Environmental Toxicology

and toxicity CHUNG S. YANG,’ JOHN F. BRADY,2 AND JUN-YAN HONG

Laboratory for Cancer Research, Department of Chemical Biologyand 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 cytochrome P450 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 xenobiotic metabolism are reviewed in the light of our current un- derstanding of the multiplicity and substrate specificity of cytochrome P450 enzymes. Although the mechanisms of action of several dietary chemicals on specific cyto- chrome P450 isozymes have been established, those for macro- and micronutrients are largely unknown. It is known, however, that specific nutrients may have varied effects on different cytochrome P450 forms and thus may affect 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, enhanced rates of metabolism of xenobiotics were observed. The effects of dietary modulation of xenobiotic metabolism on chemical toxicity and carcinogenicity are discussed. -Yang, C. S.; Brady, J. F.; Hong, J.-Y. Dietary effects on cytochromes 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 develop xenobiotic-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 years ago may correspond to the advance of animals onto land where they encountered new terrestrial plants and phyto- chemicals. The work of many investigators in the past 30 years has clearly established that various dietary factors have marked effects on the metabolism of drugs, environmental chemicals, and certain endogenous substrates. This topic has been reviewed extensively (2-9). However, only recently have we begun to understand some of these effects at the molecu- lar level. Dietary influences on xenobiotic metabolism may alter the therapeutic effects of drugs and the toxicity or car- cinogenicity of environmental chemicals. In this article, we review the mechanisms by which dietary chemicals and nutritional status affect the levels and activities of P450 en- zymes, xenobiotic metabolism, as well as chemical toxicity and carcinogenicity.

Because of space limitations, we have chosen to use selected examples to illustrate key concepts rather than to conduct an exhaustive review on this topic. Review articles are cited instead of original papers.

MODULATION OF P450 LEVELS AND XENOBIOTIC METABOLISM BY NONNUTRITIVE DIETARY CHEMICALS

Dietary chemicals may affect the levels and activities of P450 isozymes at different steps as shown inFig. 1. Dietary chem- icals may affect the levels of P450 species by altering the rates of: 1) the transcription of specific P450 genes,2) the degra- dation of specific mRNA, 3) the translation process, and 4) P450 degradation through protein turnover or by suicide inhibition. Many dietary chemicals are substrates of the P450-dependent monooxygenase system. They or their me- tabolites may inhibit or enhance the activities of this system by binding to P450 species or to NADPH:P450 reductase, by affecting 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 of Wattenberg (10), who discovered that rats on commercial rat chow had 68-fold higher intestinal benzo(a)pyrene (BP) hydroxylase activity than those on a purified diet. By adding dry 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 of cruciferous vegetables and their components on drug metab- olism have since been studied extensively. Three autolytic products 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. The structures of some of the nonnutritive dietary compounds are illustrated in Fig. 2. Indole-3-carbinol is the most potent inducing agent among these indoles. Its induction of BP hydroxylase and ethoxyresorufin dealkylase activities is be- lieved to be mainly due to the induction of P450 1A1 in the intestine 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: Laboratory for 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 reaction products of indole-3-carbinol; NDMA, N-nitrosodimethylamine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; BHA, butyl- ated hydroxyanisole; B HT, butylated hydroxytoluene.

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P450 gene Inactivation of P450 enzymes

degradation P450 mRNA

Substrates

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Flavone Tange,otin Nob,I.bn

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Figure 2. Structures of some dietary or related compounds.

738 Vol. 6 lanuarv 1992 The FASEBJournal YANC FT At

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transcription

translation

degradation .( P450 Reductase

Inactivation Inhibiton Enhancement

Figure 1. Possible sites for dietary effects onP450 enzymes. The

P450 2E1 gene is used in the illustration. In addition to affecting the rates of transcription and translation, dietary chemicals may affect the rates of degradation of P450 mRNA and protein. Dietary chemicals may serve as substrates. They may also interact with P450 enzymes and NADPH:P450 reductase, causing inactivation of P450, or inhibition or enhancement of the monooxygenase ac- tivities.

When diallyl sulfide was given orally to rats, microsomal N-

nitrosodimethylamine (NDMA) demethylase (indicative of P450 2E1 activity) decreased markedly in several hours. This was followed by a lowering of the immunodetectable P450 2El protein level in microsomes (15). The inactivation of P450 2E1 was also demonstrated in vitro when diallyl sul- fone, an oxidative metabolite of diallyl sulfide, was incubated with microsomes in the presence of an NADPH-generating system (15). The inactivation was time, concentration, and NADPH-dependent, following a typical suicide inhibition pattern. It is believed that diallyl sulfone is converted by P450 2E1 to a reactive intermediate that modifies the heme moiety of P450 2E1 (20). Phenethyl isothiocyanate, occurring as a glucusinolate in a variety of cruciferous vegetables, also inactivates 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 and BP hydroxylase activities when added to human liver microsomal incubations in the presence of NADPH (22). It was 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 the binding of the inducer to the Ah receptor. Nevertheless, indole-3-carbinol has only low binding affinity to the Ah receptor, whereas indolo[3,2-b]carbazole has a much higher affinity (12). It is suggested that under the acidic conditions in the stomach, indole-3-carbinol can be converted to indolo[3,2-b]carbazole or other acid reaction products (RXM) (13) that bind to the Ah receptor and thereby increase the transcription of the P450 1A1 gene. This suggestion is supported by the observation that the inductive effect was found when indole-3-carbinol was given to rats orally but not when 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 hepatic P450 2B1 based on the increase of immunodetectable protein and pentoxyresorufin dealkylase activity (15). Recentstudies in our laboratory indicate that this induction is accompanied by the elevation of 2B1 mRNA levels. Nuclear run-on experi- ments indicate that after administration of diallyl sulfide, the transcriptional rate of the P450 2B1 gene was increased markedly, and was observable at 4 h after treatment (16). In primary 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 in the induction of P450s by dietary factors. In the induction of P450 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 a role in this induction. In the induction of P450 2E1 by ethanol and acetone, elevation of the mRNA was not ob- served (18, 19). Protein stabilization or increased translation efficiency may be involved in this induction.

Enhancement of monooxygenase activities

Flavone, tangeretin, and nobiletin were found to increase BP hydroxylase activity and aflatoxin B, activation in human liver microsomes (4). The metabolism of antipyrine and zoxazolamine was also stimulated, but that of hexobarbital, coumarin, and 7-ethoxycoumarin was not. The stimulatory effect was P450 isozyme-specific. With purified P450 iso- zymes in a reconstituted system, the effect was observed with rabbit P450s 3A6 and 1A2 but not with P450s 2B4, 2C3, or 1AI. It was proposed that these flavonoids stimulate the monooxygenase activity by enhancing the interactions be-

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tween P450 and NADPH:P450 reductase and facilitating the flow 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. For example, diallyl sulfide and phenethyl isothiocyanate are competitive 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 the enzyme during the incubation, and P450 2Elwas 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, and naringenin inhibited nifedipen and filodipen oxidation cata- lyzed by P450 3A4 in human microsomes (24). It appears that flavones having free hydroxy groups on the A ring are inhibitors, whereas flavonoids containing no hydroxy groups (flavone, tangeretin, and nobiletin) are activators (stimula- tors) of selected monooxygenase activities.

EFFECTS OF MACRONUTRIENTS ON XENOBIOTIC METABOLISM

Compared with the effects of nonnutritive dietary chemicals, the effects of macronutrients on drug metabolism are more difficult to understand. In the former case, the actions of a compound or its metabolites can be studied, but in the latter case we have to deal with a nutritional state or status. There- fore, despite extensive investigations, our understanding of the latter subject is mostly at the descriptive level. Only re- cently have researchers begun to look at specific changes on selected 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 increase the levels of a certain group of P450s but decrease those of others. Therefore, depending on the substrates used in the study, opposite effects on drug metabolism may be observed.

Protein

Diets with low protein content or lower quality of protein usually result in lower rates of xenobiotic metabolism than a normal diet. This effect was observed in human volunteers concerning the metabolism of antipyrine and theophylline (2, 4) and in animals with a variety of xenobiotics (25). The P450 enzymes may be affected because protein synthesis is retarded under protein deficiency conditions. However, the effect on xenobiotic metabolism may be observed even without common signs of nutritional protein deficiency. It is possible that 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 oil diet to rats caused an increase in the microsomal metabolism of a variety of substrates, including aminopyrine, ethyl-

morphine, hexobarbital, heptachlor, BP, NDMA, and aniline (8, 26, 27). Generally, fat levels are important in affecting P450 levels, and those rich in polyunsaturated fatty acids are more 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 such as phenobarbital, and the effect is observable at the mRNA level (27). Although most studies suggested that the factors responsible for the increased drug metabolism were lipids, the lipid-to-carbohydrate ratio may be important. Other components or contaminants in oils such as vitamin E, cho- lesterol, and lipid peroxides may have also been contributing factors 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 a twofold higher constitutive level of P450 2E1 but did not affect the maximal acetone-inducible level of this enzyme (27). When diets containing different amounts of corn oil were fed to rats, those on the higher fat diet had higher P450

2E1 levels and higher blood acetone levels (27), consistent with the hypothesis that ketone bodies and ketosis are key factors for the regulation of P450 2E1 (28). Menhaden oil and corn oil were more effective than olive oil and lard in maintaining high levels of P450 2E1, suggesting that factors other than ketosis were involved. The total concentration of microsomal P450 was higher in rats on a diet with higher lipid content; specifically, P450s 3A and 2A1 were higher, whereas 2B1 and 2C11 were not affected (29).

Carbohydrates are usually used as variable caloric sources for isocaloric diets in studying the effects of dietary proteins or lipids on xenobiotic metabolism. The higher P450 levels observed with higher protein or lipid diets have usually been attributed 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 ketotic conditions produced. Sucrose, glucose, or fructose, when given in drinking water to mice maintained on a rodent chow, were reported to decrease drug metabolism rates in vivo and in vitro (30). The high sugar intake from the drinking water may have reduced the dietary intake of protein (or other nutrients) that affected monooxygenase activities. Alterna- tively, a high dietary or blood glucose level may inhibit the synthesis of P450s due to inhibition of y-aminolevulinic acid synthesis (31).

Fasting and dietary restriction

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

In nutritional studies with experimental animals, pair feeding is frequently used to allow the animals in the control group eating a quantity of diet equivalent to that consumed by animals in the experimental group. If the pair-fed animals consume this limited amount of food in 2 h, usually early in

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

the morning, this will create a fasting situation of 22 h in

which 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 ON

XENOBIOTIC METABOLISM

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

The induction of P450 enzymes by ethanol has long been postulated (36). The induction of P450 2E1 hassubsequently

been demonstrated in animals and humans (38, 39). This enzyme catalyzes the metabolism of small molecules such as

ethanol, 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, that chronic consumption of ethanol will increase the rate of the metabolism 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 the competitive inhibition by ethanol.

As reviewed by Lieber (36), chronic administration of ethanol increases the rate of the metabolic clearance of a variety of drugs such as meprobamate, pentobarbital,

aminopyrine, tolbutamide, propranolol, rifampicin, and

testosterone. As a result of acute ethanol consumption, however, impairment of the metabolism of drugs has been observed with meprobamate, benzodiazepines, phenothia- zine, barbiturate, morphine, methadone, warfarin, xylene, and other compounds. The induction and inhibition of P450 2E1 may account for only a small portion of these inhibitory actions. The induction of other P450s such as P450 2B1 and possible inhibition of the metabolism of these drugs by ethanol 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-

Substrates Acetone,Alcohols Aniline, Pyridine Benzene, Phenol, Styrene Alkanes CHCI3, Cd4 Vinyl chloride Dihaloethanes Trichloroethylene N-Nitrosodimethylamine N-Nitrosodiethylamine Azoxymethane t-Butylhydroperoxide Ethers Enflurane, Halothane Chlorzoxazone Acetaminophen

Figure 3. Dietary effectorsand substrates for P450 2E1.

chrome c reductase activity. In addition, increased intestinal microsomal activities of benzphetamine demethylase, 7-

ethoxycoumarin deethylase, BP hydroxylase, and aniline

hydroxylase have been observed (36). Chronic ethanol con- sumption was also shown to increase P450 content and the metabolic 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 been

observed (19, 43).

EFFECTS OF MICRONUTRIENTS ON XENOBIOTIC METABOLISM

The effects of vitamins, especially vitamin deficiencies, on drug metabolism have been investigated extensively (2, 5, 8,

9). Seemingly conflicting results in older literature may be more understandable with some new insights on this topic. 1) Whereas a general effect of all severe vitamin deficiencies is the decreased metabolic functions and lowered levels of P450-dependent metabolic activities, mild deficiency in a certain 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 effects on 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 was a 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 in depressed levels of all P450-dependent activities using sub- strateS such as BP, aminopyrine, ethylmorphine, N-methyl- aniline, aniline, and acetanilide. Therefore, mild deficiency and severe deficiency had opposite effects on the metabolism of certain drugs and carcinogens (8).

Rats fed a thiamin-deficient diet had higher concentra-

tions of P450, cytochrome b5, and NADPH:cytochrome c reductase activity in liver microsomes than those fed a diet

sufficient in thiamin. The deficient rats also had increased rates 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 laboratory indicated that thiamin deficiency increased the hepatic micro- somal P450 2E1 level (two- to fivefold) but not the P450 2Cll level (44). This observation provides an enzymatic basis for the 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.

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Vitamin A deficiency

Vitamin A-high dose

Niacin deficiency

Riboflavin deficiency

Mild

Severe

Thiamin deficiency

Vitamin C deficiency

Vitamin C - high dose

Vitamin E deficiency

Folic acid deficiency

Aluminum - high dose

Magnesium deficiency

Copper deficiency

Iron deficiency

Iron-large dose

Selenium deficiency

Zinc deficiency I Metabolism of pentobarbital, aminopyrine

Abbreviations and signs used: I, decrease; I, increase; reductase, NADPH: P450 reductase activity. Original references can be found 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

P450 Metabolism of aminopyrine, ethylmorphine, aniline, BP, 7-ethoxycoumarin

I Metabolism of aniline, 7-ethoxycoumarin

Metabolism of anesthetics

Reductase I Metabolism of NDMA, aniline, aminopyrine

Metabolism of BP, aminopyrine, ethylmorphine, N-methylaniline, aniline, acetanilide

1 P450 2E1, reductase, cytochromeb5 I NDMA, acetaminophen, aniline, aminopyrine, ethylmorphine, zoxazolamine, BP

P450, reductase Monooxygenase activities

Monooxygenase activities

Metabolism of codeine, ethylmorphine, BP

Induction of P450 2B1 by barbiturates

I Hepatic P450 I Metabolism of p-nitrophenole, ethylmorphine

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

Calcium deficiency I Monooxygenase activities

I Monooxygenase activities

I Metabolism of aniline, hexobarbital I Metabolism of BP

I Metabolism of hexobarbital, aminopyrine I Metabolism of aniline

I NADPH-dependent lipid peroxidation

I Induction of P450 by phenobarbital

DIETARY EFFECTS ON P450 TOXICITY

2E1 AND CHEMICAL

In this section, P450 2E1 is used as an example to illustrate the dietary modulation of P450 enzymes and the effect of this modulation on toxicity and carcinogenicity of chemicals. As illustrated 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 rate of enflurane metabolism in rats than a high fat/low carbohy- drate diet (29). Based on these results, it may be suggested that individuals on a low fat diet may have, on average, lower P450 2E1 levels than those on a high fat diet. Frequent con- sumption of ethanol is known to elevate the level of P450 2E1 in 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 2E1 levels in individuals undergoing weight reduction by severe fasting or a low carbohydrate diet. The induction of P450 2E1 by fasting probably also contributes to the enhanced tox- icity of acetaminophen, although other factors such as decreased glutathione levels are also important.

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

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

The inhibition of carcinogen metabolism in the liver can increase the carcinogen exposure to nonhepatic organs and thus may enhance nonhepatic carcinogenesis. This effect has been demonstrated with ethanol, which inhibited hepatocar- cinogenesis of NDMA but enhanced tumorigenesis in the nasal cavity (45). The dual effects of ethanol, i.e., an acute effect of inhibition and a chronic effect of enhancement of NDMA-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 this enzyme is more beneficial to health. A possible physiological function of P450 2E1 is in the initial step for the conversion of acetone to glucose (47), but the rate of this metabolic path- way is rather low and its physiological importance remains to be established. Lowering P450 2E1 levels may thus de- crease the susceptibility to many toxic chemicals, provided the parent compounds are not toxic and can be disposed of by other metabolic pathways or by exhalation. One exception is the dihaloethanes, which are activated by a glutathione- dependent pathway and detoxified by P450 2E1-dependent metabolism (41). A second aspect of this question is that P450 2E1 is known to be effective in causing lipid peroxida- tion (48). It remains to be determined whether higher levels

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

of P450 2El can contribute to oxidative stress in vivo. The application 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 are applications with alcoholics or patients taking isoniazid whose 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 or toxicant by the following mechanisms: 1) altering the rates of its absorption and uptake, 2) reacting or tightly binding with the drug, 3) competing with the drug for binding to plasma proteins, and 4) affecting phase I and phase II metabolism. Interfering with P450-dependent metabolism appears to be the most selective mechanism by which dietary components exert their effects on drug metabolism and car- cinogenesis. For example, the observation that the drinking of 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 in quercetin and naringenin, which are effective inhibitors of P450 3A4, the isozyme responsible for the metabolism of these dihydropyridine drugs (24). P450 3A4 is also important in the activation of aflatoxin B, in humans (50). It may be speculated that grapefruit consumption would inhibit the bioactivation 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 inhibitory actions of cruciferous vegetables against chemically induced

carcinogenesis in animals and for the association between the frequent consumption of these vegetables with lower cancer incidences at different organ sites (51). The induction of P450 1A1 in the intestine may help to metabolize and eliminate dietary polyaromatic hydrocarbons in the intes- tine, and thus may reduce the exposure of internal organs to such carcinogens. In studies of aflatoxin B1-induced hepato- carcinogenesis in the trout, the inhibitory action of the acid reaction products of indole-3-carbinol (RXM) on the meta- bolic activation of aflatoxin B, may be a major mechanism for the inhibition of carcinogenesis by indole-3-carbinol (52). On the other hand, when indole-3-carbinol was given after the aflatoxin B, treatment period, it enhanced carcinogenesis in the trout (53); the mechanisms are not known.

It has also been demonstrated that indole-3-carbinol,

when incorporated into the diet, increased the estradiol 2-hydroxylase activity in rats and humans (54), possibly due to the induction of P450s lAl and 1A2. Because 2-hydroxyl- ation converts estradiol to nonuterotropic and antiestrogenic metabolites, the increase in this metabolic activity was sug- gested to reduce the incidence of estrogen-related cancers.

Phenethyl isothiocyanate was a very potent inhibitor of NNK-induced lung tumorigenesis (55) and N-nitrosometh- ylbenzylamine-induced esophageal carcinogenesis (56). The action 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 sulfide and 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 in quantities of 3-100 ftg/g. The estimated human intake of glucosinolates through consumption of cooked vegetables is about 30 mg per day. It may be questioned whether such small quantities of dietary inhibitors can have a significant effect in inhibiting carcinogenesis. Two aspects may be perti- nent to this question: 1) Many dietary compounds may be competitive inhibitors of P450 enzymes; even when present at low concentrations, they could effectively inhibit the metabolism of low concentrations of carcinogens. 2) Many dietary 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 compounds on health have to be considered in light of the capability of the body to detoxify these dietary chemicals. Phase II metabolism, which is usually not considered in most muta- genesis assays in vitro, may be of particular importance. It

is also important to consider the doses of these chemicals re- quired to produce the possible harmful effects. Depending on 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 the commonly 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 in several animal models (59). Induction of phase II enzymes and inhibition of carcinogen activation have been proposed as the mechanisms of inhibition (8, 59). However, with 1 or 2% of BHT or BHA in the diet, respectively, tumor promo- tion activity has been demonstrated in a two-stage urinary bladder carcinogenesis model (60).

CONCLUDING REMARKS

Studies of the multiplicity and substrate specificity of P450 isozymes have contributed to our understanding of dietary

effects on xenobiotic metabolism. A dietary chemical may in- crease the level of certain P450s and decrease the levelof others; thus the rates of the metabolism of certain drugs may be 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. In addition, a nutritional deficiency may have different effects on 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 using liver microsomes from rats and mice. These results can pro- vide us with some basic understanding of the mechanisms by which a dietary factor may affect drug metabolism. Caution must be applied when extrapolating the information ob- tained from hepatic tissues to nonhepatic tissues and from animals to humans. With the understanding of human xeno-

biotic metabolism as a goal, researchers are faced with the following challenges: 1) to further elucidate the detailed mechanisms by which diet affects xenobiotic-metabolizing enzymes, 2) to understand the basis for the tissue and species specificities of xenobiotic-metabolizing enzymes, 3) to further characterize the catalytic properties of human xenobiotic- metabolizing enzymes, and 4) to pursue well-planned hu- man studies concerning the nutritional impact on drug metabolism and toxicity.

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DIETARY EFFECTS ON XENOBIOTIC METABOLISM 743

This work was supported by National Institutes of Health grants ES03938, 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 excellent secretarial assistance and Ms. Marie Leithauser for helping to pre-

pare the manuscript.

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