145] Ornithine Ornithine is synthesized endogenously via the urea cycle, requiring arginine and magnesium [144] Arginine Turkey and pork are especially rich sources; also chicken, pumpkin seeds, soybean, butternuts, egg, peanuts, walnuts, split peas, mollusks, almonds, sesame seeds, lentils, fava beans, mung beans, pine nuts, beef, sunflower seeds, and white beans [111] 2.2.6. Methyltransferases. Relatively significant attention has been given in various medical communities to this class of phase II enzymes due to the increasing importance of methylation for reducing disease risk. The conjugating donor compound in methyltransferase reactions is a methionine group from S-adenosyl-L-methionine (SAMe) [147]. Catechol O-methyltransferase (COMT) is one of the prominent methyltransferases that has received wide attention due to its role in estrogen detoxification [148]. Support for methylation consists of nutrient cofactors and methyl donors, such as methionine, vitamin B12, vitamin B6, betaine, folate, and magnesium [144]. Various foods can provide these nutrients (Table 9). Conversely, a high sucrose diet may inhibit methylation enzymes such as COMT [149].

Gene Induction of Phase II Detoxification and Antioxidant Enzymes through Nrf2
The transcription factor, Nrf2 [nuclear factor erythroid 2 (NF-E2) p45-related factor 2], is key to regulating the body's detoxification and antioxidant system. When activated, Nrf2 dissociates from the cytosolic protein, Keap1 (Kelch-like ECH associated protein 1), and translocates to the nucleus to bind to AREs in the promoter/enhancer portion of genes associated with phase II detoxification and antioxidant enzyme genes [150] (Figure 1). Nrf2-deficient animals experience increased toxicity from drugs [151], carcinogens, allergens, and environmental pollutants [152] and do not respond as well to the anti-inflammatory effects of phytochemicals [153], indicating the essentiality of these enzymes. Conversely, Nrf2 Table 9: Selected dietary sources of nutrients for methylation support (adapted from [111]).

Methionine
Meats, poultry, fish, shellfish, egg, nuts (especially Brazil nuts), seeds (especially sesame seeds and pumpkin seeds), spirulina, teff, soybeans Lower amounts found in other legumes and whole grains (especially teff and oats) Vitamin B12 Meats and meat products (especially liver and kidney), poultry, fish, shellfish, and eggs
Conversely to its role in cancer prevention, overexpression of Nrf2 is found in many cancer cells and has been shown to promote tumor growth and resistance to anticancer therapy [154]. Consequently, the inhibition of Nrf2 signaling may be clinically relevant for patients receiving cancer chemotherapy [184,185]. Overexpression of Nrf2 and CYP2E1 has also been associated with impaired GLUT4 activity and insulin resistance [68]. As noted above, supplementation (above levels normally consumed through diet) with certain phytochemicals may have inhibitory effects on Nrf2 activation, including luteolin [184] and quercetin [185] (Table 10(b)). Vitamins A, C, and E and N-acetyl cysteine have also been implicated as Nrf2 inhibitors at high doses [188]. These findings point to the need for further research to clarify outcomes as they relate to specific disease states as well as potential biphasic dose effects.

Metallothionein
Metallothionein, a cysteine-rich protein with the ability to bind divalent cations, including toxic metals such as mercury, cadmium, lead, and arsenic, is gaining recognition as an important component in heavy metal detoxification [190][191][192]. Similar to the upregulation of phase II and antioxidant enzymes, metallothionein can be induced at specific promoter regions of genes by stimuli such as heavy metals, oxidative stress, glucocorticoids, and even zinc [192]. In addition to sequestering heavy metals, it is capable of scavenging free radicals and reducing injury from oxidative stress [192], as well as inhibiting NF-B signaling [193].
Dietary patterns and nutrients may result in changes in metallothionein production. Lamb et al. (2011) reported a 54% increase in metallothionein mRNA production in a small clinical trial in women with fibromyalgia following an elimination diet in conjunction with a phytonutrientrich medical food consisting of hops, pomegranate, prune skin, and watercress [194]. Zinc supplementation (15 mg/day) to healthy men over 10 days led to significantly increased metallothionein mRNA, up to 2-fold in leukocytes and up to 4-fold from dried blood spots [195]. Metallothionein has been shown to be decreased in the intestinal mucosa of patients with inflammatory bowel disease (IBD); however, zinc supplementation (300 mg zinc aspartate, equal to 60 mg elemental zinc per day for 4 weeks) in 14 zinc-deficient patients with IBD resulted in slightly higher metallothionein concentration in the intestinal mucosa [196]. Cruciferous phytonutrients may also modulate metallothionein expression, as suggested by a 10-fold increase following a single oral dose of 50 mol sulforaphane to rats [197]. Chromium may inhibit zinc-induced metallothionein expression, according to animal studies by Kimura et al. (2011) [198]. Early-stage, in vitro studies also suggest that quercetin and Cordyceps sinensis, a mushroom native to the Himalayan region, may upregulate metallothionein expression [199,200].

Clinical Applications
With the continued emergence of data supporting the role of toxins in chronic disease processes, it is becoming increasingly necessary for clinicians to understand how to provide therapeutic modalities to reduce toxin load in patients. In this paper, several studies regarding the influence of foods and food-based nutrients on the systems of detoxification were presented. From the current information presented, listed below are some key concepts for translation into the clinical setting.

Nonclinical versus Clinical Studies.
One of the limitations that comes to the forefront in this collection of studies is how the information, in many cases, is constrained primarily to studies in cells or animals. It remains questionable as to whether similar effects would be seen in humans at moderate, reasonable doses. In the cell studies, it is difficult to anticipate findings due to the lack of pleiotropic activity that occurs in a complex, living system with multiple detoxification systems working simultaneously. Along similar lines, animal studies are often difficult to extrapolate to individuals due to the degree of variability in genotype and environmental phenotype seen in the diverse human population. Therefore, at this time, it is best to take precaution in firmly advocating foods or food-based nutrients that only have cell or animal data as support. It is best to rely on the clinical studies that have been published to date in making more firm recommendations.

Single Agent versus Lifestyle.
While this paper focuses on isolated nutrients and foods that contain those nutrients, it might be optimal from a clinical perspective to consider how an entire lifestyle might induce or inhibit the array of detoxification enzymes. For example, this paper has not addressed behaviors like smoking, physical activity, or stress. The modern clinician needs to weigh all these variables against each other. Yet, science has not fully demonstrated the individual impacts of these factors, along with all of them together. Therefore, at this time, a dietary pattern favoring whole, unprocessed, plant-based foods and the removal or reduction of toxic substances in one's environment is a twoprong approach that would seem to have the best overarching scientific underpinning.

Modulating versus Inhibiting/Inducing Effects.
In several instances, certain foods exhibited a particular activity on an enzyme, while, at higher doses, they had another, opposite effect. Essentially, many foods serve as what is commonly referred to as being "bifunctional modulators," possessing the ability to effectively induce or inhibit detoxification enzyme activity based on the dose response. Therefore, the resulting clinical takeaway might be to encourage patients to follow a mixed, varied diet, full of different plant-based, whole foods. Smaller amounts of many compounds might be more therapeutic and supportive for biochemical pathways rather than overriding signals derived from high concentrations of nutrients through high-dose supplementation or the repeat, daily ingestion of large quantities of the same food.

Polypharmacy.
For patients who are taking multiple pharmaceuticals, it is important to know which detoxification systems will be influenced by nutrients and foods so that side effects are minimized or avoided.

Dietary Supplements versus Foods.
Since there can be potent effects of food-based nutrients on detoxification pathways, it would be best for the average patient to follow, as indicated above, a mixed, complex, and whole-foods diet. Additionally, dietary supplements may be a helpful adjunct in patients in which the practitioner has information about the patient's genetic variability, so that nutrients can be tailored accordingly. Without a full understanding of a patient's SNPs (single nucleotide polymorphisms), it becomes difficult to make accurate assessments about nutrients and dosing.

Duration of Dosing.
Another factor to consider in therapeutic intervention is the timing and duration of the dose of nutrient or the food. In some of the research presented here, effects on detoxification enzymes were seen after several days of food intake or supplementation, while, in other cases, induction of an enzyme might be fairly rapid, followed by efficient adaptability. This variable needs to be considered in further clinical research and requires close monitoring in clinical practice.

Foods Known to Impact Detoxification.
Based on the four systems examined in this paper, there are several foods which seem to have demonstrated an influence on detoxification systems. Many of them have been acknowledged as part of naturopathic medicine. Hence, it would be useful to have a knowledge base of this cumulative set of foods as patients embark upon detoxification protocols. This recent scientific update notes clinical evidence of effects from cruciferous vegetables (in combination, and specifically watercress, garden cress, and broccoli), allium vegetables, apiaceous vegetables, grapefruit, resveratrol, fish oil, quercetin, daidzein, and lycopene. Many other foods, beverages, and nutrient bioactive compounds, based on this review of scientific literature, are also suggested as modulators of detoxification enzymes in vivo (Table 11).

Conclusions
Over the past decade, there has been investigation into nutrigenomic and epigenetic influences of food constituents on chronic diseases [201,202]. Similarly, studies have revealed that exposure to and accumulation of toxins play a significant role in cardiovascular disease, type 2 diabetes, and obesity [203][204][205][206][207]. Thus, one's dietary intake and environmental influences may have large bearing on the incidence of chronic disease. In fact, these influences may be significant not just for the individual, but for several generations due to the transgenerational inheritance of epigenetic changes [208,209]. Therefore, it would seem that designing clinical recommendations to maximize the effects of food and reduce the impact of toxins is essential. However, it is not without caution and critical thinking that a detoxification protocol should be assembled for patients by trained clinicians. There remain many unresolved issues regarding knowing how and what foods modulate detoxification pathways.