and toxicityCHUNG S. YANG, JOHN F. BRADY,2 AND JUN-YAN HONGLaboratory for Cancer Research, Department of Chemical Biology and Pharmacognosy, College of Pharmacy,Rutgers University, Piscataway, New Jersey 08855-0789, USA

Dietary effects on cytochromes P450, xenobiotic metabolism,

non, o/o,/nnne. n7l7Icnl en Th

ABSTRACT The levels and activities of cytochromeP450 enzymes are influenced by a variety of factors, in-cluding the diet. In this article, the effects of selected non-nutritive dietary chemicals, macronutrients, micronutri-ents, and ethanol on cytochromes P450 and xenobioticmetabolism are reviewed in the light of our current un-derstanding of the multiplicity and substrate specificityof cytochrome P450 enzymes. Although the mechanismsof action of several dietary chemicals on specific cyto-chrome P450 isozymes have been established, those formacro- and micronutrients are largely unknown. It isknown, however, that specific nutrients may have variedeffects on different cytochrome P450 forms and thus mayaffect the metabolism of various drugs differently. Nutri-tional deficiencies generally cause lowered rates of xeno-biotic metabolism. In certain cases, such as thiamin de-ficiency and mild riboflavin deficiency, however, enhancedrates of metabolism of xenobiotics were observed. Theeffects of dietary modulation of xenobiotic metabolism onchemical toxicity and carcinogenicity are discussed.-Yang, C. S.; Brady, J. F.; Hong, J. -Y. Dietary effects oncytochromes P450, xenobiotic metabolism, and toxicity.FASEBJ. 6: 737-744; 1992.

Key Words: diet . nutrition cytochromes P450 . enzyme regula-tion xenobiotics drug metabolism . toxicity . carcinogens

THE CLOSE RELATIONSHIP BETWEEN DIET and xenobiotic me-tabolism may be traced back to prehistoric days in animal-plant warfare during evolution (1). Plants synthesized chem-icals for self-protection and animals had to developxenobiotic-metabolizing enzymes such as cytochrome P450(P450)3 for the detoxication of these chemicals. The evolu-tion of the large number of P450 2 genes 400 million yearsago may correspond to the advance of animals onto landwhere they encountered new terrestrial plants and phyto-chemicals. The work of many investigators in the past 30years has clearly established that various dietary factors havemarked effects on the metabolism of drugs, environmentalchemicals, and certain endogenous substrates. This topic hasbeen reviewed extensively (2-9). However, only recently havewe begun to understand some of these effects at the molecu-lar level. Dietary influences on xenobiotic metabolism mayalter the therapeutic effects of drugs and the toxicity or car-cinogenicity of environmental chemicals. In this article, wereview the mechanisms by which dietary chemicals andnutritional status affect the levels and activities of P450 en-zymes, xenobiotic metabolism, as well as chemical toxicityand carcinogenicity.

Because of space limitations, we have chosen to useselected examples to illustrate key concepts rather than toconduct an exhaustive review on this topic. Review articlesare cited instead of original papers.

MODULATION OF P450 LEVELS AND XENOBIOTICMETABOLISM BY NONNUTRITIVE DIETARYCHEMICALS

Dietary chemicals may affect the levels and activities of P450isozymes at different steps as shown in Fig. 1. Dietary chem-icals may affect the levels of P450 species by altering the ratesof: 1) the transcription of specific P450 genes, 2) the degra-dation of specific mRNA, 3) the translation process, and4) P450 degradation through protein turnover or by suicideinhibition. Many dietary chemicals are substrates of theP450-dependent monooxygenase system. They or their me-tabolites may inhibit or enhance the activities of this systemby binding to P450 species or to NADPH:P450 reductase, byaffecting the interaction between these enzymes, or by affect-ing key steps in the catalytic cycle.

Induction of P450-dependent activities

The effect of diet on P450-dependent monooxygenase activi-ties was clearly demonstrated in the pioneering work ofWattenberg (10), who discovered that rats on commercial ratchow had 68-fold higher intestinal benzo(a)pyrene (BP)hydroxylase activity than those on a purified diet. By addingdry vegetable powder to the purified diet, Brussels sprouts,cabbage, turnips, and other vegetables were found to be in-ducers of intestinal BP hydroxylase in rats. The effects ofcruciferous vegetables and their components on drug metab-olism have since been studied extensively. Three autolyticproducts of indolylmethyl glucosinolate (glucobrassicin):indole-3-carbinol, indole-3-acetonitrile, and indole-3-carboxy-aldehyde, isolated from Brussels sprouts, were found to in-duce intestinal and hepatic BP hydroxylase in rats. Thestructures of some of the nonnutritive dietary compoundsare illustrated in Fig. 2. Indole-3-carbinol is the most potentinducing agent among these indoles. Its induction of BPhydroxylase and ethoxyresorufin dealkylase activities is be-lieved to be mainly due to the induction of P450 1A1 in theintestine and P450s IA1 and 1A2 in the liver (11).

It was demonstrated that after an oral dose of indole-3-carbinol to rats, P450 lAl mRNA was elevated severalfold

To whom correspondence should be addressed, at: Laboratoryfor Cancer Research, Department of Chemical Biology and Phar-macognosy, College of Pharmacy, Rutgers University, Piscataway,NJ 08855-0789, USA.

2Present address: Chemistry Department, University of Califor-nia, Davis, CA95616, USA.

3Abbreviations: P450(s), cytochromes P450; BP, benzo(a)pyrene;Ah receptor, aromatic hydrocarbon receptor; RXM, acid reactionproducts of indole-3-carbinol; NDMA, N-nitrosodimethylamine;NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; BHA, butyl-ated hydroxyanisole; B HT, butylated hydroxytoluene.

P450 gene Inactivation of P450 enzymes

degradation P450 mRNA

Substrates

c)OO\PS

Flavone Tange,otin Nob,I.bn

Quercebn Kaenpfeoi

Figure 2. Structures of some dietary or related compounds.

738 Vol. 6 lanuarv 1992 The FASEBJournal YANC FT At

II III I III

transcription

translation

degradation .( P450 Reductase

Inactivation Inhibiton Enhancement

Figure 1. Possible sites for dietary effects on P450 enzymes. TheP450 2E1 gene is used in the illustration. In addition to affectingthe rates of transcription and translation, dietary chemicals mayaffect the rates of degradation of P450 mRNA and protein. Dietarychemicals may serve as substrates. They may also interact withP450 enzymes and NADPH:P450 reductase, causing inactivationof P450, or inhibition or enhancement of the monooxygenase ac-tivities.

When diallyl sulfide was given orally to rats, microsomal N-nitrosodimethylamine (NDMA) demethylase (indicative ofP450 2E1 activity) decreased markedly in several hours. Thiswas followed by a lowering of the immunodetectable P4502El protein level in microsomes (15). The inactivation ofP450 2E1 was also demonstrated in vitro when diallyl sul-fone, an oxidative metabolite of diallyl sulfide, was incubatedwith microsomes in the presence of an NADPH-generatingsystem (15). The inactivation was time, concentration, andNADPH-dependent, following a typical suicide inhibitionpattern. It is believed that diallyl sulfone is converted byP450 2E1 to a reactive intermediate that modifies the hememoiety of P450 2E1 (20). Phenethyl isothiocyanate, occurringas a glucusinolate in a variety of cruciferous vegetables, alsoinactivates P450 2E1 by a suicide mechanism (21). Psoralens,which are found in edible plants such as figs, celery, parsley,and parsnip, were shown to decrease 7-ethoxycoumarin andBP hydroxylase activities when added to human livermicrosomal incubations in the presence of NADPH (22). Itwas suggested that methoxsalen (8-methoxypsoralen), ber-gapten (5-methoxypsoralen), and psoralen are suicide inhi-bitors of P450 enzymes; P450 lAl or 1A2 may be involved.

in the liver and colon, and P450 1A2 mRNA was also ele-vated in the liver (11). The induction of P450 IAI involves thebinding of the inducer to the Ah receptor. Nevertheless,indole-3-carbinol has only low binding affinity to the Ahreceptor, whereas indolo[3,2-b]carbazole has a much higheraffinity (12). It is suggested that under the acidic conditionsin the stomach, indole-3-carbinol can be converted toindolo[3,2-b]carbazole or other acid reaction products(RXM) (13) that bind to the Ah receptor and thereby increasethe transcription of the P450 1A1 gene. This suggestion issupported by the observation that the inductive effect wasfound when indole-3-carbinol was given to rats orally but notwhen given intraperitoneally (13). Acid-treated indole-3-carbinol was much more effective than untreated indole-3-carbinol in inducing ethoxyresorufin activity in primary cul-tures of rat hepatocytes (14). Certain flavones may also in-duce P450 IA1 by binding to the Ah receptor and subse-quently activating the P450 1A1 gene.

A second example of transcriptional regulation is the in-duction of P450 2B1 by diallyl sulfide. Diallyl sulfide, a com-ponent of garlic oil, has been shown to induce rat hepaticP450 2B1 based on the increase of immunodetectable proteinand pentoxyresorufin dealkylase activity (15). Recent studiesin our laboratory indicate that this induction is accompaniedby the elevation of 2B1 mRNA levels. Nuclear run-on experi-ments indicate that after administration of diallyl sulfide, thetranscriptional rate of the P450 2B1 gene was increasedmarkedly, and was observable at 4 h after treatment (16). Inprimary culture of rat hepatocytes, P450 2Bl was not in-duced by diallyl sulfide but by its metabolite diallyl sulfone(unpublished results).

Posttranscriptional mechanisms may also be involved inthe induction of P450s by dietary factors. In the induction ofP450 2E1 by fasting, elevation of P450 2E1 mRNA was ob-served (17) but transcriptional activation could not be con-vincingly demonstrated by nuclear run-on experiments (un-published results). Stabilization of the mRNA may play arole in this induction. In the induction of P450 2E1 byethanol and acetone, elevation of the mRNA was not ob-served (18, 19). Protein stabilization or increased translationefficiency may be involved in this induction.

Enhancement of monooxygenase activities

Flavone, tangeretin, and nobiletin were found to increase BPhydroxylase activity and aflatoxin B, activation in humanliver microsomes (4). The metabolism of antipyrine andzoxazolamine was also stimulated, but that of hexobarbital,coumarin, and 7-ethoxycoumarin was not. The stimulatoryeffect was P450 isozyme-specific. With purified P450 iso-zymes in a reconstituted system, the effect was observed withrabbit P450s 3A6 and 1A2 but not with P450s 2B4, 2C3, or1AI. It was proposed that these flavonoids stimulate themonooxygenase activity by enhancing the interactions be-

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tween P450 and NADPH:P450 reductase and facilitating theflow of electrons to P450. Administration of flavone to neo-natal rats also increased the rate of zoxazolamine metabo-lism several-fold (4).

Inhibition of monooxygenase activities

Dietary compounds can bind to the active sites of P450 en-zymes, serving as substrates or competitive inhibitors. Forexample, diallyl sulfide and phenethyl isothiocyanate arecompetitive inhibitors of P450 2El-catalyzed reactions (20, 21).In addition, phenethyl isothiocyanate also competitively in-hibits the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco carcinogen, in lung micro-somes (23). In this case, a purely competitive mode of inhibi-tion was not observed, possibly due to the inactivation of theenzyme during the incubation, and P450 2El was not involved.

Many dietary flavonoids have been shown to inhibit mono-oxygenase activities. For example, quercetin, kaempferol,morin, and chrysin inhibited BP hydroxylase activity in hu-man liver microsomes (4). Quercetin, kaempferol, andnaringenin inhibited nifedipen and filodipen oxidation cata-lyzed by P450 3A4 in human microsomes (24). It appearsthat flavones having free hydroxy groups on the A ring areinhibitors, whereas flavonoids containing no hydroxy groups(flavone, tangeretin, and nobiletin) are activators (stimula-tors) of selected monooxygenase activities.

EFFECTS OF MACRONUTRIENTS ON XENOBIOTICMETABOLISM

Compared with the effects of nonnutritive dietary chemicals,the effects of macronutrients on drug metabolism are moredifficult to understand. In the former case, the actions of acompound or its metabolites can be studied, but in the lattercase we have to deal with a nutritional state or status. There-fore, despite extensive investigations, our understanding ofthe latter subject is mostly at the descriptive level. Only re-cently have researchers begun to look at specific changes onselected enzymes and the molecular mechanisms involved.Many previously seemingly contradictory results may be in-terpretable based on our new knowledge on P450 isozymes.A dietary manipulation of nutritional status may increasethe levels of a certain group of P450s but decrease those ofothers. Therefore, depending on the substrates used in thestudy, opposite effects on drug metabolism may be observed.

Protein

Diets with low protein content or lower quality of proteinusually result in lower rates of xenobiotic metabolism thana normal diet. This effect was observed in human volunteersconcerning the metabolism of antipyrine and theophylline(2, 4) and in animals with a variety of xenobiotics (25). TheP450 enzymes may be affected because protein synthesis isretarded under protein deficiency conditions. However, theeffect on xenobiotic metabolism may be observed even withoutcommon signs of nutritional protein deficiency. It is possiblethat dietary protein level influences the physiological state,such as hormone levels, which affect the level of P450 enzymes.

Lipid and carbohydrate

In comparison to a fat-free diet, feeding a 3-10% corn oildiet to rats caused an increase in the microsomal metabolismof a variety of substrates, including aminopyrine, ethyl-

morphine, hexobarbital, heptachlor, BP, NDMA, and aniline(8, 26, 27). Generally, fat levels are important in affectingP450 levels, and those rich in polyunsaturated fatty acids aremore effective than those rich in saturated and monounsatu-rated fatty acids. Dietary lipids are also essential in produc-ing an optimal induction of P450 enzymes by inducers suchas phenobarbital, and the effect is observable at the mRNAlevel (27). Although most studies suggested that the factorsresponsible for the increased drug metabolism were lipids,the lipid-to-carbohydrate ratio may be important. Othercomponents or contaminants in oils such as vitamin E, cho-lesterol, and lipid peroxides may have also been contributingfactors in certain experiments (26).

Recent results from our laboratory indicate that, in com-parison to a fat-free diet, a 20% corn oil diet produced atwofold higher constitutive level of P450 2E1 but did notaffect the maximal acetone-inducible level of this enzyme(27). When diets containing different amounts of corn oilwere fed to rats, those on the higher fat diet had higher P4502E1 levels and higher blood acetone levels (27), consistentwith the hypothesis that ketone bodies and ketosis are keyfactors for the regulation of P450 2E1 (28). Menhaden oiland corn oil were more effective than olive oil and lard inmaintaining high levels of P450 2E1, suggesting that factorsother than ketosis were involved. The total concentration ofmicrosomal P450 was higher in rats on a diet with higherlipid content; specifically, P450s 3A and 2A1 were higher,whereas 2B1 and 2C11 were not affected (29).

Carbohydrates are usually used as variable caloric sourcesfor isocaloric diets in studying the effects of dietary proteinsor lipids on xenobiotic metabolism. The higher P450 levelsobserved with higher protein or lipid diets have usually beenattributed to the protein or lipids. However, metabolic glu-cose deprivation, seen in cases of fasting and diabetes,caused induction of P450 2E1, possibly due to the ketoticconditions produced. Sucrose, glucose, or fructose, whengiven in drinking water to mice maintained on a rodent chow,were reported to decrease drug metabolism rates in vivo andin vitro (30). The high sugar intake from the drinking watermay have reduced the dietary intake of protein (or othernutrients) that affected monooxygenase activities. Alterna-tively, a high dietary or blood glucose level may inhibit thesynthesis of P450s due to inhibition of y-aminolevulinic acidsynthesis (31).

Fasting and dietary restriction

During fasting, microsomal aminopyrine N-demethylaseand hexobarbital hydroxylase activities were decreased, butaniline p-hydroxylase and p-nitroanisole O-demethylase ac-tivities were increased in male rats (32). The induction ofP450 2E1 by fasting (17) can account for the increased ani-line hydroxylase and NDMA demethylase activities. Fastingfor 2 or 3 days caused a 50% decrease in the level of themale-specific P450 2C11 (33), and this may account for thepreviously observed decrease in aminopyrine demethylaseactivity. During fasting, the mRNA for P450 2E1 was sig-nificantly elevated, a situation similar to diabetes (34), butsuch elevation was not observed during the induction ofP450 2E1 by acetone pretreatment (19). The results Suggestthat there are differences in the mechanisms of P450 2E1 in-duction under these conditions.

In nutritional studies with experimental animals, pairfeeding is frequently used to allow the animals in the controlgroup eating a quantity of diet equivalent to that consumedby animals in the experimental group. If the pair-fed animalsconsume this limited amount of food in 2 h, usually early in

740 Vol. 6 Ianuarv 1992 Th cAcIR In, ,n.I VAklt CT Al

the morning, this will create a fasting situation of 22 h inwhich increases in P450 2E1 and its activities can be ob-served (35). The practice of overnight fasting before an ex-periment may also cause an increase in the P450 2E1 level.

EFFECT OF ALCOHOL CONSUMPTION ONXENOBIOTIC METABOLISM

Consumption of alcoholic beverages is widespread and canaccount for a significant portion of caloric intake of certainindividuals. Ethanol can affect the absorption, plasma pro-tein binding, blood flow, and distribution of xenobiotics aswell as have profound influence on phase I and phase IImetabolism of xenobiotics (36, 37).

The induction of P450 enzymes by ethanol has long beenpostulated (36). The induction of P450 2E1 has subsequentlybeen demonstrated in animals and humans (38, 39). Thisenzyme catalyzes the metabolism of small molecules such asethanol, other alcohols, NDMA, acetone, aniline, enflurane,ether, acetaminophen, benzene, chloroform, carbon tetra-chloride, dihaloethanes, and other small halogenated hydro-carbons (Fig. 3) (40, 41). It is understandable, therefore, thatchronic consumption of ethanol will increase the rate of themetabolism and the toxicity of the aforementioned com-pounds. On the other hand, the acute effect of alcohol con-sumption is an inhibition of P450 2E1 function due to thecompetitive inhibition by ethanol.

As reviewed by Lieber (36), chronic administration ofethanol increases the rate of the metabolic clearance of avariety of drugs such as meprobamate, pentobarbital,aminopyrine, tolbutamide, propranolol, rifampicin, andtestosterone. As a result of acute ethanol consumption,however, impairment of the metabolism of drugs has beenobserved with meprobamate, benzodiazepines, phenothia-zine, barbiturate, morphine, methadone, warfarin, xylene,and other compounds. The induction and inhibition of P4502E1 may account for only a small portion of these inhibitoryactions. The induction of other P450s such as P450 2B1 andpossible inhibition of the metabolism of these drugs byethanol remain to be studied.

Chronic ethanol administration to rats caused a prolifera-tion of the endoplasmic reticulum of the upper small intes-tine along with increases in P450 content and NADPH:cyto-

SubstratesAcetone,AlcoholsAniline, PyridineBenzene, Phenol, StyreneAlkanesCHCI3, Cd4Vinyl chlorideDihaloethanesTrichloroethyleneN-NitrosodimethylamineN-NitrosodiethylamineAzoxymethanet-ButylhydroperoxideEthersEnflurane, HalothaneChlorzoxazoneAcetaminophen

Figure 3. Dietary effectorsand substrates for P450 2E1.

chrome c reductase activity. In addition, increased intestinalmicrosomal activities of benzphetamine demethylase, 7-ethoxycoumarin deethylase, BP hydroxylase, and anilinehydroxylase have been observed (36). Chronic ethanol con-sumption was also shown to increase P450 content and themetabolic activation of N-nitrosopyrrolidine in rat esopha-gus (42). The extent to which these effects are due to the in-duction of P450 2E1 or its activity remains to be determined.The induction of P450 2E1 in kidney and lung has also beenobserved (19, 43).

EFFECTS OF MICRONUTRIENTS ON XENOBIOTICMETABOLISM

The effects of vitamins, especially vitamin deficiencies, ondrug metabolism have been investigated extensively (2, 5, 8,9). Seemingly conflicting results in older literature may bemore understandable with some new insights on this topic.1) Whereas a general effect of all severe vitamin deficienciesis the decreased metabolic functions and lowered levels ofP450-dependent metabolic activities, mild deficiency in acertain nutrient may enhance P450-dependent activities.Therefore, depending on the severity of the deficiency, oppo-site effects on xenobiotic metabolism may be observed.2) Deficiency in a single nutrient may produce varied effectson the metabolism of different xenobiotics due to the differ-ent effects on specific P450 isozymes. These points are illus-trated in the cases of riboflavin and thiamin deficiencies.

During the development of riboflavin deficiency, there wasa gradual decrease in the activity of NADPH:P450 reductase,possibly due to the lowered levels of cellular FAD and FMN.On the other hand, during early stages of riboflavin deficiency,the levels of some P450s may be increased, possibly to com-pensate for the decreased NADPH:P450 reductase levels;some of the monooxygenase activities such as NDMA de-methylase, aniline hydroxylase, and aminopyrine demethyl-ase activities were elevated. When the deficiency was pro-longed, the level of P450 enzymes decreased, resulting indepressed levels of all P450-dependent activities using sub-strateS such as BP, aminopyrine, ethylmorphine, N-methyl-aniline, aniline, and acetanilide. Therefore, mild deficiencyand severe deficiency had opposite effects on the metabolismof certain drugs and carcinogens (8).

Rats fed a thiamin-deficient diet had higher concentra-tions of P450, cytochrome b5, and NADPH:cytochrome creductase activity in liver microsomes than those fed a dietsufficient in thiamin. The deficient rats also had increasedrates in the metabolism of acetaminophen, NDMA, amino-pyrine, ethylmorphine, zoxazolamine, heptachlor, aniline,N-methylaniline, acetanilide, and BP, but not in the metabo-lism of hexobarbital (8). Recent studies from our laboratoryindicated that thiamin deficiency increased the hepatic micro-somal P450 2E1 level (two- to fivefold) but not the P450 2Clllevel (44). This observation provides an enzymatic basis forthe enhanced rate of in vivo metabolism of aniline, NDMA,and acetaminophen, all of which are substrates for P450 2El.Thiamin deficiency was shown to increase cytosolic glutathi-one S-transferase activity moderately but not steroid isomer-ase activity (44). The mechanisms of these effects on drug-metabolizing enzymes remain to be elucidated.

Mechanistic information on the effects of other micro-nutrients on xenobiotic metabolism is lacking. These effects,together with the aforementioned effects on xenobiotic me-tabolism by riboflavin and thiamin nutrition, are summa-rized in Table 1.

Vitamin A deficiency

Vitamin A-high doseNiacin deficiency

Riboflavin deficiencyMild

SevereThiamin deficiency

Vitamin C deficiency

Vitamin C - high dose

Vitamin E deficiency

Folic acid deficiencyAluminum - high dose

Magnesium deficiencyCopper deficiency

Iron deficiency

Iron-large doseSelenium deficiencyZinc deficiency I Metabolism of pentobarbital, aminopyrine

Abbreviations and signs used: I, decrease; I, increase; reductase, NADPH: P450 reductase activity. Original references can befound in reviews (2, 3, 5, 8, 9).

TABLE 1. Effects of micronutrients on xenobiotic metabolism

DIETARY EFFECTS ON XENORIOTIC MFTAROI 1cM 741

Nutritional status Xcnobiotic metabolism and enzymes

P450Metabolism of aminopyrine, ethylmorphine, aniline, BP, 7-ethoxycoumarin

I Metabolism of aniline, 7-ethoxycoumarinMetabolism of anesthetics

ReductaseI Metabolism of NDMA, aniline, aminopyrine

Metabolism of BP, aminopyrine, ethylmorphine, N-methylaniline, aniline, acetanilide1 P450 2E1, reductase, cytochrome b5I NDMA, acetaminophen, aniline, aminopyrine, ethylmorphine, zoxazolamine, BP

P450, reductaseMonooxygenase activitiesMonooxygenase activitiesMetabolism of codeine, ethylmorphine, BPInduction of P450 2B1 by barbiturates

I Hepatic P450I Metabolism of p-nitrophenole, ethylmorphine

Cadmium, cobalt, and otherheavy metals-high dose 1 P450 and related activities

Calcium deficiency I Monooxygenase activities

I Monooxygenase activitiesI Metabolism of aniline, hexobarbitalI Metabolism of BPI Metabolism of hexobarbital, aminopyrineI Metabolism of anilineI NADPH-dependent lipid peroxidationI Induction of P450 by phenobarbital

DIETARY EFFECTS ON P450TOXICITY

2E1 AND CHEMICAL

In this section, P450 2E1 is used as an example to illustratethe dietary modulation of P450 enzymes and the effect of thismodulation on toxicity and carcinogenicity of chemicals. Asillustrated in Fig. 3, diet may provide inducers, suppressors,inhibitors, and substrates for P450 2El. In addition to affect-ing the hepatic microsomal P450 2E1 level measured in vitro,a low fat/high carbohydrate diet also resulted in a lower rateof enflurane metabolism in rats than a high fat/low carbohy-drate diet (29). Based on these results, it may be suggestedthat individuals on a low fat diet may have, on average, lowerP450 2E1 levels than those on a high fat diet. Frequent con-sumption of ethanol is known to elevate the level of P450 2E1in humans, and is believed to be a key mechanism for the en-hanced toxicity of acetaminophen, CCI,, and other chemi-cals. Fasting can be an important factor in raising P450 2E1levels in individuals undergoing weight reduction by severefasting or a low carbohydrate diet. The induction of P4502E1 by fasting probably also contributes to the enhanced tox-icity of acetaminophen, although other factors such asdecreased glutathione levels are also important.

The activity of P450 2E1 is also modulated by dietary inhi-bitors and suppressors of this enzyme. The consequence ofthis inhibition and suppression of P450 2E1 is the inhibitionof metabolism and toxicity of certain xenobiotics. The inhi-

bition of enflurane metabolism by diallyl sulfide andphenethyl isothiocyanate, which are competitive and suicideinhibitors of P450 2E1, has been demonstrated in rats (un-published results).

The inhibition of carcinogen metabolism in the liver canincrease the carcinogen exposure to nonhepatic organs andthus may enhance nonhepatic carcinogenesis. This effect hasbeen demonstrated with ethanol, which inhibited hepatocar-cinogenesis of NDMA but enhanced tumorigenesis in thenasal cavity (45). The dual effects of ethanol, i.e., an acuteeffect of inhibition and a chronic effect of enhancement ofNDMA-induced carcinogenesis, have also been observed (46).

Knowing that many dietary factors can modulate P450 2E1,it may be questioned whether a higher or lower level of thisenzyme is more beneficial to health. A possible physiologicalfunction of P450 2E1 is in the initial step for the conversionof acetone to glucose (47), but the rate of this metabolic path-way is rather low and its physiological importance remainsto be established. Lowering P450 2E1 levels may thus de-crease the susceptibility to many toxic chemicals, providedthe parent compounds are not toxic and can be disposed ofby other metabolic pathways or by exhalation. One exceptionis the dihaloethanes, which are activated by a glutathione-dependent pathway and detoxified by P450 2E1-dependentmetabolism (41). A second aspect of this question is thatP450 2E1 is known to be effective in causing lipid peroxida-tion (48). It remains to be determined whether higher levels

742 Vol. 6 January 1992 The FASEB lournal YANC FT Al

of P450 2El can contribute to oxidative stress in vivo. Theapplication of dietary-derived P450 2E1 inhibitors and sup-pressors for the prevention of acetaminophen toxicity is be-ing explored in our laboratory. Of particular importance areapplications with alcoholics or patients taking isoniazidwhose P450 2El levels are known to be elevated.

DIETARY EFFECTS ON DRUG METABOLISM,TOXICITY, AND CARCINOGENESIS

Food and dietary components may affect the fate of a drug ortoxicant by the following mechanisms: 1) altering the ratesof its absorption and uptake, 2) reacting or tightly bindingwith the drug, 3) competing with the drug for binding toplasma proteins, and 4) affecting phase I and phase IImetabolism. Interfering with P450-dependent metabolismappears to be the most selective mechanism by which dietarycomponents exert their effects on drug metabolism and car-cinogenesis. For example, the observation that the drinkingof 200 ml of grapefruit juice markedly inhibited the oxida-tion of nifedipine and felodipine in human volunteers (49)may be interpreted on the basis that grapefruit is rich inquercetin and naringenin, which are effective inhibitors ofP450 3A4, the isozyme responsible for the metabolism ofthese dihydropyridine drugs (24). P450 3A4 is also importantin the activation of aflatoxin B, in humans (50). It may bespeculated that grapefruit consumption would inhibit thebioactivation of aflatoxin B, and could inhibit hepatocar-cinogenesis in populations exposed to rather high concentra-tions of this carcinogen.

Indoles and isothiocyanates are two major classes of com-pounds that occur as glucosinolates in cruciferous vegeta-bles. The actions of indole-3-carbinol and phenethyl isothio-cyanate may account for some of the reported inhibitoryactions of cruciferous vegetables against chemically inducedcarcinogenesis in animals and for the association betweenthe frequent consumption of these vegetables with lowercancer incidences at different organ sites (51). The inductionof P450 1A1 in the intestine may help to metabolize andeliminate dietary polyaromatic hydrocarbons in the intes-tine, and thus may reduce the exposure of internal organs tosuch carcinogens. In studies of aflatoxin B1-induced hepato-carcinogenesis in the trout, the inhibitory action of the acidreaction products of indole-3-carbinol (RXM) on the meta-bolic activation of aflatoxin B, may be a major mechanismfor the inhibition of carcinogenesis by indole-3-carbinol (52).On the other hand, when indole-3-carbinol was given afterthe aflatoxin B, treatment period, it enhanced carcinogenesisin the trout (53); the mechanisms are not known.

It has also been demonstrated that indole-3-carbinol,when incorporated into the diet, increased the estradiol2-hydroxylase activity in rats and humans (54), possibly dueto the induction of P450s lAl and 1A2. Because 2-hydroxyl-ation converts estradiol to nonuterotropic and antiestrogenicmetabolites, the increase in this metabolic activity was sug-gested to reduce the incidence of estrogen-related cancers.

Phenethyl isothiocyanate was a very potent inhibitor ofNNK-induced lung tumorigenesis (55) and N-nitrosometh-ylbenzylamine-induced esophageal carcinogenesis (56). Theaction can be attributed largely to its inhibition of carcino-gen activation (23). Probably via a similar mechanism, di-allyl sulfide was also an effective inhibitor in both carcino-genesis models (unpublished results; ref 57). Diallyl sulfideand related organosulfur compounds may be partially re-sponsible for the negative association between the consump-tion of allium vegetables and incidence of gastric cancer (58).

The amount of diallyl sulfide derived from garlic is only inquantities of 3-100 ftg/g. The estimated human intake ofglucosinolates through consumption of cooked vegetables isabout 30 mg per day. It may be questioned whether suchsmall quantities of dietary inhibitors can have a significanteffect in inhibiting carcinogenesis. Two aspects may be perti-nent to this question: 1) Many dietary compounds may becompetitive inhibitors of P450 enzymes; even when presentat low concentrations, they could effectively inhibit themetabolism of low concentrations of carcinogens. 2) Manydietary chemicals can inhibit carcinogen activation. Al-though most of them are present only in small concentra-tions, in combination their actions can be significant.

The human diet is also known to contain mutagens, carcin-ogens, and tumor promoters. The effects of these compoundson health have to be considered in light of the capabilityof the body to detoxify these dietary chemicals. Phase IImetabolism, which is usually not considered in most muta-genesis assays in vitro, may be of particular importance. Itis also important to consider the doses of these chemicals re-quired to produce the possible harmful effects. Dependingon the dose, a compound can have either beneficial or harm-ful effects, and sometimes the effects depend on the experi-mental model used. This point can be illustrated with thecommonly used food additives, butylated hydroxyanisole(BHA) and butylated hydroxytoluene (BHT). These antiox-idants may be present at a total concentration of up to 0.02%in certain food items. When added to the diet at a concentra-tion of 0.5%, BHA and BHT inhibited carcinogenesis inseveral animal models (59). Induction of phase II enzymesand inhibition of carcinogen activation have been proposedas the mechanisms of inhibition (8, 59). However, with 1 or2% of BHT or BHA in the diet, respectively, tumor promo-tion activity has been demonstrated in a two-stage urinarybladder carcinogenesis model (60).

CONCLUDING REMARKS

Studies of the multiplicity and substrate specificity of P450isozymes have contributed to our understanding of dietaryeffects on xenobiotic metabolism. A dietary chemical may in-crease the level of certain P450s and decrease the level ofothers; thus the rates of the metabolism of certain drugs maybe enhanced and those of others lowered. The distinction be-tween an inhibitory effect after an acute dose and an induc-tion effect after a treatment also helps to explain the diver-gent effects of dietary chemicals on drug metabolism. Inaddition, a nutritional deficiency may have different effectson the metabolism of a certain drug; the rate may be en-hanced in mild deficiency but decreased in severe deficiency.

Most of the studies reviewed herein were carried out usingliver microsomes from rats and mice. These results can pro-vide us with some basic understanding of the mechanisms bywhich a dietary factor may affect drug metabolism. Cautionmust be applied when extrapolating the information ob-tained from hepatic tissues to nonhepatic tissues and fromanimals to humans. With the understanding of human xeno-biotic metabolism as a goal, researchers are faced with thefollowing challenges: 1) to further elucidate the detailedmechanisms by which diet affects xenobiotic-metabolizingenzymes, 2) to understand the basis for the tissue and speciesspecificities of xenobiotic-metabolizing enzymes, 3) to furthercharacterize the catalytic properties of human xenobiotic-metabolizing enzymes, and 4) to pursue well-planned hu-man studies concerning the nutritional impact on drugmetabolism and toxicity.

DIETARY EFFECTS ON XENOBIOTIC METABOLISM 743

This work was supported by National Institutes of Health grantsES03938, CA46535, and CA37037, a grant from the American In-stitute for Cancer Research, and NIEHS Center grant ES05022.The authors wish to thank Ms. Dorothy Wong for her excellentsecretarial assistance and Ms. Marie Leithauser for helping to pre-pare the manuscript.

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