A role for dietary lipids and antioxidants in the activation of carcinogens

Free Radical Biology & Medicine, Vol. 5, pp. 95-111, 1988 Printed in the USA. All rights reserved.

0891-5849/88 $3.00+ .00 © 1988 Pergamon Press plc

Review Article A ROLE FOR DIETARY LIPIDS AND ANTIOXIDANTS IN THE ACTIVATION OF CARCINOGENS JON D. GOWER Division of Comparative Medicine, Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ, U.K. (Received 5 August 1987; Accepted 21 October 1987)

Abstract--The ways in which dietary polyunsaturated fats and antioxidants affect the balance between activation and detoxification of environmental precarcinogens is discussed, with particular reference to the polycyclic aromatic hydrocarbon benzo(a)pyrene. The structure and composition of membranes and their susceptibility to peroxidation is dependent on the polyunsaturated fatty acid (PUFA) content of the cell and its antioxidant status, both of which are determined to a large degree by dietary intake of these compounds. An increase in the PUFA content of membranes stimulates the oxidation of precarcinogens to reactive intermediates by affecting the configuration and induction of membrane-bound enzymes (e.g., the mixed-function oxidase system and epoxide hydratase); providing increased availability of substrates (hydroperoxides) for peroxidases that cooxidise carcinogens (e.g., prostaglandin synthetase and P-450 peroxidase); and increasing the likelihood of direct activation reactions between peroxyl radicals and precarcinogens. Antioxidants, on the other hand, protect against lipid peroxidation, scavenge oxygenderived free radicals and reactive carcinogenic species. In addition some synthetic antioxidants exert specific effects on enzymes, which results in increased detoxification and reduced rates of activation. The balance between dietary polyunsaturated fats, antioxidants and the initiation of carcinogenesis is discussed in relation to animal models of chemical carcinogenesis and the epidemiology of human cancer. Keywords--Lipids, Antioxidants, Carcinogens, Benzo(a)pyrene, Diet

INTRODUCTION

the presence of direct-acting carcinogens and their precursors in food, or the production of these during food preparation, it is also evident that there are both microand macro-constituents present in the diet which influence the likelihood o f cancer caused by other agents. Evidence for these modifying agents, which can either be protective or may increase cancer risk, is so strong that scientific bodies such as the National A c a d e m y of Sciences U S A and the European Organization o f Cancer Prevention Studies, have drawn up a series o f dietary guidelines. 3,4 These include recommendations to decrease the amount of fat in the diet as this is associated with increased cancer incidence, particularly of the intestine and the breast, and to ensure an adequate intake of vitamins and minerals, some of which appear to have a protective role. Evidence, however, does not support the current faddism that all food additives are bad for you and that megadoses of vitamins guarantee protection from the harmful effects of the environm e n t - - i n fact the reverse may actually be true in some circumstances. Carcinogenesis involves an initiating event followed

Since Percival Pott found an unusually high incidence of skin and scrotal cancers among chimney sweeps in 1775,1 it has become increasingly clear that a considerable number of cancers, perhaps up to 90%, have important environmental factors in their etiologies. 2 It is these factors that are thought to be responsible for wide differences in the incidence of various types of cancer between countries. It has been concluded that dietary factors exert the greatest environmental influence on carcinogenesis and Doll and Peto have estimated that diet is responsible for approximately 35% o f the total cancer deaths in the USA. 2 In addition to Jon Gower was born in London and is 30 years old. After obtaining a biochemistry degree at Bristol University, he worked with Professor Eric Wills in the biochemistry department at the Medical College of St. Bartholomew's Hospital, London. He completed his PhD in 1982 on the effects of dietary lipids on the metabolism of benzo(a)pyrene in the intestine. Jon Gower is now with the Surgical Research Group at the Clinical Research Centre in Harrow and is investigating the role of free radicals in the pathogenesis of ischaemic damage. 95

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by promotion, and the ways in which dietary lipids and antioxidants might influence these stages are likely to be highly dependent upon the type and site of the tumour and the initiating carcinogen. Possible mechanisms include effects on hormonal 5 and immune status, 6 changes in the levels of endogenous promotors, 7 alterations in intestinal microflora 7 and effects on the nutritional requirements of the tumour. 8 Many studies have demonstrated that dietary fat can exert profound effects on the promotional phase of carcinogenesis, that is, the period following the administration of the carcinogenic agent. In addition there is substantial evidence that dietary lipids and antioxidants may affect the initiation of carcinogenesis by influencing the nature of the chemical initiator (Figure 1). The majority of environmental agents are not directly carcinogenic but must first be converted to reactive electrophilic derivatives which modify DNA. 9 This activation process is therefore obligatory for these agents to initiate carcinogenesis, and the effectiveness of such agents will depend on the balance between those processes that activate the xenobiotic and those that are responsible for its detoxification. This article will concern itself purely with the ways in which dietary fats and antioxidants can alter this balance (Figure 1). The polycyclic aromatic hydrocarbon benzo(a)pyrene (BP) is a widely occurring environmental pollutant formed by the combustion of organic materials including cigarettes. It is present in smoked foods and as a contaminant in a wide range of crops, particularly vegetables. 10It induces cancer of the skin, respiratory tract and stomach in animals and may be a cause of cancer in humans. A considerable amount of investigation has been directed towards this compound and its requirement for metabolic activation and it will therefore be

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Fig. 2. Activation and detoxification pathways of benzo(a)pyrene.

used predominantly in this review as an example of the wide range of precarcinogenic compounds to which we are exposed. The activation of BP, which has been described in detail in several reviews,~'12 is summarised in Figure 2. The initial step is the formation of BP-7,8-epoxide by the mixed-function oxidase system (MFO), which is present in the endoplasmic reticulum of virtually all mammalian tissues. It consists of an electron transport system, the terminal oxidases of which are a group of closely related cytochromes known generally as cytochrome P-450. Two further metabolic steps result in the formation of BP-7,8-diol, 9,10-epoxides (BPDEs), which bind to DNA very strongly, are highly mutagenic and carcinogenic and are considered to be the ultimate carcinogenic forms of BP. 13 BPDEs exist in two stereoisomeric f o r m s - - B P D E I, which is the most mutagenic and carcinogenic, and BPDE II. They are very unstable and decompose spontaneously to noncarcinogenic tetrols. Quinones are formed from BP by an MFO-catalysed reaction involving the generation of 6-hydroxy-BP (Fig.

Lipids, antioxidants, and carcinogenactivation 2), and it has been suggested that these metabolites may play a role in BP-induced carcinogenesis.14 BP quinones are readily converted by one electron steps to diols, which are reoxidised in the presence of 02 to quinones via semiquinone radicals and this results in the production of H202. The free radical species involved in this redox cycle are able to induce singlestranded DNA scissions, which may lead to somatic mutation and neoplastic transformation. Recently it has been discovered that the oxidation of a wide variety of xenobiotics including BP can take place by mechanisms which do not involve the MFO system. In 1975, Marnett and coworkers demonstrated the conversion of BP-7,8-diol to BPDEs during the formation of prostaglandins from arachidonic acid by the prostaglandin synthetase system. 15 A fatty acidderived peroxy radical is believed to be responsible for this epoxidation, 16,17 and other peroxidases including horseradish peroxidase 18 the cytochrome P-450 system,19 and lipoxygenase 2° can also catalyse these types of reaction. The formation of the ultimate carcinogenic form of BP from BP-7,8-diol also takes place during lipid peroxidation of rat hepatic 21 and intestinal microsomes 22 initiated by ADP, Fe 2÷ and ascorbate. In addition, mutagenic derivates are formed from BP in the presence of peroxidising polyunsaturated fatty acids 23 and from BP-7,8-diol during the stimulation of polymorphonuclear leukocytes, which involves the production of oxidants by myeloperoxidase.24 The systems described above will also catalyse the formation of BPquinones from BP. 25'15 There are several enzyme systems which convert metabolites of xenobiotics such as BP to conjugates that are inactive and easily excreted. 12 These include glutathione-S-transferases (GSTs), which are a group of closely related enzymes found predominantly in the cytosol, the microsomal enzyme UDP-glucuronyltransferase (UDPGT), and sulphotransferase (ST), a soluble enzyme which catalyses the formation of sulphate conjugates (Fig. 2). Epoxide hydratase, which is closely associated with the MFO system, inactivates reactive epoxides such as BP-4,5-epoxide by hydrating them to dihydrodiols. However, epoxide hydratase catalyses the conversion of BP-7,8-epoxide to the corresponding diol, which is an important step in the activation pathway of BP to BPDEs, and these highly carcinogenic epoxides are poor substrates for this enzyme. Thus, epoxide hydratase may be regarded as an activating enzyme in BP carcinogenesis and several lines of evidence support this assumption. 26 From this brief summary it is evident that there are several possible routes by which precarcinogens such as BP may be activated to true carcinogenic agents and several ways in which they may be detoxified. Some steps necessitate the involvement of enzymes, whereas

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others may also proceed by reaction with species such as peroxy radicals or oxygen-derived free radicals. The relative importances of these different pathways in vivo remain to be established; however, by using the highly stereospecific nature of some of these reactions, progress is being made in this area. 15 It is likely that the rates of various steps in these pathways will be dependent on the nature of the xenobiotic and the tissue in which it is situated, and will also be affected by the level of enzyme induction, oxidative stress, antioxidant potential and peroxide tone of the environment. These factors will in turn be influenced by the nature and quantity of dietary polyunsaturated fats and antioxidants. I. DIETARY FAT

The nature of dietary fat must be considered both in terms of the total quantity of fat ingested and its fatty acid composition. Some polyunsaturated fatty acids (PUFAs) cannot be synthesised, and inclusion of a small proportion of these in the diet is an essential nutritional requirement. Linoleic acid (Cls:2 ~6) is the major essential fatty acid (EFA) and this can be convetted to arachidonic acid (C2o:4), which because it provides the substrate for prostaglandin synthesis, is an important membrane constituent. Linolenic acid (C1s:3 co3) is a minor essential fatty acid, which is poorly incorporated into phospholipids but which is converted to membrane constituents such as C20:5 and C22:6. ~6 fatty acids, particularly C~s:2, are found in abundance in oils produced from cereals such as corn oil and sunflower seed oil, whilst fish oils are rich in the to3 PUFAs especially C20:5 and C22:6. Animal fats such as lard contain smaller proportions of these PUFAs and higher concentrations of monounsaturated and saturated fatty acids such as CIs:I. Other oils such as coconut oil contain very small proportions of unsaturated fatty acids and are rich in saturates such as Cl2:0, C14:0 and C16:0. It is therefore apparent that the intake of dietary fat may vary widely both in terms of quantity and quality and recently Western societies are cutting down on the intake of saturated fats in dairy products and replacing them with polyunsaturated fats in, for example, margarine. Epidemiological data collected for over a decade has associated high levels of dietary fat intake in man with an increased incidence of cancer at a number of sites, particularly the breast and the intestine 2,27,28 and possibly the lung 2s and pancreas. 29 The results from these population studies have stimulated a considerable amount of investigation into the effects of dietary fat in experimental models of carcinogenesis and it is now well-established that the incidence of a wide variety of chemically induced tumours in animals are increased

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when diets containing high levels of fat and especially polyunsaturated fat are given. 3° For instance, in one study only 40% of rats fed a low fat diet (2%) developed dimethylbenzanthracene-(DMBA)-induced mammary tumours whereas tumours were found in 78% of the animals fed a 20% saturated fat diet (18% coconut oil and 2% linoleic acid) and in all the animals fed a highly unsaturated 20% corn oil diet.3~ Polyunsaturated fatty acids other than linoleic acid, such as those found in fish oils (C20:5 and C22:6), are also effective at increasing the number of DMBA-induced mammary tumours 32 although, in another study, rats fed high levels (22.5%) of dietary fish oils developed less azoxymethane-induced colon cancers than those fed equivalent amounts of corn oil or low levels of these oils. 33 An increase in both the number of tumour-bearing animals and the multiplicity of respiratory tract tumours induced by BP has been demonstrated when hamsters were fed high fat diets and this effect was most pronounced in the group fed unsaturated fat (sunflower oil). 34The number of dimethylhydrazine-induced large bowel tumours in rats was also significantly greater when the animals were maintained on a 20% safflower oil diet (high in polyunsaturates) than when diets containing 5% or 20% coconut oil were given. 35 Dietary fat has also been shown to modify the development of pancreatic, 36 skin and liver tumours in experimental a n i m a l s . 31,27

There are a number of ways in which dietary fat may influence the carcinogenicity of environmental agents. Gross nutritional factors involving imbalances in a number of nutrients including fat are known to

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affect carcinogen metabolism 37 and fat may influence the bioavailability of ingested hydrophobic carcinogens. 38 One widely reported observation is that the fatty acid composition of cellular membranes and particularly the PUFA content of the microsomal fraction is highly dependent on the composition of fat in the diet. Thus when rats are removed from normal stock diet and fed for 2 0 - 3 0 days on synthetic diets containing no fat or 10% saturated coconut oil, the microsomal levels of C~8:2, C20:5 and C22:6 fall significantly in both the liver and intestinal mucosa (Figure 3 ) . 39-42 Similarly, feeding corn oil that is rich in C~8:2 (58%) results in an increased incorporation of this fatty acid into these fractions whereas lard (10% C]8:2) is less effective. 42 In the liver, the increased levels of C,8:2 were accompanied by elevations in C20:4 but this fatty acid was more resistant to change in the intestine. 41 Microsomes prepared from the livers of rats fed a highly polyunsaturated cod liver oil or herring oil diet contained significant proportions of C2o:5 and C22:6, which were present only in low or undetectable levels when the other synthetic diets w e r e f e d . 39"42 The microsomal content of C20:5 in the intestinal mucosa was similar to that of the liver, but less C 2 2 : 6 w a s incorporated into the membranes of this tissue. 42 The proportion of C~8:2 decreased to a low level when rats were fed cod liver oil and this demonstrates that to3 PUFAs are incorporated into the endoptasmic reticulum in preference to ~o6 C~8:2 when this fatty acid is in short supply. 42 A good correlation has also been found between the PUFA content of several diets (6% coconut oil, corn

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Fig. 3. The proportion of linoleic acid (C~:2), eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6) in the microsomal fractions of the liver and intestinal mucosa of rats fed diets containing different lipids (10% of diet). Values represent the mean + S.E.M. of 6 determinations.

Lipids, antioxidants, and carcinogenactivation oil and herring oil) and the PUFA composition of microsomal phosphatidylcholines from rat liver. 43 The fatty acid composition of the total lipids of lungs from rats is similarly affected when diets containing corn oil (7%), coconut oil (7%), safflower oil (10%) or butter (10%) are fed. 44 Thus the dependence of PUFA composition of microsomal membranes on the fatty acid composition of our diet appears to be a general e f f e c t - after all we are what we eat! There are two important consequences of this phenomenon that are relevant to the activation of carcinogens. First, changes in the PUFA content of the endoplasmic reticulum result in an alteration of its structure, which may in turn affect the carcinogen metabolising systems that are present in these membranes (discussed below). Secondly, the double bonds of PUFAs are susceptible to attack by free radicals, which results in their peroxidation, and this process may interact with carcinogen activation in a number of ways (see Section III).

A. Carcinogen activation A number of studies have been conducted in which the effect of dietary fat on the activity of the MFO system has been investigated in relation to both the metabolism of drugs and carcinogens. 45 The majority of these studies have been conducted on the liver, which has a high level of MFO activity, and it is now well established that dietary fat is required to maintain both the normal activity of this microsomal enzyme system and its inducibility,aS although the P-450 system of the nuclear envelope seems less affected by diet. 46 When rats are transferred to a fat-free diet, the levels of hepatic microsomal P-450 decrease significantly 46-4gand this is accompanied by decreases in many MFO activities, 45 including the hydroxylation of BP. 49 Furthermore, the inducibility of MFO activities by phenobarbitone 5° and 3-methylcholanthrene(3-MC) 46 is highly impaired when rats are maintained on a fat-free diet. A significant fall of 24% in the rate of BP hydroxylation has also been observed in the intestinal mucosa of rats when the stock diet was replaced by a fat-free d i e t f although the rate of BP hydroxylation in the lung has been reported to be unchanged when a 10% corn oil diet was replaced by a fat-free diet) 1 The addition of fats containing high proportions of C18:2 (such as corn oil and sunflower oil) to fat-free diets restores both the levels of P-450 and the carcinogen metabolising capacity of the hepatic endoplasmic reticulum, 45,49,51,52 as well as the MFO-catalysed metabolism of BP in the rat intestine 41and it also increases the formation of BP-DNA adducts formed by both the microsomal and nuclear fractions of the rat liver) 1 However, highly saturated fats such as coconut oil are

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considerably less effective at maintaining P-450 levels and MFO activity in the liver of rats 5°'52'53 and mice 54 and in the intestinal mucosa of rats.41 Thus a key factor appears to be the availability of essential fatty acids. The amount of dietary fat is also an important determinant. For example, increasing the amount of corn oil in the diet to levels up to 20% (w/w) has been shown to progressively increase P-450 levels and the rates of drug and BP metabolism in the rat liver, 5° P450 levels and BP metabolism in the rat small intestine 4~'55 and P-450 levels in the rat c o l o n ) 5 In addition, increasing the content of sunflower oil (70% C18:2) in the diet from 10 to 30% resulted in elevated rates of aniline hydroxylation and aminopyrine demethylation and increased levels of hepatic microsomal P-450 in young rats) 6 Feeding diets containing fish oils (10%) which are rich in C2o:5 and C22:6 results in higher levels of P-450 in the rat liver compared to animals given the same quantity of c o r n oil 43'47,52 and in high rates of BP hydroxylation in both the rat liver4°,49 and intestinal mucosa. 4J The MFO system is affected only marginally by the geometry of the dietary polyunsaturated f a t s , 45,57 that is to say the ratio of cis-isomers, which occur naturally, to trans-isomers which are produced in variable amounts during the hydrogenation of edible oils for the manufacture of margarine. Taken together, these studies demonstrate that the oxidation of carcinogens catalysed by the MFO system in most tissues studied is dependent upon the presence of dietary EFAs and in particular is stimulated when the dietary content of C18:2, C20:5, and C22:6 is increased. The close correlation between the rate of BP hydroxylation and the microsomal content of C18:2 + C20:5 + C22:6in both rat liver 4° and intestinal mucosa 41 strongly suggests that this effect is mediated by changes in the lipid composition of the endoplasmic reticulum. There is considerable interest in the role of the lipid microenvironment in modulating the activity and specificity of P-450. In vitro studies have shown that membrane integrity is an essential requirement for function of the MFO system and treatment with phospholipases or detergents inactivates this s y s t e m : 8 Reconstitution of P-450 activity requires the presence of phospholipid, and phosphatidylcholine (PC) is particularly effective in this respect whilst phosphatidylethanolamine (PE) antagonises) 9 Several lines of evidence support the conclusion that a membrane of suitable composition and configuration may allow the synthesis of P-450 to be stimulated or allow more of the cytochrome to be accommodated. In support of this hypothesis it has been observed that: 1. The induction of P-450 cytochromes by phenobarbitone and methylcholanthrene is impaired when the

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microsomal content of PUFAs is l o w , 45 and it is perhaps significant that phenobarbitone induction of P-450 involves the proliferation of the endoplasmic reticulum and an increase in the hepatic content of phosphatidylcholine, a phospholipid which contains a large amount of C18:2 .60 2. There is a good correlation between P-450 levels in human liver and high levels of phospholipids and low levels of triglycerols. 6~ 3. The rising capacity of liver microsomes to metabolise substrates and the increase in P-450 levels during the first half of the lifespan of the mouse is associated with an increase in the PC/PE ratio and hence an increase in the degree of polyunsaturation of the membrane fatty acids. 62 Changes in the rate of carcinogen metabolism however are not always parallelled by changes in the P450 content of the microsomes. For example, depriving rats of food for 72 hours results in a dramatic decrease in BP hydroxylase activity in the rat intestinal mucosa but levels of P-450 and NADPH-cytochome C reductase remain unchanged. 63 This may be due to the inability to measure the specific cytochrome involved in BP-metabolism, but there are several studies that have shown that changes in the lipid microenvironment of the MFO system can affect its ability to metabolise substrates. For instance, an increased phospholipid/ cholesterol ratio in liver microsomal membranes brought about by feeding rats a high lipid diet results in a greater degree of fluidity of the membrane and an increase in the specific activity of P-450 to hydroxylate aniline. 64 Feeding rats a low protein diet has the opposite effect on these parameters. Mechanisms by which the microenvironment may modulate MFO activity include: 1. The substrate binding site of P-450 is embedded in the hydrophobic interior of the phospholipid bilayer, 65 and it is therefore possible that dietary fat may alter substrate-enzyme interactions. Ebel and coworkers have shown that substrate binding to P450 is influenced by the fluidity of the microsomal membrane lipids, 66 which is in turn affected by its fatty acid composition. The contribution of this effect to the dietary fat-induced increase in carcinogen metabolism has been found to be minimal in a number of studies,45 although Cheng and coworkers showed that dietary corn oil increased the 3-MC induction of substrate-binding per unit of P-450 in both the microsomal and nuclear fractions of the rat liver. 46 2. The low-spin/high-spin equilibrium of P-450 is modulated by the lipids of the membrane, and the addition of free unsaturated fatty acids to partially purified P-450 causes a shift in this equilibrium,

which allows the iron of the cytochrome to be more easily reduced. 67 A recent report has concluded that the major contribution of phospholipids in the reconstitution of P-450 enzyme systems in vitro is the facilitation of an active P-450:NADPH-P-450 reductase complex. 68 Thus, increases in the fluidity of the membrane by dietary induced changes in its PUFA content may allow a more rapid transfer of electrons between these two sites within the membrane. The close association of epoxide hydratase with the MFO system in the endoplasmic reticulum stimulated us to investigate the effect of dietary fats on this enzyme. We found that the activity of epoxide hydratase (measured as the rate of hydration of BP-4,5-epoxide to the corresponding diol) in the rat liver exhibited a similar dependency on dietary fat as the MFO system and was closely correlated to the proportion of CI8:2 -~- C20:5 -~- C22:5 -~ C22:6 in the microsomal fraction. 42 Thus its activity was lowest when a fat-free diet was fed and increased significantly when 10% corn oil (2.3-fold) or cod liver oil (3.0-fold) was added to this diet. Similar results have also been obtained by another group who compared the rate of BP-4,5-epoxide hydration in hepatic microsomes of rats fed a 2% corn oil diet and diets containing 6% coconut, peanut, corn and herring oils. 52 Epoxide hydratase in the intestinal mucosa was also stimulated (1.6-fold) by the addition of cod liver oil to a fat-free diet but was not enhanced when a corn oil diet w a s f e d . 42

B. Carcinogen detoxification Few studies have been carried out to investigate the effect of dietary fat on the enzymes involved in the conjugation of carcinogens. A recent study has shown that the hepatic microsomal UDPGT activity towards group I substrates (the conjugation of 4-nitrophenol and 2-napthol was studied but BP-metabolites are also group I substrates) was lowest in rats fed a low lipid diet (2% corn oil) and a 6% saturated coconut oil diet and was significantly higher in the group given a 6% corn oil diet. 52 Feeding a highly polyunsaturated herring oil diet resulted in the highest rate of glucuronidation, which was 2.3-fold greater than in the low lipid diet group. UDPGT activity towards group II substrates, e.g. chloramphenicol, was unaffected by these dietary manipulations. 52 The author has also found that addition of corn oil (10%) to a fat-free diet significantly increases the UDPglucuronidation of 4-nitrophenol in the rat intestinal mucosa by 2-fold (unpublished observation). Glutathione transferases and sulphotransferases, which are principally cytosolic, are unlikely to be affected by dietary fat.

Lipids, antioxidants, and carcinogenactivation II. DIETARY SYNTHETIC ANTIOXIDANTS

Antioxidants are used widely to prevent the autoxidation of unsaturated fatty acids and therefore increase the shelf-life of fat-containing foods. Vitamin E, which is an extremely efficient antioxidant in lipid membranes, is often used, although synthetic antioxidants such as butylated hydroxyanisole (BHA or E320), butylated hydroxytoluene (BHT or E3 21) and propyl gallate are more commonly added to foodstuffs. The human consumption of synthetic phenolic antioxidants is in the order of 10 mg/day, 69 which is similar to the Recommended Dietary Allowance for vitamin E. 7° BHT and BHA are generally considered to be nontoxic, although some tumour-promoting activity has been observed when these substances are fed in high doses (around 20 g/kg diet). 71 BHA and BHT (generally given at 5g/kg diet) are very effective at inhibiting chemically induced carcinogenesis at a number of sites in experimental animals. This includes the inhibition of BP and BP-7,8-diol induced neoplasia of the forestomach and the lung and the carcinogenic effect of benzo(a)anthracenes and 2-acetylaminofluorene. 72 Of particular note is the very effective inhibition of pulmonary adenoma by BHA when given by oral intubation 4 hours before the administration of Bp. 73 The well-established anticancer properties of synthetic antioxidants has stimulated a great deal of research into the mechanisms of their action. This has demonstrated highly specific effects on enzymes involved in carcinogen metabolism, which are discussed below. Involvement of the more general properties of natural and synthetic antioxidants such as the stabilisation of membranes, inhibition of lipid peroxidation and scavenging of free radicals is discussed in section III.

A. Carcinogen activation The effects of synthetic phenolic antioxidants on the MFO system are complex. There are marked differences between species, probably due to variations in the metabolism of these compounds; and small differences in the structure of antioxidants can profoundly alter their effects. Further complications arise from the fact that these compounds alter the composition of P450 isozymes--hence various MFO activities and measurements of P-450 content are often unrelated. TM The effect of antioxidants on BP metabolism has been most thoroughly studied in the mouse liver where they have profound effects. Although there are some conflicting reports, probably due to differences in the methods for measuring BP metabolism, most studies have shown that hepatic microsomes have a decreased capacity to convert BP to its activation products when

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BHA or BHT are included in the diet. 74'75 By hplc analysis of BP metabolites, it has been found that hepatic microsomes from BHA fed mice produced much smaller amounts of 9-hydroxy BP from the parent molecule than microsomes from control animals. 76 A dramatic decrease in the 9-hydroxylation of BP and an increase in the level of BP-4,5-diol has also been observed in hepatocytes prepared from BHA-fed mice. 77 Feeding rats BHT, BHA, and ethoxyquin (10g/kg diet) had similar effects on the hepatic microsomal metabolism of BP and a decrease in BP-7,8-diol levels was also observed. 78,79This change in the specificity of the epoxidation from the 9,10- to the 4,5- positions is likely to be important as this would shift the further metabolism of BP-7,8-diol away from the carcinogenic BP7,8-diol-9,10-epoxides. This has been demonstrated in hepatic microsomes from BHA-fed mice 76,8°and is further supported by decreases in the extent of BP metabolite binding both to exogenous DNA 75'8° and to the DNA of isolated hepatocytes. 77 In addition, inclusion of BHA in the diet has been found to halve the binding of BP metabolites to nuclear macromolecules when mouse hepatic nuclei, which contain P-450 and epoxide hydratase, were incubated in the presence of BP. 8L The altered metabolism of BP demonstrated in these studies is thought to be due to the induction of specific P-450 isozymes by dietary BHA. In addition, BHA and BHT added to microsomal or nuclear fractions in vitro have been shown to directly inhibit the metabolism of BP 82 and BP-7,8-diol 8° and to decrease the binding of their products to DNA. 82 This may be due to the binding of these phenols to P-450 and possibly to the cytochrome-substrate complex. Alternatively, the inhibitory properties of these compounds may be the result of their ability to act as electron donors and discharge the active hydroxylating species of P-450. 75 Other tissues in the mouse which are targets for BPinduced neoplasms have varying responses to antioxidants. When BHA and BHT were added in vitro, BP metabolism was inhibited to a lesser extent in lung microsomes than in liver and neither of these compounds had any effect on aryl hydrocarbon hydroxylase activities in skin homogenates. 82 It was concluded that these differences were due to the exact nature of the cytochromes involved. One study found that dietary BHA had no effect on the binding of BPDEs to DNA when BP was incubated in the presence of microsomes prepared from the lung, forestomach, and liver of mice.83 However, another report did find that lung microsomes from mice responded to dietary BHA (5g/kg) in a similar wayto liver, producing less 9-hydroxy-BP and BP7,8-diol from BP and less BPDEs from BP-7,8-diol but without a change in the gross microsomal P-450 content. 84Most significantly, dietary BHA (5g/kg) has been shown to inhibit the formation of BPDE:DNA

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adducts in vivo in lung, liver and forestomach of mice exposed to BP at doses similar to the level of human contact. 85 Epoxide hydratase is sensitive to induction by synthetic antioxidants, and increases in its activity of between 1.4 and 6.5-fold in the mouse and rat liver have been observed when BHA, BHT and ethoxyquin were added to the diet in levels of around 5 g/kg. 74'75'78~79"86 Feeding BHA (7.5 g/kg) also increased epoxide hydratase levels by about 2-fold in mouse stomach, colon, and thymus and in the small intestine by 6-fold although in rats only a 2-fold increase was observed in this organ. 87 This study found no effect of BHA on epoxide hydratase activity in the lung or kidney in these species; however in another report a 1.6-fold enhancement of activity occurred in the lung when either BHT or ethoxyquin were added to the diet (5 g/kg) and a 1.8-fold increase was induced in the kidney by dietary ethoxyquin. 88

B. Carcinogen detoxification Synthetic antioxidants induce many of the enzymes involved in the detoxification of carcinogenic intermediates. Hepatic glutathione transferase activity in both mice and rats is induced by a number of phenolic antioxidants, dietary BHA being particularly effective, and this results in increased induction of up to 9 - f o l d . 74'75'86 This induction by BHA is the result of increased synthesis of the enzyme. 89 GSTs are a family of cytosolic enzymes with differing substrate specificities and the level of induction depends to some extent on the substrate. When the production of glutathione conjugates of BP-4,5-oxide was examined, dietary BHA (8 g/kg) elevated the rate of this reaction some 2-fold in the rat liver. 79 There is also a microsomal form of GST which was found to be less suspectible to induction by BHA. 75 GST activities in extrahepatic tissues of the mouse and rat such as the lung, kidney, colon 87 and forestomach 86 were elevated to a lesser extent by BHA feeding (approximately 2-fold) than in the liver although the rate of 1,2-dichloro-4-nitrobenzene conjugation in the mouse small intestine increased by 15-fold. 87 Glutatione reductase which regenerates GSH, the substrate for GSTs, from its oxidised form (GSSG) is also induced in mouse and rat liver by approximately 2-fold when BHA or BHT are added to the diet (7.5 g and 5 g/kg respectively) 74 and this is accompanied by an increase in the level of non-protein thiols in several tissues when BHA is added to the d i e t . 74'87 Dietary BHA and BHT elevate the activity of UDPglucuronyl transferase in the mouse and rat l i v e r . 74'75 This enzyme utilises UDP-glucuronic acid (UDPGA)

which is maintained at low concentrations in the cell and is produced by a NAD-requiring dehydrogenase which is also stimulated by dietary BHA and BHT. 74 Furthermore these compounds also elevate the activity of quinone reductase (DT-diaphorase) which would, in the presence of quinones, generate NAD from NADH and therefore stimulate the production of UDPGA. 74 The stimulation of DT-diaphorase by phenolic antioxidants may also provide increased protection against the carcinogenic effects of quinones as this enzyme catalyses the 2-electron reduction of quinones to metabolites which cannot redox cycle and form mutagenic oxygen radicals. 9° It can be concluded that synthetic dietary antioxidants increase the capacity of cells in a variety of organs to conjugate many of the intermediates formed during the metabolism of carcinogens. This is probably a spin-off from BHA and BHT excretion as glucuronide conjugates or mercapturic acid derivatives (metabolites of GSH-conjugates) and the increase in activity of these conjugating enzymes is probably designed to accelerate elimination of the antioxidants themselves. The increased capacity for conjugation may be of only minimal importance to the anticarcinogenic properties of these synthetic compounds. Thus although Dock and coworkers found a 5-fold increase in GSHtransferase activity towards BPDEs in the livers of mice treated with BHA, it was concluded from kinetic data that this would have little effect because the unstimulated GSH-conjugating capacity was unlikely to be saturated during BP-7,8-diol activation. 9~ In addition this author found no increase in glucuronic acid, sulphate or GSH conjugates in BP-treated hepatocytes from BHA-fed mice despite elevations in the activities of the enzymes involved and a marked decrease in the level of BP metabolites bound to intracellular D N A . 77 Thus the conjugating capacity of normal hepatocytes appeared to be quite sufficient to deal with the reactive intermediates formed and the major effect of BHA was therefore considered to take place at the point of BP activation.

III. LIPID PEROXIDATION

The double bonds of PUFAs are suspectible to attack by free radicals and this results in the formation of lipid radicals which combine with oxygen to yield peroxy radicals. These species may then react with another molecule of an unsaturated fatty acid resulting in a chain reaction and the formation of lipid hydroperoxides which break down in the presence of transition metals to a complex mixture of short-chain molecules including aldehydes and hydrocarbon gases.

Lipids, antioxidants, and carcinogen activation Lipid peroxidation has been studied in vitro using isolated hepatocytes as well as subcellular fractions such as microsomal suspensions. There is considerable evidence that lipid peroxidation occurs in vivo and plays a role in the normal functioning of the cell such as the synthesis of prostaglandins. A number of studies have demonstrated a deleterious increase in the rate of lipid peroxidation in vivo in a number of situations including iron-overload 92and the exposure to toxic agents such as CC14 93 and cigarette smoke. 94 The susceptibility of a tissue to lipid peroxidation will be dependent on the level of oxidative stress to which it is subjected, the amount of substrate present, that is the PUFAs, and the level of antioxidant defence mechanisms. These last two factors are modulated by the levels of various constituents in the diet. Thus, in two studies addition of corn oil (rich in C18:2) to a fatfree diet increased the microsomal content of lipid peroxidation products 10-fold in the liver and doubled the in vitro rate of peroxidation catalysed by NADPH or ascorbate. 39,48 The susceptibilities of C20:5 and C22:6 to peroxidation are considerably greater than those of fatty acids with only two double bonds; as an example, rats fed a 10% herring oil diet exhibited levels of hepatic microsomal peroxidation which were 2.6-fold greater than in animals given corn oil. 48 The microsomal fraction of rat small intestinal mucosa peroxidised at a very low rate compared with the liver but addition of cod liver oil to the diet elevated the rate of peroxidation by 20-fold. 22 Higher levels of lipid peroxides have also been observed in lung tissue of rats fed diets containing corn oil or safflower oil (75% C18:2) than in those fed hydrogenated coconut oil or butter and the differences between these groups became more pronounced when the rats were exposed to hyperbaric oxygen. 44 A number of defence mechanisms have been developed to protect cellular constituents from uncontrolled attack by free radicals. Vitamin E is the most important antioxidant which is incorporated into membranes and protects the PUFAs from peroxidation. 95 Ascorbic acid is a water-soluble reducing agent which scavenges free radicals and protects against lipid peroxidation in vivo, probably by reducing vitamin E radicals back to vitamin E. 96 Primates and guinea-pigs are unable to synthesise ascorbic acid which must therefore be supplied in the diet as vitamin C. The enzyme glutathione peroxidase (GSH-Px) converts H202 and fatty acid hydroperoxides to harmless hydroxy acids and specifically requires GSH as substrate which is converted in this reaction to its oxidised form (GSSG).97 GSH-Px contains selenium at its active centre and this has been known for many years to be an important dietary antioxidant. Selenium deficiency causes a drop in GSH-Px activity and renders animals

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more susceptible to a number of situations (for example, elevated oxygen concentrations) involving increased oxidative stress. This effect can often be reversed by additional vitamin E intake and several of the problems resulting from vitamin E deficiency can be offset by increasing dietary selenium levels which, in turn result in a progressive increase in GSH-Px activity. 98 It is therefore apparent that several dietary microconstituents are necessary for the body to cope with living in an oxygen-rich environment and a number of epidemiological studies have indicated a protective effect for several antioxidants against cancer. Increased dietary intake of ascorbic acid and vitamin E are associated with a decrease in the incidence of gastrointestinal cancers, the protective role of ascorbic acid possibly resulting from the inhibition of in vivo nitrosation. 3°,37 The consumption of B-carotene is also inversely correlated with cancer risk, particularly of the lung, oral, and gastrointestinal tract and this may possibly be due to its ability to quench singlet oxygen. 99 Low serum selenium concentrations have been associated with an increased cancer risk, particularly of the gastrointestinal tract, and in one study the impact of selenium deficiency was greater when combined with low serum vitamin E levels. 1°° In another study, increased blood levels of vitamin E appeared to have a protective effect against breast cancer. 101 Although epidemiological data should be treated with caution, further weight is provided by a number of studies involving experimental animals. The anticarcinogenic effect of vitamin E has been demonstrated by Newmark and Mergens who have shown that vitamin E administration inhibits tumour formation caused by polynuclear aromatic hydrocarbons and is also effective at blocking the formation of carcinogenic nitrosamides. 102 Several studies have shown that increasing the amount of selenium given to experimental animals inhibits carcinogenesis of the colon, liver, skin, and mammary gland induced by a number of different agents. 103,104In one study, the increase in the number of DMBA-induced mammary tumours as a result of selenium deficiency was particularly pronounced when the diet was rich in polyunsaturated fats 105and chemoprevention by increasing the intake of selenium was potentiated by additional vitamin E intake. 1°3 However, in another study, selenium included either in a low-fat or highfat diet failed to influence BP-induced tumour response in any organs of the hamster. 106 Differences between studies of this nature are thought to be due to complex interactions between selenium and other dietary factors. 1°6 In another report, it was concluded that the effect of selenium appeared to be exerted mainly at the

104

J.D. GOWER dation in hepatocytes by Fe 2+ , ADP, and NADPH also results in the degradation of P-450 and this is prevented by the addition of phenolic antioxidants. H2 BHT also prevents the loss of P-450 in adrenal cortex mitochondria subjected to NADPH-dependent lipid peroxidation but superoxide dismutase is ineffective.l J3 It is possible that changes in the configuration of the membrane as a result of lipid peroxidation were responsible for the loss of P-450 and MFO activities in these in vitro experiments. Studies using fluorescence anisotropy have shown that lipid peroxidation increases the order of the acyl chains of microsomal phospholipids probably due to formation of covalent bonds between adjacent lipid radicals. This results in a decrease in the rotational mobility of P-450 probably as a result of slow protein aggregation. 114 In addition, it has been demonstrated that peroxidation of adrenal microsomal membranes decreases the binding of substrates such as benzphetamine to the P-450 complex. ~ While it is difficult to be certain that lipid peroxidation causes the destruction of MFO activity in vivo, several studies have demonstrated a relationship between these two events:

promotional stage of carcinogenesis and did not affect the formation of DMBA-DNA adducts.m7 The possible interactions between lipid peroxidation and the activation of BP are summarised in Figure 4 which also shows points at which antioxidants may exert effects. A. Enzymes involved in carcinogen metabolism The peroxidation of hepatic microsomes in vitro by both enzymic and non-enzymic mechanisms results in a loss of several of the enzyme activities associated with this membrane system. P-450 levels rapidly diminish 1°9 with a concomitant decrease in MFO activities such as the oxidative demethylation of aminopyrine ~m and this is associated with a loss of microsomal PUFAs and the formation of malonaldehyde.m9 Addition of inhibitors of lipid peroxidation prevents these changes. 109 Similar findings have been demonstrated in adrenal microsomes where peroxidation dramatically decreased the rate of hydroxylation of BP and steroids and the levels of P-450 and NAD(P)Hcytochrome c reductase.ll~ Inhibition of lipid peroxidation by increasing the microsomal protein concentration prevented these changes and malonaldehyde had no direct inhibitory effect. Initiation of lipid peroxi-

Membrane

1. Depletion of hepatic glutathione by administration of phorone to rats leads to an increase in lipid per-

PUFA PUFA

• A I

# I

," //

/

PRE-CARCINOGEN

Radical initiator

~, ~/" " ipid Peroxidation

I

Direct Reactions -~

Q

I Semiquinone Fenton-Type ~-GSH-Px--~-[ Reactions BP-diols

EH -Epoxide hydratase PRE-CARCINOGEN

Hydrop!roxJdes

PUFA -Polyunsaturated fatty acids MFO -Mixed-function oxidase system

Peroxyl radicals

METABOLITE e.g. BP-Quinones

Key:.

Peroxidases

UDPGT -UDP-glucuronyl transferase GSH-Px -Glutathione peroxidase -Steps inhibitable

ACTIVATED CARCINOGEN e.g. BP-diol-epoxides

Hydroxy acids

02

OH"

Q

]r [ MUTATIONS DNA I

Fig. 4. Possible interactionsbetween lipid peroxidationand the activationof benzo(a)pyrene.

by antioxidants

Lipids, antioxidants, and carcinogenactivation oxidation (measured in vitro) and to a depression in the P-450 catalysed metabolism of N,N-dimethyl4-amino-azobenzene in vivo (measured as excreted N-demethylated metabolites in the bile). H5 These events occurred in parallel and it was concluded that glutathione may contribute to the regulation of P-450 activity, possibly by sequestering the amount of iron available for lipid peroxidation or by interactions with free radicals either directly or through glutathione peroxidase. . Treatment of mice and rats with polyribocytidylic acid increases the activity of xanthine oxidase in the liver which results in an enhanced rate of lipid peroxidation and this is paralleled by a decrease in the content of P-450.116 P-450-independent xenobiotic-metabolising enzymes and cytochrome c reductase activities were not affected. . The degradation of P-450 and P-448 in the rat liver following a period of induction by phenobarbitone and 20-methylcholanthrene is associated with an increase in lipid peroxide levels and could be prevented by the intraperitoneal administration of BHT (100 mg/kg body weight). ~17Further studies showed that lipid peroxidation may act as a triggering mechanism which makes P-450 more accessible to endogenous proteases. ~18 This circumstantial evidence linking lipid peroxidation and decreased P-450 levels must however be treated with caution. For instance, it is known that CC14 administration, which is very effective at initiating lipid peroxidation, results in decreases in the activities of glucose-6-phosphatase, BP-hydroxylase and P-450 levels in the endoplasmic reticulum both in vivo and in vitro. 119,12°However, CCI4 induced damage to P-450 in isolated hepatocytes still takes place when lipid peroxidation is blocked by promethazine although this drug protects glucose-6-phosphatase activity.l~9 From this and similar studies it can be concluded that lipid peroxidation is not obligatory for the CC14 mediated destruction of P-450 but instead the covalent binding of the CCI~ radical to P-450 may be responsible. 119,12° Rowe and Wills showed that the addition of vitamin E (120 mg/kg diet) to a 10% lard diet fed to rats resulted in increased levels of P-450 in microsomes and this was associated with a decreased microsomal lipid peroxide content. 48They concluded that a delicate balance between vitamin E and polyunsaturated fat is essential for maximum MFO activities and the prevention of membrane damage by lipid peroxidation. BHT (250 mg/kg diet) had no effect on either of these parameters and this was probably due to the inability of this antioxidant to become localised in the microsomal membrane. 48

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Other studies have shown that vitamin E deficiency in rats decreases the activity of the hepatic microsomal MFO system, the rate of BP hydroxylation and the P450 content but not the level of cytochrome b5 and cytochrome c reductase. 9s,~2t Enhanced vitamin E intake in rats (40 mg/kg daily) produced a 6-fold increase in hepatic BP hydroxylase activity and this was associated with a marked decrease in microsomal lipid peroxidation. 122 An adequate intake of dietary vitamin E has also been found to be essential for the protection of P-450 following the administration to rats of methyl ethyl ketone peroxide which is a potent inducer of lipid peroxidation.~Z3 Vitamin C deficiency depresses MFO-catalysed reactions for a number of substrates and this appears to be the result of a decrease in the content of specific P450 cytochromes. 37 Different responses have been found in various tissues but it can generally be concluded that ascorbic acid has little effect on BP-hydroxylation or epoxide hydratase activity. 37 The presence of lipid peroxidation products in the diet has been shown to have little effect on hepatic MFO activities including BP hydroxylation, the levels of P-450 and cytochrome b5 and the activity of UDPGT. 124,~25However, in this study the authors reported a significant decrease in the P-450 content of the small intestine as a result of subjecting the corn oil diet to oxidation. Thus it appears that the effect of dietary lipid peroxides on carcinogen metabolism is restricted to the gastrointestinal tract and this is almost certainly due to the poor absorption of these molecular species from the lumen or to their breakdown in intestinal cells. The effect of lipid peroxidation and the products of this oxidative chain-reaction on carcinogen metabolising enzymes other than the mixed-function oxidase system has not been studied in any depth. It is possible that some carcinogen-metabolising enzymes may be affected because various products of lipid peroxidation such as 4-hydroxyenals are known to inactivate many enzymes and block protein and DNA synthesis.~°8 In one study the activities of hepatic GSH-transferases and epoxide hydratase were unaffected by increased rates of lipid peroxidation as a result of administering polyribocytidylic acid to rats and mice. 116UDPGT activity in the rat colon has been reported to be increased by some 50% when diets containing oxidised corn oil were fed. 125 B. One electron reactions

The dramatic increase in the rate of lipid peroxidation in microsomes prepared from the intestinal mucosa of rats fed diets rich in polyunsaturated fats led

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J.D. GOWER

us to investigate the effect of dietary lipids on the oxidation of BP-7,8-diol mediated by lipid peroxidation of this fraction in vitrofl 2 Similar rates of BP-tetrol production (hydrolysis products of BPDEs) were found when intestinal microsomes prepared from rats fed diets which were fat-free, or contained 10% lard or corn oil were incubated in the presence of Fe 2+, ascorbate and ADP. However, addition of 10% cod liver oil to the fat-free diet increased the rate of BP-7,8-diol activation 4-fold to a level which was greater than the MFOcatalysed reaction in all parts of rat intestine. By hplc analysis, it was found that the conversion of BP-7,8diol to the more carcinogenic of the BPDEs (BPDE I) was favoured during lipid peroxidation of the intestinal fractions. The rate of BP oxidation to BP-quinones catalysed by hepatic microsomes undergoing lipid peroxidation has also been found to be similarly affected by changing the lipid composition of the diets fed to rats (McNeill, personal communication). Another pathway which may contribute towards the activation of BP in vivo has been demonstrated by Nemoto who has shown that in the presence of unsaturated fatty acids and heme-compounds, BP is converted to products, probably quinones, which bind to proteins. 126 The extent to which BP may encounter unsaturated fatty acids in the blood stream and thus the possible level of oxidation by this pathway is likely to be highly dependent upon the nature of the dietary fat intake. Lipid peroxidation may play an important role in one-electron transfer reactions involving carcinogens and catalysed by peroxidases, and there is increasing evidence that these systems contribute towards the activation of carcinogens in vivo. 15,t9The peroxidase activities of P-450 (or P-420) and prostaglandin synthetase require hydroperoxides as substrates and, although the prostaglandin synthetase system can make its own hydroperoxide (PGG2), this can be replaced by other fatty acid hydroperoxides such as 13-OOH-C18:2 produced by non-specific reactions. Thus an increase in the level of hydroperoxides as a result of increased lipid peroxidation may stimulate the peroxidase-catalysed activation of carcinogens. A dietary-induced increase in the membrane content of C20:4 and CI8:2 which are substrates for the prostaglandin synthetase system and lipoxygenase respectively may also increase the cooxidation of carcinogens by these enzymes 127 although the rate-limiting step in these pathways is the phospholipase-catalysed removal of these fatty acids from the membrane. Antioxidants can interact with peroxidases in a number of ways which have opposite effects. Thus BHA and BHT, added in vitro, inhibit the P-450-peroxidasecatalysed oxidation of BP, probably by acting as elec-

tron donors and discharging the active hydroxylating complex of P-450. 75 However, the peroxidase activity of P-450, like its MFO activity, is sensitive to the damaging effects of lipid peroxidation and is severely lowered when vitamin E-deficient rats are administered methyl ethyl ketone peroxide which is a potent inducer of lipid peroxidation.12s Feeding diets adequate in vitamin E protected this microsomal enzyme activity from peroxidative damage. 123BHA and BHT also inhibit the in vitro cooxidation of carcinogens by prostaglandin synthetase and hematin which is thought to be due to the scavenging of peroxyl radicals. 128On the other hand, prostaglandin synthetase and lipoxygenase are irreversibly inactivated by the oxygen radicals which they produce and this product-inhibition phenomenon may be prevented in vivo by radical scavengers such as vitamin E and glutathione peroxidase. 2° The ability of the glutathione peroxidase system to remove hydroperoxides may be an important determinant of the rate of peroxidase-catalysed carcinogen metabolism. In addition, GSH-Px removes H202 which is generated during the redox cycling of quinones and will therefore protect DNA from hydroxyl radical damage induced by these agents. In vitamin E-deficient rats, which exhibit increased rates of lipid peroxidation, decreased levels of GSH-Px activity were found in the mitochondrial matrix and in mitochondrial and microsomal membranes of the liver.121 This may have been the result of attack by toxic free radicals or of changed selenium metabolism. The combined effects of an increased level of hydroperoxides and a decreased activity of GSH-Px resulting from vitamin E-deficiency suggests that the rate of peroxidase mediated carcinogen activation may be stimulated under these conditions. GSH-Px activity also fell in EFA-deficient animals and this may have been due to the adaptation of this enzyme to decreased peroxide levels. ~2~ Oral administration of BHA and retinol acetate (vitamin A) has also been shown to increase GSH-Px activity in rat hepatic tissue but increased vitamin E intake had no effect. ~22 The effect of dietary polyunsaturated fats and antioxidants on peroxidase-mediated carcinogen activation merits considerable further investigation. In one study the activation of the carcinogen N-hydroxy-2acetylaminofluorene in cultured mammary cells, which is thought to proceed by a peroxidase-dependent mechanism, was stimulated 7-fold when rats were fed a selenium deficient diet and this was thought to be due to decreased glutathione peroxidase activity.129 Decreased levels of selenium-dependent GSH-Px activity can be compensated for by an increase in GSHtransferases which also possess peroxidase activity.13° This family of enzymes constitute an extraordinarily

Lipids, antioxidants, and carcinogen activation

high proportion (between 3 and 10%) of the cytosolic proteins and may serve as an expendable protective " b u f f e r " which detoxifies carcinogens by reacting covalently with their electrophilic intermediates.131 There is also the possibility that antioxidants may protect against carcinogenesis by reacting directly with reactive carcinogenic species and by preventing the interaction of epoxides with DNA, thus preventing mutational changes and the initiation of carcinogenesis. Vitamin E and BHT have been shown to significantly reduce BP-induced chromosomal aberrations and mutations in Chinese hamster cells in vitro both in the absence and presence of an $9 activating mix. 132,133In another study, ascorbic acid, 13-carotene, tocopherol acetate and BHA had no effect on the mutagenicity of cigarette-smoke condensate or BP in the Salmonella TA98 tester strain in the presence of rat-liver homogenate.134 The induction of nuclear damage to mouse colonic epithelial cells following administration of BP intrarectally was significantly reduced when the mice were fed a number of plant phenols or BHA (2%) for a week prior to this challenge. 135While the mechanism of action of BHA may have been through its effect on carcinogen metabolising enzymes (Section II), the inhibitory nature of the other phenols, such as caffeic and ellagic acids was considered to be due to their aromatic structures which can ring-stack with planar carcinogens such as BPDEs through pi-bonding and therefore antagonise the mutagenicity of these intermediates, m It has also been shown that antioxidants such as BHA can interact with the 6-oxy-BP radical ~36 which is the precursor to BP-quinones, and antioxidants will also scavenge the oxygen-derived free radicals produced during redox cycling of these metabolites. Finally, the effect of dietary antioxidants and polyunsaturated fats may not be restricted to activation of carcinogens within cells. Precarcinogens such as BP may be oxidised to mutagenic and toxic species in foodstuffs which contain polyunsaturated fats and are subjected to conditions which induce peroxidation, such as production of free radicals by ~-irradiation, heat, u.v. light or traces of metals. ~37 In addition, BP quinones may be formed in the presence of peroxidising polyunsaturated fats during the passage of BP along the digestive tract. CONCLUSIONS AND SUMMARY

It is clear that dietary polyunsaturated fats and antioxidants interact with the activation and detoxification of carcinogens in a complexity of ways. Dietary fats supply the building blocks for membranes, and changes in the polyunsaturated fat content of the diet,

107

especially if highly polyunsaturated fats are included, dramatically affects the composition of membranes over a short time interval of days to weeks. An increase in the PUFA content results in more fluid membranes and an increase in the MFO-catalysed oxidation of precarcinogens to activated products. This is not accompanied by an increase in the activity of cytosolic conjugating enzymes which remove these harmful products and thus the balance may be tipped towards an increase in the carcinogenicity of environmental agents. Epoxo ide hydratase activity is also increased although it is difficult to assess the effect which this may have on the overall process of carcinogen activation and detoxification. For protection against peroxidative damage, an increase in the PUFA content of cells must be accompanied by increased protective mechanisms through intake of antioxidants and in particular vitamin E. m Furthermore as tissue PUFAs have longer halflives than tocopherol, ingestion of polyunsaturated foodstuffs well protected by this vitamin may not continue to be protected at a later time. 138 Unprotected PUFAs undergo peroxidation which can be initiated by intracellular free radicals produced in small quantities under normal circumstances or by a wide variety of environmental toxins which pose a high level of oxidative stress. This leads to changes in the membrane structure and a decrease in MFO-activity but may lead to an increase in the activation of carcinogens via direct reactions with peroxidation products and by cooxidation catalysed by a wide variety of peroxidases. The timing of antioxidant intake with respect to carcinogen exposure is worth considering. Immediate effects of antioxidants include the scavenging of both the peroxyl radicals which are responsible for the formation of carcinogenic electrophiles as well as the carcinogenic products themselves. Longer term effects of antioxidants include the stabilisation and protection of membranes and membrane-bound enzymes and also the maintenance of GSH-Px activity which removes H202 and the substrates for peroxidases. Synthetic antioxidants given in the diet exert profound effects on carcinogen metabolising enzymes and inhibit BP-DNA adduct formation in vivo which may account for their anticarcinogenic properties. While blocking agents of this type may eventually be used specifically for this purpose, at present the very low consumption of these agents by man (approximately 0.1 mg/kg/day 69 compared to doses of several hundred mg/kg/day fed to mice) is well below the level at which they exert any effect. The evidence for a role of dietary lipids and antioxidants in the modification of the carcinogenic potential of precarcinogens presented in this article has been derived mainly from in vitro studies. The chal-

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lenge remains to define more fully the mechanisms of carcinogen activation, particularly the role of the relatively newly discovered one-electron reactions, and the effects which dietary constituents have on these processes in vivo. Acknowledgements--First and foremost, I would like to remember Professor Eric Wills who died on 6th April 1985. His pioneering work on lipid peroxidation is part of the foundations on which much of todays free-radical biochemistry is based. He is sadly missed. I would like to thank Dr. Colin Green for helpful comments and Sylvia Sanderson for preparation of the manuscript. REFERENCES

1. Pott, P. Chirurgical observations relative to the cancer of the scrotum. London. 1775. Reprinted in Natl. Cancer Inst. Monograph 10:7; 1963. 2. Doll, R.; Peto, R. The causes of cancer: Quantitative estimates of avoidable risks of cancer in the United States today. J. Natl. Cancer Inst. 66:1191-1308; 1981. 3. Palmer, S.; Bakshi, K. Diet, nutrition, and cancer: Interim dietary guidelines. J. Natl. Cancer Inst. 70:1152-1170; 1983. 4. Consensus Statement on Provisional dietary guidelines. In: Joossens, J. V.; Hill, M. J.; Geboers, J.; eds. Diet and human carcinogenesis. Amsterdam: Excerpta Medica; 1985:341- 342. 5. Welsch, C. W.; Aylsworth, C. F. Enhancement of murine mammary tumorigenesis by feeding high levels of dietary fat: A hormonal mechanism? J. Natl. Cancer Inst. 70:215-221; 1983. 6. Vitale, J. J.; Broitman, S. A. Lipids and immune function. Cancer Res. 41:3706-3710; 1981. 7. Reddy, B. S. Dietary fat and its relationship to large bowel cancer. Cancer Res. 41:3700-3705; 1981. 8. Hopkins, G. J.; West, C. E. Effect of dietary polyunsaturated fat on the growth of a transplantable adenocarcinoma in C3HA mice. J. Natl. Cancer Inst. 58:753-756; 1977. 9. Miller, E. C.; Miller, J. A. Searches for ultimate chemical carcinogens and their reactions with cellular macromolecules. Cancer 47:2327-2345; 1981. 10. Preussmann, R. A. W. The role of food contaminants and additives in human carcinogenesis. In: Joossens, J. V.; Hill, M. J.; Geboers, J.; eds. Diet and human carcinogenesis. Amsterdam: Excerpta Medica; 1985:35-48. 11. Pelkonen, O.; Nebert, D. W. Metabolism of polycyclic aromatic hydrocarbons: Etiologic role in carcinogenesis. Pharmacol. Rev. 34:189-220; 1982. 12. Gelboin, H. V. Benzo(a)pyrene metabolism, activation and carcinogenesis: Role and regulation of mixed-function oxidases and related enzymes. Physiol. Rev. 60:1107-1166; 1980. 13. Sims, P.; Grover, P. L.; Swaisland, A.; Pal, K.; Hewer, A. Metabolic activation of benzo(a)pyrene proceeds by a diolepoxide. Nature 252:326-328; 1974. 14. Lesko, S. A.; Ts'o, P. O. P.; Yang, S.-U.; Zheng, R. Benzo(a)pyrene radicals and oxygen radical involvement in DNA damage, cellular toxicity and carcinogenesis. In: McBrien, D. ; Slater, T. F., eds. Free radicals, lipidperoxidation and cancer. New York: Academic Press; 1982:401-414. 15. Krauss, R. S.; Eling, T. E. Arachidonic acid-dependent cooxidation. A potential pathway for the activation of chemical carcinogens in vivo. Biochem. Pharmacol. 33:3319-3324; 1984. 16. Dix, T. A.; Marnett, L. J. Free radical epoxidation of 7,8dihydroxy-7,8-dihydrobenzo(a)pyrene by hematin and polyunsaturated fatty acid hydroperoxides. J. Am. Chem. Soc. 103:6744-6746; 1981. 17. Marnett, L. J. Hydroperoxide dependent oxidation during prostaglandin biosynthesis. In: Pryor, W. A., ed. Free radicals in biology, Vol. 6. New York: Academic Press; 1977:63-94. 18. Mason, R. P.; Chignell, C. F. Free radicals in pharmacology and toxicology-selected topics. Pharmacol. Rev. 33:189-211 ; 1982.

19. O'Brien, P. J. Peroxide-mediated metabolic activation of carcinogens. In: Yagi, K., ed. Lipid peroxides in biology and medicine. New York: Academic Press; 1980:317-338. 20. Nemoto, N.; Takayama, S. Arachidonic acid-dependent activation of benzo(a)pyrene to bind to proteins with cytosolic and microsomal fractions from rat liver and lung. Carcinogenesis 5:961-964; 1984. 21. Dix, T. A.; Marnett, L. J. Metabolism of polycyclic aromatic hydrocarbon derivatives to ultimate carcinogens during lipid peroxidation. Science 221:77-79; 1983. 22. Gower, I. D.; Wills, E. D. The oxidation of benzo(a)pyrenc7,8-dihydrodiol mediated by lipid peroxidation in the rat intestine and the effect of dietary lipids. Chem.-Biol. Interact. 63:63-74; 1987. 23. McNeill, J. M.; Wills, E. D. The formation of mutagenic derivatives of benzo(a)pyrene by peroxidising fatty acids. Chem. Biol. Interact. 53:197-207; 1985. 24. Trush, M. A.; Seed, J. L.; Kensler, T. W. Oxidant-dependent metabolic activation of polycyclic aromatic hydrocarbons by phorbol ester-stimulated human polymorphonuclear leukocytes: Possible link between inflammation and cancer. Proc. Natl. Acad. Sci. USA 82:5194-5198; 1985. 25. Morgenstern, R.; DePierre, J. W.; Lind, C.; Guthenberg, C.: Mannervik, B.; Ernster, L. Benzo(a)pyrene quinones can be generated by lipid peroxidation and are conjugated with glutathione. Biochem. Biophys. Res. Commun. 99:682-690; 1981. 26. Alexandrov, K.; Dansette, P. M.; Frayssinet, Ch. Effect of purified epoxide hydrolase on metabolic activation and binding of benzo(a)pyrene to exogenous DNA. Shift of the activation pathway. Biochem. Biophys. Res. Commun. 93:611-616; 1980. 27. Carroll, K. K. Lipids and carcinogenesis. J. Environ. Pathol. Toxicol. 3:253-271; 1980. 28. Kroes, R.; Beems, R. B.; Bosland, M. C.; Bunnik, G. S.: Sinkeldam, E. J. Nutritional factors in lung, colon, and prostate carcinogenesis in animal models. Fed. Proc. 45:136-141 ; 1986. 29. Carroll, K. K.; Khor, H. T. Dietary fat in relation to tumorigenesis. Prog. Biochem. Pharmacol. 10:308-353; 1975. 30. Weisburger, J. H. Role of fat, fiber, nitrate, and food additives in carcinogenesis: A critical evaluation and recommendations. Nutr. Cancer 8:47-62; 1986. 31. McCay, P. B.; King, M.; Rikans, L. E.; Pitha, J. V. Interactions between dietary fats and antioxidants on DMBA-induced mammary carcinomas and on AAF-induced hyperplastic nodules and hepatomas. J. Environ. Pathol. Toxicol. 3:451465: 1980. 32. Hopkins, G. J.; Kennedy, T. G.; Carroll, K. K. Polyunsaturated fatty acids as promoters of mammary carcinogenesis induced in Sprague-Dawley rats by 7,12-dimethylbenz(a)anthracene. J. Nat. Cancer Inst. 66:517-522; 1981. 33. Reddy, B. S.; Maruyama, H. Effect of dietary fish oil on azoxymethane-induced colon carcinogenesis in male F344 rats. Cancer Res. 46:3367-3370; 1986. 34. Beems, R. B.; van Beek, L. Modifying effect of dietary fat on benzo(a)pyrene-induced respiratory tract tumours in hamsters. Carcinogenesis 5:413-417; 1984. 35. Broitman, S.; Vitale, J. J.; Vavrousek-Jakuba, E.; Gottlieb, L. S. Polyunsaturated fat, cholesterol and large bowel tumorigenesis. Cancer 40:2455-2463; 1977. 36. Roebuck, B. D.; Longnecker, D. S.; Baumgartner, K. J.; Thron, C. D. Carcinogen-induced lesions in the rat pancreas: Effects of varying level of essential fatty acid. Cancer Res. 45:5252 5256; 1985. 37. Campbell, T. C. Influence of nutrition on metabolism of carcinogens. Adv. Nutr. Res. 2:29-55; 1979. 38. Vetter, R. D.; Carey, M. C.; Patton, J. S. Coassimilation of dietary fat and benzo(a)pyrene in the small intestine: An absorption model using the killifish. J. Lipid Res. 26:428-434; 1985. 39. Hammer, C. T.; Wills, E. D. The role of lipid components of the diet in the regulation of the fatty acid composition of the rat liver endoplasmic reticulum and lipid peroxidation. Biochem. J. 174:585-593; 1978.

Lipids, antioxidants, and carcinogen activation 40. Hammer, C. T.; Wills, E. D. Dependence of the rate of metabolism of benzo(a)pyrene on the fatty acid composition of the liver endoplasmic reticulum and on the dietary lipids. Nutr. Cancer 2:113-118; 1980. 41. Gower, J. D.; Wills, E. D. The dependence of the rate of BP metabolism in the rat small intestinal mucosa on the composition of the dietary fat. Nutr. Cancer 8:151-161; 1986. 42. Gower, J. D.; Sayer, J. W.; Wills, E. D. The effect of dietary lipids and antioxidants on the activity of epoxide hydratase in the rat liver and intestine. Biochem. Pharmacol. 35:19651969; 1986. 43. Mounie, J.; Bereksi-Reguig, K.; Goudonnet, H.; Escousse, A.; Truchot, R. Liver microsome phospholipids and cytochrome P-450 concentration in phenobarbital treated rats fed on different diets. Life Sci. 32:503-510; 1983. 44. Alam, S. Q.; Alam, B. S. Pulmonary lipid peroxides and fatty acids of rats fed different lipids and exposed to oxygen at hyperbaric pressure. Lipids 20:248-251; 1985. 45. Wade, A. E. Effects of dietary fat on drug metabolism. J. Environ. Pathol. Toxicol. Oncol. 6:161-189; 1986. 46. Cheng, K. C.; Ragland, W. L.; Wade, A. E. Dietary fat and 3-MC induction of hepatic nuclear and microsomal cytochrome P-450. Drug Nutr. Interact. 1:63-74; 1981. 47. Smith, M. T.; Wills, E. D. Effects of dietary lipid and phenobarbitone on the distribution and concentration of cytochrome P-450 in the liver studied by quantitative cytochemistry. FEBS Lett. 127:33-36; 1981. 48. Rowe, L.; Wills, E. D. The effect of dietary lipids and vitamin E on lipid peroxide formation, cytochrome P-450 and oxidative demethylation in the endoplasmic reticulum. Biochem. Pharmacol. 25:175-179; 1976. 49. Lambert, L.; Wills, E. D. The effect of dietary lipids on 3,4,benzo(a)pyrene metabolism in the hepatic endoplasmic reticulum. Biochem. Pharmacol. 26:1423-1427; 1977. 50. Wade, A. E.; White, R. A.; Walton, L. C.; Bellows, J. T. Dietary fat--a requirement for induction of mixed-function oxidase activities in starved-refed rats. Biochem. Pharmacol. 34:3747-3754; 1985. 51. Baker, M. T.; Karr, S. W.; Wade, A. E. The effects of dietary corn oil on the metabolism and activation of benzo(a)pyrene by the benzo(a)pyrene metabolizing enzymes of the mouse. Carcinogenesis 4:9-15; 1983. 52. Mounie, L; Faye, B.; Magdalou, J.; Goudonnet, H.; Truchot, R.; Siest, G. Modulation of UDP glucuronosyl-transferase activity in rats by dietary lipid. J. Nutr. 116:2034-2043; 1986. 53. Wills, E. D. The role of the polyunsaturated fatty acid composition of the endoplasmic reticulum in the regulation of the rate of oxidative drug and carcinogen metabolism. In: Coon, M. J.; Conney, A. H.; Estabrook, R. W.; Gelbion, H. V.; Gillette, J. R.; O'Brien, P. J., eds. Microsomes, drug oxidations and chemical carcinogenesis. New York: Academic Press; 1980:545-548. 54. Spector, A.; Baron, J.; Fontenelle, L. J. Effects of special lipid diets on the mixed function oxygenase enzyme system in the livers of mice. Proc. West. Pharmacol. Soc. 27:495-499; 1984. 55. Smith-Barbaro, P.; Hanson, D.; Reddy, B. S. Effect of fat and microflora on hepatic, small intestinal and colonic HMG CoA reductase, cytochrome P450 and cytochrome B5. Lipids 16:183188; 1981. 56. Antal, M.; Nagy, K.; Bedo, M. B. Effect of dietary protein and lipid on the activity of hepatic mixed-function oxidase system in young and adult rats. Ann. Nutr. Metab. 26:393399; 1982. 57. Nishiyama, K.; Ide, T.; Sugano, M. Activities of liver mixed function oxidase system in rats fed trans fat. Lipids 20:325327; 1985. 58. Chapman, M. D.; Marmering, G. J. Role of phospholipids in the hepatic microsomal drug-metabolizing system. Mol. Pharmacol. 6:631-640; 1970. 59. Strobel, M. W.; Lu, A. Y. H.; Heidema, J.; Coon, M. J. Phosphatidyl-choline requirement in the enzymatic reduction

60.

61.

62.

63.

64.

65. 66. 67.

68.

69. 70. 71. 72. 73.

74.

75.

76. 77.

78. 79. 80.

109

of hemoprotein P-450 and in fatty acid, hydrocarbon and drug hydroxylation. J. Biol. Chem. 245:4851-4854; 1970. Davison, S. C.; Wills, E. D. Studies on the lipid composition of the rat liver endoplasmic reticnlum after induction with phenobarbital and 20-methylcholanthrene. Biochem. J. 140:461468; 1974. Savolainen, M. J.; Arranto, A. J.; Hassinen, I. E.; Luoma, P. V.; Pelkonen, R. O.; Sotaniemi, E. A. Relationship between lipid composition and drug metabolizing capacity of human liver. Eur. J. Clin. Pharmacol. 27:727-732; 1985. Hawcroft, D. M.; Jones, T. W.; Martin, P. A. Studies on age related changes in cytochrome P-450, cytochrome b-5 and mixed function oxidase activity in mouse liver microsomes, in relation to their phospholipid composition. Arch. Gerontol. Geriatr. 1:55-74; 1982. Stohs, S. J.; Grafstrom, R. C.; Burke, M. D.; Moldeus, P. W.; Orrenius, S. G. The isolation of rat intestinal microsomes with stable cytochrome P-450 and their metabolism of benzo(a)pyrene. Arch. B iochem. B iophys. 177:105-116; 1976. Narbonne, J. F.; Pelissier, M. A.; Bonnamour, D.; Borin, C.; Albrecht, R. Lipid composition and monooxygenase relationship in liver microsomal membranes from rats fed unbalanced diets. Toxicol. Lett. 23:73-77; 1984. Taniguchi, H.; Imai, Y.; Sato, R. Substrate binding site of microsomal cytochrome P-450 directly faces membrane lipids. Biochem. Biophys. Res. Commun. 118:916-922; 1984. Ebel, R. E.; O'Keefe, D. H.; Peterson, J. A. Substrate binding to hepatic microsomal cytochrome P450. Influence of the microsomal membrane. J. Biol. Chem. 253:3888-3897; 1978. Gibson, G. G.; Cinti, D. L.; Sligar, S. G.; Schenkman, J. B. The effect of microsomal fatty acids and other lipids on the spin state of partially purified cytochrome P-450. J. Biol. Chem. 255:1867-1873; 1980. Muller-Enoch, D.; Churchill, P.; Fleischer, S.; Guengerich, F. P. Interaction of liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in the presence and absence of lipid. J. Biol. Chem. 259:8174-8182; 1984. Collings, A. J.; Sharratt, M. The BHT content of human adipose tissue. Food Cosmet. Toxicol. 8:409-412; 1970. Vitamin E. Nutr. Rev. 35:57-62; 1977. Ito, N.; Fukushima, S.; Tsuda, H. Carcinogenicity and modification of the carcinogenic response by BHA, BHT, and other antioxidants. CRC Crit. Rev. Toxicol. 15:109-150; 1985. Wattenberg, L. W. Inhibitors of chemical carcinogenesis. J. Environ. Pathol. Toxicol. 3:35-52; 1980. Speier, J. L.; Lam, L. K. T.; Wattenberg, L. W. Effects of administration to mice of BHA by oral intubation of BP-induced pulmonary adenoma formation and metabolism of BP. J. Natl. Cancer Inst. 60:605-609; 1978. Cha, Y. N.; Heine, H. S. Comparative effects of dietary administration of 2(3)-tert-butyl-4-hydroxyanisole and 3,5-di-tertbutyl-4-hydroxytoluene on several hepatic enzyme activities in mice and rats. Cancer Res. 42:2609-2615; 1982. Rahimtula, A. D.; Jernstrom, B.; Dock, L.; Moldeus, P. Effect of dietary and in vitro 2(3)-t-butyl-4-hydroxy-anisole and other phenols on hepatic enzyme activities in mice. Br. J. Cancer 45:935-944; 1982. Sydor, W., Jr.; Chou, M. W.; Yang, S. K.; Yang, C. S. Regioselective inhibition of benzo(a)pyrene metabolism by butylated hydroxyanisole. Carcinogenesis 4:131-136; 1983. Dock, L.; Cha, Y. N.; Jernstrom, B.; Moldeus, P. Effect of 2(3)-tert-butyl-4-hydroxyanisole on benzo(a)pyrene metabolism and DNA-binding of benzo(a)pyrene metabolites in isolated mouse hepatocytes. Chem. Biol. Interact. 41:25-37; 1982. Kahl, R.; Kahl, G. F. Effect of dietary antioxidants on benzo(a)pyrene metabolism in rat liver microsomes. Toxicology 28:229-233; 1983. Aspry, K. E.; Bjeldanes, L. E Effects of dietary broccoli and butylated hydroxyanisole on liver-mediated metabolism of benzo(a)pyrene. Food Chem. Toxicol. 21:133-142; 1983. Dock, L.; Rahimtula, A.; Jernstrom, B.; Moldeus, P. Metabolism of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene in mouse liver microsomes and the effect of

110

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95. 96. 97.

J.D. GOWER 2(3)-tert-butyl-4-hydroxyanisole. Carcinogenesis 3:697-701; 1982. Hennig, E. E.; Demkowicz-Dobrzanski, K. K.; Sawicki, J. T.; Mojska, H.; Kujawa, M. Effect of dietary butylated hydroxyanisole on the mouse hepatic monooxygenase system of nuclear and microsomal fractions. Carcinogenesis 4:1243-1246; 1983. Yang, C. S.; Sydor, W., Jr.; Martin, M. B.; Lewis, K. F. Effect of butylated hydroxyanisole on the aryl hydrocarbon hydroxylase of rats and mice. Chem. Biol. Interact. 37:337350; 1981. Ioannou, Y. M.; Wilson, A. G.; Anderson, M. W. Effect of butylated hydroxyanisole on the metabolism of benzo(a)pyrene and the binding of metabolites to DNA, in vitro and in vivo, in the forestomach, lung, and liver of mice. Carcinogenesis 3:739-745; 1982. Sydor, W., Jr.; Lewis, K. F.; Yang, C. S. Effects of butylated hydroxyanisole on the metabolism of benzo(a)pyrene by mouse lung microsomes. Cancer Res. 44:134-138; 1984. Adriaenssens, P. I.; White, C. M.; Anderson, M. W. Doseresponse relationships for the binding of benzo(a)pyrene metabolites to DNA and protein in lung, liver, and forestomach of control and butylated hydroxyanisole-treated mice. Cancer Res. 43:3712-3719; 1983. Lam, L. K.; Sparnins, V. L.; Hochalter, J. B.; Wattenberg, L. W. Effects of 2- and 3-tert-butyl-4-hydroxyanisole on glutathione S-transferase and epoxide hydrolase activities and sulfhydryl levels in liver and forestomach of mice. Cancer Res. 41:3940-3943; 1981. Benson, A. M.; Cha, Y-N.; Bueding, E.; Heine, H. S.; Talalay, P. Elevation of extrahepatic glutathione S-transferase and epoxide hydratase activities by BHA. Cancer Res. 39:29712977; 1979. Kahl, R. Enhancement of epoxide hydratase activity in rat lung, kidney and liver by dietary antioxidants. Cancer Len. 8:323-328; 1980. Pearson, W. R.; Windle, J. J.; Morrow, J. F.; Benson, A. M.; Talalay, P. Increased synthesis of glutathione-S-transferases in response to anticarcinogenic antioxidants. J. Biol. Chem. 258:2052-2062; 1983. Chesis, P. L.; Levin, D. E.; Smith, M. T.; Ernster, L.; Ames, B. N. Mutagenicity of quinones: Pathways of metabolic activation and detoxification. Proc. Natl. Acad. Sci. USA 81:16961700; 1984. Dock, L.; Martinez, M.; Jernstrom, B. Induction of hepatic glutathione S-transferase activity by butylated hydroxyanisole and conjugation of benzo(a)pyrene diol-epoxide. Carcinogenesis 5:841-844; 1984. Bacon, B. R.; Tavill, A. S.; Brittenham, G. M.; Park, C. H.; Recknagel, R. O. Hepatotoxicity of chronic iron overload: Role of lipid peroxidation. In: Poli, G.; Cheeseman, K. H.; Dianzani, M. U.; Slater, T. F., eds. Free radicals in liver injury. Oxford: IRL Press; 1985:49-57. Recknagel, R. O.; Glende, E. A.; Hruszkewycz, A. N. Chemical mechanisms in carbon tetrachloride toxicity. In: Pryor, W. A., ed. Free radicals in biology, Vol. II1. New York: Academic Press; 1977:97-132. Pryor, W. A.; Lightsey, J. W.; Prier, D. G. The production of free radicals in vivo from the action of xenobiotics: The initiation of autoxidation of polyunsaturated fatty acids in nitrogen dioxide and ozone. In: Yagi, K., ed. Lipid peroxides in biology and medicine. New York: Academic Press; 1982:122. Porter, R.; Whelan, J., eds. Biology of vitamin E. Ciba Foundation Symp. 101, London: Pitman; 1983. Kunert, K-J.; Tappel, A. L. The effect of vitamin C on in vivo peroxidation in guinea pigs as measured by pentane and ethane production. Lipids 18:271-274; 1983. McCay, P. B.; Gibson, D. D.; Fong, K. L.; Hornbrook, K. R. Effect of glutathione peroxidase activity on lipid peroxidation in biological membranes. Biochim. Biophys. Acta 431:459-468; 1976.

98. Chen, J.; Goetchius, M. P.; Campbell, T. C.; Combs, G. F.. Jr. Effects of dietary selenium and vitamin E on hepatic mixedfunction oxidase activities and in vivo covalent binding of aflatoxin BI in rats. J. Nutr. 112:324-331; 1982. 99. Wolf, G. Is dietary [3-carotene an anti-cancer agent? Nutr. Rev. 40:257-261; 1982. 100. Salonen, J. T.; Salonen, R.; Lappetelainen, R.; Maenpaa, P. H.; Alfthan, G.; Puska, P. Risk of cancer in relation to serum concentrations of selenium and vitamins A and E: Matched case-control analysis of prospective data. Br. Med. J. 290:417 420; 1985. 101. Wald, N. J.; Boreham, J.; Hayward, J. L.; Bulbrook, R. D. Plasma retinol, 13-carotene and vitamin E levels in relation to the future risk of breast cancer. Br. J. Cancer 49:321-324; 1984. 102. Newmark, H. L.; Mergens, W. J. Alpha-tocopherol and its relationship to tumor induction and development. In: Zedeck, M.; Lipkin, M., eds. Inhibition of tumor induction and development. New York: Plenum; 1981:127-168. 103. Horvath, P. M.; Ip, C. Synergistic effect of vitamin E and selenium in the chemoprevention of mammary carcinogenesis in rats. Cancer Res. 43:5335-5341; 1983. 104. Griffin, A. C. Role of selenium in the chemoprevention of cancer. Adv. Cancer Res. 29:419-442; 1979. 105. Ip, C.; Sinha, D. K. Enhancement of mammary tumorigenesis by dietary selenium deficiency in rats with a high polyunsaturated fat intake. Cancer Res. 41:31-34; 1982. 106. Beems, R. B. Dietary selenium- and benzo(a)pyrene-induced respiratory tract tumours in hamsters. Carcinogenesis 7:485489; 1986. 107. Ip, C.; Daniel, F. B. Effects of selenium on 7,12-dimethytbenz(a)anthracene-inducedmammary carcinogenesis and DNA adduct formation. Cancer Res. 45:61-65; 1985. 108. Esterbauer, H. Lipid peroxidation products: Formation, chemical properties and biological activities. In: Poli, G.; Cheeseman, K. H.; Dianzani, M. U.; Slater, T. F., eds. Free radicals in liver injury. Oxford: IRL Press; 1985:29-47. 109. Levin, W.; Lu, A. Y. H.; Jacobson, M.; Kuntzman, R. Lipid peroxidation and the degradation of cytochrome P-450 heme. Arch. Biochem. Biophys. 158:842-852; 1973. 110. Wills, E. D. Effects of lipid peroxidation on membrane-bound enzymes of the endoplasmic reticulum. Biochem. J. 123:983 991; 1971. 111. Brogan, W. C.; Miles, P. R.; Colby, H. D. Effects of lipid peroxidation on adrenal microsomal monooxygenases. Biochim+ Biophys. Acta 758:114-120; 1983. 112. Novikov, K.; Kagan, V. Stabilization of cytochrome P-450 in hepatocytes by free radical scavengers of different nature. Acta Physiol. Pharmacol. Bulg. 11:61-69; 1985. 113. Klimek, J.; Schaap, A. P.; Kimura, T. The relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in adrenal cortex mitochondria. Biochem. Biophys. Res. Commun. 110:559-566; 1983. 114. Gut, J.; Kawato, S.; Cherry, R. J.; Winterhalter, K. H.: Richter, C. Lipid peroxidation decreases the rotational mobility of cytochrome P-450 in rat liver microsomes. Biochim. Biophys. Acta 817:217-228; 1985. 115. Levine, W. G. Glutathione, lipid peroxidation and regulation of cytochrome P-450 activity. Life Sci. 31:779-784; 1982. 116. Koizumi, A.; Walford, R. L.; Imamura, T. Treatment with poly I.C. enhances lipid peroxidation and the activity of xanthine oxidase, and decreases hepatic P-450 content and activities in mice and rats. Biochem. Biophys. Res. Commun. 134:632-637; 1986. 117. Kagan, V. E.; Savov, V. M.; Serbinova, E. A. Mechanisms of disassembly of a mixed function oxygenase system in liver endoplasmic reticulum: I. The role of peroxidation of membrane phospholipids. Acta Physiol. Pharmacol. Bulg. 10:73 81; 1984. 118. Kagan, V. E+; Savov, V. M.; Serbinova, E. A. Mechanisms of disassembly of mixed function oxygenase system in liver endoplasmic reticulum: II. The interrelation between lipid per-

Lipids, antioxidants, and carcinogen activation

119.

120.

121.

122.

123. 124.

125. 126. 127.

128.

129.

130.

131.

132.

oxidation and proteolytic degradation of cytochrome P-450. Acta Physiol. Pharmacol. Bulg. 10:82-89; 1984. Poli, G.; Cheeseman, K.; Slater, T. E; Dianzani, M. U. The role of lipid peroxidation in CC14-induced damage to liver microsomal enzymes: comparative studies in vitro using microsomes and isolated liver cells. Chem. Biol. Interact. 37:1324; 1981. Colby, H. D.; Brogan, W. C.; Miles, P. R. Carbon tetrachloride-induced changes in adrenal microsomal mixed-function oxidases and lipid peroxidation. Toxicol. Appl. Pharmacol. 60:492-499; 1981. Jensen, G. E.; Clausen, J. Subcellular distribution of the cytochrome P-450 complex and glutathione peroxidase activity in vitamin E and essential fatty acid deficiency. Lipids 16:137141; 1981. Stohs, S. J.; Hassan, M. Q.; Murray, W. J. Effects of BHA, d-alpha-tocopherol and retinol acetate on TCDD-mediated changes in lipid peroxidation, glutathione peroxidase activity and survival. Xenobiotica 14:533-537; 1984. Ando, M.; Tappel, A. L. Effect of dietary vitamin E on methyl ethyl ketone peroxide damage to microsomal cytochrome P450 peroxidase. Chem. Biol. Interact. 55:317-326; 1985. Lambert, L.; Wills, E. D. The effect of dietary lipid peroxides, sterols and oxidised sterols on cytochrome P450 and oxidative demethylation in the endoplasmic reticulum. Biochem. Pharmacol. 26:1417-1421; 1977. Perciballi, M.; Pintauro, S. J. The effects of fractionated thermally oxidized corn oil on drug-metabolizing enzyme systems in the rat. Food Chem. Toxicol. 23:737-740; 1985. Nemoto, N. Marked activation of benzo(a)pyrene to proteinbinding forms in the presence of unsaturated fatty acids and heme-compounds. Carcinogenesis 7:267-271; 1986. Craven, P. A.; DeRubertis, E R. Fatty acid induced drug and carcinogen metabolism in rat and human colon. A possible link to the association of high dietary fat intake and colonic cancer. Biochem. Biophys. Res. Commun. 94:1044-1051; 1980. Dix, T. A.; Fontana, R.; Panthani, A.; Marnett, L. J. Hematin-catalyzed epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene by polyunsaturated fatty acid hydroperoxides. J. Biol. Chem. 260:5358-5365; 1985. Wong, P. K.; Hampton, M. J.; Floyd, R. A. In: Powles, T.; Bockman, R.; Honn, K.; Ramwell, P., eds. Prostaglandins and cancer: First International Conference. New York: Alan R. Liss; 1982:167-179. Lawrence, R. A.; Parkhill, L. K.; Burk, R. F. Hepatic cytosolic non-selenium-dependent glutathione peroxidase activity: Its nature and the effect of selenium deficiency. J. Nutr. 108:981987; 1978. Litwack, G.; Ketterer, B.; Arias, I. M. Ligandin: A hepatic protein which binds steroids, bilirnbin, carcinogens and a number of exogenous organic anions. Nature (Lond.) 234:466467; 1971. Smalls, E.; Patterson, R. M. Reduction of benzo(a)pyrene

133. 134.

135.

136.

137. 138.

111

induced chromosomal aberrations by DL-alpha-tocopherol. Eur. J. Cell. Biol. 28:92-97; 1982. Paschin, Y.; Bahitova, L. M. Inhibition of the mutagenicity of benzo(a)pyrene in the V79/HGPRT system by bioantioxidants. Mutat. Res. 137:57-59; 1984. Terwel, L.; van der Hoeven, J. C. Antimutagenic activity of some naturally occurring compounds towards cigarette-smoke condensate and benzo(a)pyrene in the salmonella/microsome assay. Mutat. Res. 152:1-4; 1985. Wargovich, M. J.; Eng, V. W.; Newmark, H. L. Inhibition by plant phenols of benzo(a)pyrene-induced nuclear aberrations in mammalian intestinal ceils: A rapid in vivo assessment method. Food Chem. Toxicol. 23:47-49; 1985. Sullivan, P. D.; Calle, L. M.; Shafer, K.; Nettleman, M. Effect of antioxidants on benzo(a)pyrene free radicals. In: Jones, P. W.; Freudenthal, R. I., eds. Carcinogenesis, Vol. 3. New York: Raven Press; 1978:1-8. Gower, J. D.; Wills, E. D. The oxidation of benzo(a)pyrene mediated by lipid peroxidation in irradiated synthetic diets. Int. J. Radiat. Biol. 49:471-484; 1986. Mead, J. F. Nutrients with special functions: Essential fatty acids. In: Alfin-Slater, R. B.; Kritchevsky, D., eds. Human nutrition, Vol. 3A. New York: Plenum Press; 1980:213-238.

LIST OF ABBREVIATIONS

--Butylated hydroxyanisole --Butylated hydroxytoluene --Benzo(a)pyrene BPDE --Benzo(a)pyrene-diol-epoxide DMBA --Dimethylbenzanthracene --Essential fatty acid EFA GSH --Glutathione (reduced) GSSG --Glutathione (oxidised) GSH-Px --Glutathione peroxidase --Glutathione-S-transferase GST HPLC --High Pressure Liquid Chromatography 3-MC --3-Methylcholanthrene --Mixed-function oxidase MFO --Phosphatidylcholine PC --Phosphatidylethanolamine PE PUFA --Polyunsaturated fatty acid ST --Sulphotransferase UDPGA--UDP-Glucuronic acid UDPGT --UDP-Glucuronyl-transferase BHA BHT BP