Dietary modification of intestinal and pulmonary aryl hydrocarbon hydroxylase activity

TOXICOLOGY

AND

APPLIED

23,741-748

PHARMACOLOGY

Dietary Modification Aryl Hydrocarbon

(1972)

of Intestinal Hydroxylase

and Pulmonary Activity1

LEE W. WATTENBERG Department

of Pathology, University of Minnesota Minneapolis, Minnesota 55455

Received

August

Medical

School,

8,1972

The issue to which this presentation is ultimately addressed is the potential role of the mixed-function oxidase system as a survival mechanism in an environment contaminated with numerous noxious chemicals. Only a segment of this problem will be dealt with in any detail, namely those aspects having to do with the component part of the mixed function oxidase system designated as aryl hydrocarbon hydroxylase (AHH). At the present time the full range of substrates metabolized by AHH is not known. However, this system does metabolize an extremely important group of compounds, the polycyclic hydrocarbons, which include a large number of carcinogens. The polycyclic hydrocarbons can be formed from combustion of almost any organic material, e.g., wood, gasoline, coal, tobacco, and are ubiquitous in urban areas. Because of the almost inevitable exposure of urban dwellers to these compounds, biochemical systems metabolizing them are of importance. A factor which enhances the interest in AHH in this regard is that the activity of this system in the major portals of entry, i.e., intestines and lung, is largely and possibly entirely the result of induction by exogenous inducers (Wattenberg, 1970, 1971). The implication of this finding is that AHH activity will be determined in these tissues by environmental factors, and the result of such contact will control the capacity to metabolize this important group of compounds. The vast majority of studies of the mixed-function oxidase system have been devoted to the liver. The liver has traditionally been thought of as the principal site of drug metabolism, and in fact microsomal mixed-function oxidase activity is high in this organ. However, an accumulating body of knowledge indicates that mixed-function oxidase activity occurs in a wide variety of other tissues including the major portals of entry, i.e., intestinal tract and lung (Wattenberg and Leong, 1962; Wattenberg et al.. 1962; Gelboin and Blackburn, 1964; Jellinck and Goody, 1967; Wattenberg, 1970). For demonstrating the full range of tissue distribution of these enzymes, AHH employing benzo[a]pyrene BP as a representative substrate has been of considerable importance. Two aspects of this reaction are particularly favorable for distribution studies. The first is that a highly sensitive fluorometric technique is available for the qualitative determination of BP hydroxylation (Wattenberg et al., 1962; Wattenberg and Leong, 1 This study was supported by USPHS Grant CA 09599 from the National Cancer Institute and a grant from the American Medical Association Education and Research Foundation. Copyright rights

All

0 1972 by Academic Press, Inc. of reproduction in any form reserved.

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WATTENBERG

1965). The second is that there is a morphologic histochemical procedure for demonstrating the localization of this reaction in tissue sections (Wattenberg and Leong, 1962). In this paper the main focus is on AHH activity in the intestinal tract and lung. In the initial section quantitative and histochemical studies of the localization of AHH are presented. This is followed by a discussion of factors, particularly dietary ones, which affect the level of AHH activity in these tissues. The final section deals with data relating to the role of AHH in protecting against chemical carcinogens. LOCALIZATION OF AHH ACTIVITY GASTROINTESTINAL TRACT AND

IN THE LUNG

Quuntz’tatiue studies. AHH activity occurs in tissues of the gastrointestinal tract and lung. The activity in the gastrointestinal tract has been studied most thoroughly in the rat. In this species, the highest activity is found in the proximal portion of the small intestine. The level of activity in this region of the small intestine is similar to that of liver. There is a gradient of activity with a progressive decrease occurring as the ileocecal junction is approached (Wattenberg et al., 1962). After feeding inducers, AHH activity increases throughout the entire length of the small intestine, but the activity in the proximal portion is still considerably higher than that in the more distal regions. In the large bowel, the activity is considerably lower than in the small intestine. However, as in the small intestine, a gradient of activity is found in which the AHH activity progressively decreases from the proximal portion to the distal portion. Both the forestomach and glandular stomach show a low activity. Following the feeding of inducers, the activity increases in both portions of the stomach. AHH activity has been found in the proximal portion of the small intestine of all species studied thus far. This includes mouse, guinea pig, rabbit, hamster, dog, bull, monkey, baboon and man (surgical specimens) (Wattenberg et al., 1962). AHH activity has been found in lung of the rat, mouse, hamster, rabbit, bull and man (Wattenberg et al., 1968a; Wattenberg and Leong, 1962; Wattenberg, 1970). Investigations with the human lung have been carried out only on surgically resected specimens from older individuals. Some specimens showed activity, and others did not. Studies of AHH activity have been performed on the tracheal mucosa of the rabbit and the bull. In both, a very weak activity was found (Wattenberg, 1970). Histochemical studies. AHH activity in the small intestine is located in the columnar cells covering the villi both in untreated animals and in animals fed various inducers, but the activity is considerably higher in the latter (Wattenberg et al., 1962). The stomach of the rat has two parts, a forestomach lined by squamous epithelium, and a glandular portion lined by a glandular epithelium. In the forestomach, activity is found in the squamous epithelium of the mucosa. In the glandular regions, it occur in the epithelial cells of the surface and in a few cell layers in the adjacent superficial portion of the gastric glands. In the large intestine, activity is most intense in the columnar cells on the surface of the mucosa. A somewhat erratic activity is found in crypt cells. AHH activity has been demonstrated in the lung. Activity is found in alveolar walls. It has not been detected in tracheal or bronchial mucosa. As mentioned above, quantitative studies indicate that there is in fact some activity present in the tracheal mucosa; however, it is very low and less than the sensitivity of the histochemical method. The

DIET

AND

ARYL

HYDROCARBON

HYDROXYLASE

743

precise identification of the cell type or types in the alveolar walls in which the enzyme activity occurs has not been made. FACTORS EFFECTING THE LEVEL OF AHH ACTIVITY OF THE INTESTINE AND LUNG Investigations have been carried out to determine chemical characteristics necessary for compounds to act as inducers of increased AHH activity. In early experiments, it was found that a number of polycyclic hydrocarbons, such as 3-methylcholanthracene and BP are potent inducers of increased AHH activity in the intestinal tract and lung as well as in the liver, (Conney et al., 1957; Gelboin and Blackburn, 1964; Wattenberg ef al., 1962; Jellinck and Goody, 1967). The next group of compounds found to be inducers were the phenothiazines (Wattenberg and Leong, 1965). Subsequently, a series of substituted five-membered heterocyclic compounds such as 2,5-bis-(4’pyridyl)-1,3,4-thiadiazole were found to have inducing activity and also several 2phenylbenzothiazoles. (Wattenberg et al., 1968b). An additional group of inducers are the flavones and related compounds (Wattenberg et al.. 1968a). A large number of flavonoid compounds occur in nature in plants and are consumed in the diet. Flavone itself is an inducer. Halogen substitution into the 4’ position results in a marked increase in inducing activity. Hydroxy groups reduce the inducing capacity of flavone. Polyhydroxylated compounds such as quercetin are without any inducing effect. In contrast, conversion of hydroxy to methoxy groups results in restoration of inducing activity. The effects of hydroxy groups on inducing activity is of importance since many of the naturally occurring flavonoid compounds are polyhydroxyl derivatives. Some are not. Two such compounds, tangeretin (5,6,7.8,4’pentamethoxyflavone) and nobiletin (5,6,7,8,3’-4’-hexamethoxyflavone), which occur in citrus fruits, are moderately potent inducers. A synthetic compound, /3-naphthoflavone is an exceedingly potent inducer and has been particularly useful in studies of the effects of high levels of microsomal enzyme activity on carcinogenesis. In studies of the AHH activity of the small intestine and lung, it had been assumed that a normal level of activity of substantial magnitude exists. This assumption now appears to be incorrect, and in fact most if not all the activity in these two structures results from exposure to exogenous inducers. The first evidence for this came from studies of starved female Sprague-Dawley rats (Wattenberg et al., 1962). These animals showed almost total loss of AHH activity in the small intestine. It was later found that the lung behaved in the same manner. A similar loss of AHH activity was observed in rats fed a fat free diet. Supplementation of this diet with a variety of lipid materials including crude vegetable oils and linoleic acid did not result in restoration of AHH activity. These findings suggested that it was the purified nature of the diet that was the critical factor. Subsequently, studies were carried out employing a balanced purified diet, i.e., vitamin free casein 27 y/,, starch 59 ‘/“, corn oil lOoA, salt mix 4 yd,plus a complete vitamin supplement (Normal Protein Test Diet, Nutritional Laboratories, Cleveland). Again there was almost a total loss of AHH activity in the small intestine and lung (Wattenberg, 1970,197l). Determination of the effects of starvation and feeding a purified diet have also been carried out on 3-methyl-4-methylaminoazobenzene N-demethylase activity of the small intestine. Both of these regimens result in a profound

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decrease in the activity of this reaction as compared to the level of activity in animals fed Purina Rat Chow (Billings and Wattenberg, 1972). A series of experiments was subsequently performed in which several crude diets were fed to rats. All these resulted in substantial levels of AHH activity in the small intestine and lung of the rat (Wattenberg, 1972). The diets were Purina Rat Chow, Charles River Rat Diet, Cargill Rabbit Diet, and Rockland Rat Diet. Additional work done with Purina Rat Chow showed that the level of AHH activity in the two tissues was related to the amount of diet consumed. Diet studies have also been carried out employing other species. Mice, hamsters and rabbits have been fed Purina Rat Chow or purified diet or have been starved. In each instance almost total loss of AHH activity occurred in animals which have been starved or fed a purified diet, whereas, those fed Purina Rat Chow had substantial levels of activity. The results of all the findingsin the feedingexperiments indicate that most and possibly all of the AHH activity of the small intestine and lung is due to an exogenous inducer or inducers. Under the particular experimental conditions employed, the inducer or inducers were present in the crude diets. Work has begun in an effort to identify the inducer(s). Initially, various components of crude diets were tested. Considerable inducing activity was found in the vegetable component, which consists of alfalfa meal. Commercial alfalfa meal was found to have inducing activity. Subsequently, the plant itself was obtained from a farm in which no chemical treatment of the soil or plant had been carried out. This alfalfa also had inducing activity. Because of the results obtained with alfalfa, an investigation was begun of the inducing activity of other vegetables. Many vegetables have inducing activity, some do not. The most potent found thus far are members of the Brassicaceae family including Brussels sprouts, cabbage, turnips, broccoli and cauliflower. Inducing activity has also been found in spinach, dill and celery (Wattenberg, 1971, 1972). Further studies with Brussels sprouts and cabbage showed that quite marked differences in inducing activity occur with different varieties of the vegetable. Determinations of the distribution of inducing activity throughout the Brussels sprouts plant have been performed. The inducing activity is not uniformly distributed, but is largely confined to the sprouts, themselves. Studies aimed at isolating and identifying the active compound(s) are currently in progress. MIXED

FUNCTION CHEMICAL

OXIDASE ACTIVITY CARCINOGENESIS

AND

A number of studies have demonstrated that it is possible to protect against the carcinogenic effects of chemical carcinogens by inducing increased mixed function oxidase activity (Table 1). In early studies it was shown that administration of polycyclic hydrocarbon inducers markedly reduced the incidence of hepatic cancer which resulted from feeding 3’-methyl-4-dimethylaminoazobenzene (Richardson et al., 1952; Meechan, 1953; E. C. Miller et al., 1958). The mixed function oxidase system inactivated this carcinogen by demethylation and also by azo dye reduction (Conney et al., 1956). Other studies demonstrated that polycyclic hydrocarbon inducers can markedly reduce the incidence of tumors of the liver, mammary gland, ear duct and small intestine in rats fed 2-acetylaminofluorine or 7-fluoro-2-acetylaminofluorene (Miller et al., 1958).

DIETANDARYLHYDROCARBONHYDROXYLASE

745

TABLE 1 INHIBITIONOF CARCINOGENESISBY INDUCTION OF INCREASED MICROSOMALENZYMEACTIVITY

Carcinogen _- ._.~..__--.___

Inducer

Rat

Liver

2-Acetylaminofluorene Polycyclic hydrocarbons

Rat

Liver,

Urethane

Pentobarbital

Mouse

Urethane

P-Naphthoflavonechlordane, phenobarbital Phenothiazine

3’-Methyl-4-dimethylaminoazobenzene

Bracken fern carcinogen Benzo[u]pyrene

Polycyclic hydrocarbons

Species Organ

/%Naphthoflavone

Benzo[a]pyrene

fl-Naphthoflavone

7,12-Dimethylbenz[a]anthracene

Polycyclic hydrocarbons

7,12-Dimethylbenz[a]- Phenothiazines anthracene 7,12-Dimethylbenz[a]- fi-Naphthoflavone anthracene 7,12-Dimethylbenz[a]- P-Naphthoflavone anthracene

References Richardsonet al. (1952), Miller et al. (1958) Miller et al. (19%)

breast Lung Silva (1967), Adenis et al. (1970) Mouse Lung Yamamoto ef al. (1971) Rat Intestine, Pamukcuet al. bladder (1971) Mouse Lung Wattenbergand Leong (1970) Mouse Skin Wattenberg and Leong( 1970) Rat Breast Hugginset ul. (1964),Wheatley (1968) Rat Breast Wattenberg and Leong(1968) Rat Breast Wattenberg and Leong( 1968) Mouse Lung Wattenberg and Leong(1968)

In this instance the inactivation of the carcinogens resulted from ring hydroxylation (Miller and Miller, 1965). More recently, studies have been carried out which have shown that it is possible to protect against the carcinogenic effects of polycyclic hydrocarbon carcinogens by inducing increasedAHH activity. One of the experimental modelswhich has been used for this purpose is the formation of mammary tumors in rats given large oral dosesof 7,12-dimethylbenz[cc]anthracene (DMBA). Induction of increased AHH activity by a number of different types of inducers prior to administration of the DMBA will inhibit tumor formation. The inducers employed include the following: polycyciic hydrocarbons (Huggins et al., 1964; Wheatley, 1968); phenothiazines (Wattenberg and Leong, 1967) and flavones (Wattenberg and Leong, 1968). A second test system which has been employed is the formation of pulmonary adenomas in the mouse subjected to exposuresto polycyclic hydrocarbons. With this system,it hasbeen shown that flavone inducers will inhibit pulmonary adenoma formation resulting from oral administration of DMBA or BP (Wattenberg and Leong, 1968, 1970). In the tumor inhibition studies which have been carried out in the rat, it is difficult to assigna preciserole to different tissuesin the overall carcinogen detoxification process.

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The reason for this is that administration of inducers results in an increase in microsomal enzyme activity in several tissues including the liver. In the mouse studies, the flavone inducers which are employed produced no effect or trivial effects on the AHH activity of the liver whereas a marked induction occurred in the small bowel and lung. These data strongly suggest that the small bowel and lung played a major or possibly the total role in the process of inhibition. Studies of inhibition of skin tumor formation in the mouse by induction of increased AHH activity have been carried out. In this experimental model, the carcinogen is applied directly to the target tissue. Accordingly, it is possible to study the effect of induction of increased microsomal enzyme activity under conditions in which no tissue is interposed between the site of carcinogen application and the target tissue. Employing fl-naphthoflavone as an inducing agent and BP as the carcinogen, experiments were carried out in which it was found that induction of increased AHH activity in the skin protects against the carcinogenic effects of BP (Wattenberg and Leong, 1970). Having presented these experiments showing a protective effect of enhanced mixed function oxidase activity, I would like to discuss an apparently conflicting set of data. It has been demonstrated for many chemical carcinogens that the microsomal mixed function oxidase system converts these compounds to a proximate carcinogenic form (Miller, 1970). A common situation appears to be one in which the carcinogen is metabolized at two or more sites on the molecule. One or more of these results in detoxification, and one is an activation step. The classic example of this is the aromatic amines. With these compounds, ring hydroxylation results in detoxification whereas hydroxylation of the nitrogen is an activation reaction (Miller and Miller, 1969). A similar situation, i.e., both microsomal detoxification and activation has been described for the polycyclic hydrocarbon carcinogens (Grover et al., 1971; Harper, 1959; Schlede et al., 1970). With some carcinogens only activation reactions appear to occur. In spite of the known mixed-function oxidase activation pathways, there are no reported data which clearly indicate that induction of increased mixed function oxidase activity causes an increase in carcinogenicity of a chemical carcinogen in an in vivo experiment. There are several possible explanations for this. The first is that under conditions where a dual attack occurs, the detoxification reactions predominate. The second is that slow activation of a carcinogen is as effective as rapid activation. In fact one might anticipate that it might be even more effective in terms of providing the active chemical species at a critical time or times in the cell cycle and/or preventing loss of activated species due to excess amount over that most effective for the number of critical binding sites available at a particular time. A third possibility is that the proper experiment for demonstrating that induction of increased mixed-function oxidase activity results in an increased tumor incidence in vivo has not been done. But let us suppose for the moment that such an experiment was reported. What would the implications be? This question really bears on the larger one of the overall role of the mixed-function oxidase system as a broad range survival mechanism (Wattenberg, 1966). This is an old concept which has been repeatedly brought up and discarded (Williams, 1959). The basis for discarding it is that instances can be cited in which the mixed-function oxidase system activates compounds to more toxic forms. The validity of discarding the mixed-function oxidase system as an overall survival

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system because of its capacity to produce some adverse effects is open to serious question. Broad range survival systems simply cannot be dealt with on this basis. If one looks at a well established broad range survival system such as the immunologic system, this point is clearly illustrated. With immunologic deficiencies, the survival of the organism is threatened. If the deficiency is severe enough, death is a virtual certainty. Yet there are a whole series of pathologic entities, some resulting in death, which result from various

untoward

immunologic

reactions.

However

in balance, this defense

mechanism is of enormous advantage to the organism. Evaluation of the overall implications of systems with very large numbers of component reactions is somewhat unusual for many biological scientists who more commonly deal with specific critical experiments. However, in considering the role of a system as complex as the mixed-function oxidase system, it is necessary to evaluate the overall

balance between protective effects and potentially

noxious ones. At the present time.

this is extremely difficult if not impossible to do. However, as more data become available, it would seem worthwhile to continually reevaluate the concept of the mixedfunction oxidase system as apossiblesurvival systemagainst foreign organiccompounds. This could be of considerable importance in an increasingly contaminated environment. The level of activity of this system may hold the key to the health or even survival of man and other species. At least one component of the mixed-function oxidase system, AHH is directly controlled in the tissues of the major portals of entry by exogenous inducers, and this makes it readily affected by chance environmental contacts or potentially by overt manipulation. REFERENCES L., VLAEMINCK, M. N., and DRIESSENS, J. (1970). L’adenome Pulmonaire de la Souris Swiss recevant de l’urethane. VIII. Action du phenobarbital. C. R. Sot. Biol. 164, 560-652. BILLINGS, R. and WATTENBERG, L. W. (1972).The effectsof dietary alterationson 3-methyl4-methylaminoazobenzene N-demethylaseactivity. Proc. Sot. Exp. Biol. Med. 139,865-867. CONNEY. A. H., MILLER, E. C., and MILLER, J. A. (1956). The metabolismof methylated aminoazo dyes. V. Evidence for induction of enzyme synthesisin the rat by 3-methylcholanthrene.Cancer Res. 16, 450-459. CONNEY, A. H., MILLER, E. C., and MILLER, J. A. (1957).Substrate-inducedsynthesisand other propertiesof benzpyrenehydroxylase in rat liver. J. Biol. Chem. 228, 753-766. GELBOIN, H. V., and BLACKBURN, N. R. (1964).Thestimulatory effectof 3-methylcholanthrene on benzpyrenehydroxylase activity in severalrat tissues:inhibition by actinomycin D and Puromycin. Cancer Res. 24, 356-360. GROVER, P. L., SIMS, P., HUBERMAN, E., MARQUARDT, H., KUROKI, T., and HEIDELBERGER, C. (1971).In vitro transformation of rodent cellsby K-region derivativesof polycyclic hydrocarbons.Proc. Nat. Acad. Sci. U.S. 68,1098-1101. HARPER, K. H. (1959). The intermediary metabolismof polycyclic hydrocarbons. Brit. J. ADENIS,

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