Metabolism of dietary genotoxins by the human colonic microflora; the fecapentaenes and heterocyclic amines

Mutation Research, 238 (1990) 209-221

209

Elsevier MUTREV 02806

Metabolism of dietary genotoxins by the human colonic microflora; the fecapentaenes and heterocyclic amines R.L. Van Tassell a, D.G.I. Kingston b and T.D. Wilkins

a

a Department of Anaerobic Microbiology, b Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 (U.S.A.)

(Accepted 12 October 1989) Keywords: Genotoxins, dietary, metabolism; Colonic microflora; Fecapentaenes; Heterocyclic amines; Anaerobic bacteria; Plas-

malogens Summary The microflora of the human colon is a complex ecosystem of anaerobic bacteria which have the capability of enzymatically transforming a variety of dietary (or biliary) compounds to genotoxic metabolites. In the past, most investigators studying the interplay between diet and colonic flora and its role in the etiology of cancers focused on the reductive and glycosidic potential of the bacterial enzymes - many of which reverse the oxidative and conjugative reactions performed by the liver. Recent work in our laboratory has focused on the metabolism of two relatively new classes of genotoxins, the fecapentaenes and the heterocyclic amines (pyrolysis carcinogens). The fecapentaenes (conjugated ether lipids) are produced in the colon by Bacteroides spp. from polyunsaturated ether phospholipids (plasmalogens) whose natural origin and function are unknown. The fecapentaenes are potent direct-acting genotoxins that are detected in the feces of most individuals on normal western diets. The heterocyclic amines, which originate from fried or broiled proteinaceous foods, normally require activation by the liver before being potent mutagens or carcinogens. However, the " I Q " subclass (e.g. IQ and MelQ) can be activated in the colon by Eubacterium and Clostridium species to a 7-hydroxy form which is directly mutagenic in Salmonella. Although there is no direct evidence that the fecapentaenes or the 7-hydroxy " I Q " compounds influence risk for colon cancer, the potency and prevalence of these bacterial metabolites is cause for concern.

Human population and animal studies strongly support the contention that our diet is a major contributing factor in the complex etiology of cancer of the large bowel. However, these studies

This work was supported by PHS grants CA-23857 and CA40821 from the National Cancer Institute of the National Institutes of Health. Correspondence: Dr. R.L. Van Tassell, Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University,Blacksburg, VA 24061 (U.S.A.).

also show that the effect of diet on colon carcinogenesis probably is indirect. That is, the diet provides the host with the promutagens and procarcinogens from which the "ultimate" genotoxins are generated as well as affects the manner in which the host metabolizes these compounds. Furthermore, diet plays a significant role in tumor p r o m o t i o n b y inducing cocarcinogens and bacterial metabolism which enhances turnover of colonic cells thus increasing D N A synthesis and the likelihood of mutations. That tumors are a hundred times more common in the colon than in

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210 the small intestines suggests that potential genotoxins which originate from the diet are activated in vivo after absorption by the small intestines or, are formed endogenously after passage into the colon. However, it is not clear to what extent activated dietary genotoxins cause DNA damage within colonic mucosal cells and whether this putative genotoxicity arises from compounds present in the colonic cavity or in the blood stream. In addition, it is not known whether the same compounds are responsible for initiating tumors in both the proximal and distal regions of the colon for which different risk factors have been described (Weisburger, 1987). Generally, host-mediated activation of compounds to genotoxins and their subsequent dissemination in vivo is considered to be a function of the liver and enterohepatic circulation. However, in the case of colon cancer, we believe that the metabolic activity of the colonic microflora also plays a major role. The human colonic microflora is among the most complex bacterial ecosystem in nature comprising more than 400 species, primarily anaerobes, which usually exceeds 101° organisms per gram of colon contents and accounts for over 30% of the total fecal mass (Moore et al., 1978; Drasar, 1988). Consequently, while the liver is metabolizing xenobiotics for subsequent elimination, the coIonic microflora is "re-metabolizing" some of these hepatic metabolites as well as unabsorbed dietary components in order to maintain the steady state of their bacterial ecosystem. While hepatic metabolism is generally oxidative (often involving "activation" of compounds by the addition of molecular oxygen), metabolism of the colonic flora is generally reductive (often using the same compounds as final electron acceptors). While the liver is busy conjugating compounds to molecules such as glucuronic acid and sulphate, the bacteria in the colon are busy deconjugating these compounds which have reached the colon through bile (Rowland, 1988). Thus, from a chemical perspective, the colonic flora tends to reverse the metabolism of the liver, often resulting in reabsorption of compounds which were originally directed toward excretion. Consequently, the colonic microflora must always be considered when studying the "host" metabolism of endogenous or exogenous

compounds, particularly those compounds which may cause human cancers (Hennegan, 1988). Historically, studies on the activation of compounds by the bacteria in the colon have focused on characterizing the metabolism of compounds in bacterial cultures or comparing their metabolism in conventional and germ-free animals (Hill, 1989). The main drawbacks to "closed" (in vitro) culturing of the colonic microflora are the severe depletion of nutrients and accumulation of toxic waste products as well as the likelihood that the diversity of bacterial species will be quickly diminished by overgrowth of a few hardy species. Although attempts have been made to model the colon by continuously culturing the microflora in chemostats, these systems also suffer from selective overgrowth as well as the lack of a growth "medium" which accurately reflects the ileocecal effluent and mucosal secretions to which the microflora has become adapted (Coates et al., 1988). Consequently the experimental animals, particularly mice and rats, have become the standard "continuous-flow" models for studying the bacterial metabolism of xenobiotics. If a metabolite is found in the feces of conventional animals and not in the feces of germ-free animals, the metabolite is probably a product of the colonic flora. Although this simplistic premise is not in itself false, interpretation of results from such animal studies - - as well as studies with bacterial cultures - - must be tempered with caution when extrapolated to the microflora of man (Wilkins, 1983). There are considerable differences between the basal metabolism of conventional and gnotobiotic rodents, as well as the intestinal metabolism of experimental animals and man (Rowland, 1986; Heneghan, 1988). Significant differences in basal metabolism also exist between bacteria in broth culture and bacteria in the "colloidal" colonic environment. Nevertheless, several classical studies in vitro and in vivo have shown that a wide range of bacterial reactions may occur which can lead to the activation or production of intestinal mutagens and carcinogens. Whether or not these "activation" studies reflect reactions for which cancer researchers should show concern is still debated. The bacterial reactions reported in previous studies may be grouped into several broad classes including hydrolysis, dehydroxylation, dehydro-

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genation and reduction. The hydrolytic reactions involve a variety of glycosidases, such as fl-glucosidases, fl-galactosidases, fl-glucuronidases, sulfatases and lyases (Brown, 1988; Larson, 1988). Glycosides generally reach the colon as dietary (plant) flavanoids or hepatic glucuronides. By hydrolysis of certain glycosides the bowel flora may release genotoxic aglycones which include the classic methylazoxymethanol, quercetin and benzo[a]pyrene. The bacterial reactions involving dehydroxylation and dehydrogenation are relevant to cancer in that they affect metabolism of primary to secondary bile acids. The secondary bile acids have been shown to be co-carcinogens and promoters of tumors in experimental animals. Although there is controversy as to what role these compounds may play in colon carcinogenesis in humans, epidemiological and carcinogenicity studies have convinced many researchers that the bile acids and their bacterial metabolites are involved. Once tumor initiation occurs, the secondary bile acids may "promote" the transformed cells to a fully progressed neoplasia, a process which may be as important in colon carcinogenesis as initial mutations or other transforming events. Bacterial reductions are performed primarily by the nitroreductases and azoreductases. It has been shown that the reduction of nitrates and aromatic amines by the colonic bacteria can result in formation of reactive N-nitroso compounds and substituted aromatic amines (Mallet, 1988). Similarly, reductive cleavage of the azo dyes found in foods and cosmetics can result in formation of toxic metabolites such as naphthylamines and phenylamines. Recently, bacterial deamination of amino acids (Wrong, 1988), biotransformation of toxic metals (Rowland, 1988) and hydrolysis of phytooestrogens (Setchell and Caenepeel, 1988) also have been discussed as being potentially important in carcinogenesis. Once again however, whether or not these metabolites play a role in human cancers remains speculative. The bacteria in the human colon primarily subsist on the hosts' mucins and secretions and certain components of dietary fiber such as hemicellulose and mucins (McCarthy and Salyers, 1988). Consequently, changes in the diet do not significantly alter the types and numbers of bacterial species which comprise the human microflora

(Mallet and Rowland, 1988). However, the metaboric activity of these bacteria may be changed by diet without changing the bacterial species themselves. The manner in which diet influences bacterial metabolism within the colonic milieu is complex and a detailed account is beyond the scope of this minireview. Several reviews are available for the interested reader (Wilkins and Van Tassell, 1983; Goldman, 1983; Drasar and Barrow, 1985; Mallett and Rowland, 1988). However, since the diet not only is a major source of colonic promutagens and procarcinogens but also affects enzyme expression of colonic bacteria in general, mention of the two major dietary components associated with colon cancer, fiber and fat, and their interaction with the colonic microflora is in order. Dietary fibers, those poorly defined and highly variable complex carbohydrates, can dictate which of the degradative bacterial enzymes involved in carbon and energy metabolism will be induced or repressed and which bacterial products including volatile fatty acids and ammonia will be generated. It is the degradative enzymes (e.g., glycosidases, cellulases, lyases, etc.) which create a "fermentative" environment which is more or less conductive to the generation of DNA-damaging substances from dietary components. Unfortunately, studies using experimental animals and humans which try to determine which "fibers" effect the expression of which enzymes have provided conflicting results, thus definitive conclusions cannot be made. In addition to directly affecting bacterial metabolism, there is also evidence that dietary fibers also can affect the interaction of bacteria with potential genotoxins by binding or diluting the compounds and decreasing transit time thereby limiting their concentrations or periods of exposure to the microflora. Dietary fat, on the other hand, also can affect bacterial metabolism of genotoxins, primarily by inducing increased biliary secretion of primary bile acids. As more bile enters the intestines, particularly the proximal region of the colon, the levels of certain "nondegradative" enzymes (e.g. glucuronidases, reductases, etc.) are increased thereby enhancing the potential of the microflora to deconjugate or reduce hepatic metabolites, whose colonic levels also have increased as a result of increased biliary

212 secretion. In addition, the increased levels of primary bile acids result in a colonic environment in which certain genotoxins are more soluble, as well as result in greater colonic levels of the bacterially-derived tumor promoting secondary bile acids. Although the forementioned effects of fiber and fat are but a few examples of how diet can alter the metabolism of the colonic microflora, there are many other contributing dietary factors which affect colonic metabolism, both host-derived (Laitinen and Watkins, 1986) and bacterial (Tannock, 1983) as well as affect colon carcinogenesis (Weisburger, 1989). Most of the conclusions on the effects of diet are based on products excreted in, (i) feces of animals during feeding studies or (ii) feces of individuals in populations with different diets, at different risks for colon cancer or both (Reddy, 1989). Subsequently, few conclusions can be made as to the direct effects of diet in vivo. Without opening a virtual "Pandora's box" concerning the role of fiber, fat, and other dietary constituents in colon carcinogenesis, for which "Western" man is at highest risk, we would like to reiterate a simple premise - - regardless of how one views the relationship between "diet" and colon cancer, the colonic microflora can and does act as an important intermediary. In our laboratory, we are currently studying the metabolism of two classes of compounds produced in vivo by the colonic microflora and the potential involvement of these compounds in human colorectal cancer. These are the fecapentaenes and the heterocyclic amines (a.k.a. the pyrolysis carcinogens). The remainder of this review will focus on our research efforts on the fecapentaenes and the heterocyclic amines, with emphasis on their bacterial metabolism and the consequences thereof.

Fecapentaenes, historical perspectives Since their discovery in 1977 by Bruce et al. (1977) the fecapentaenes have been the focus of much multidisciplinary international research. Most of the early work concentrated on their (i) isolation from feces, (ii) structures, (iii) chemical synthesis and (iv) biological effects. Two forms of these polyunsaturated 1-O-(1-alkenyl)glycerols

have been isolated and described (Hirai et al., 1982; Baptista et al., 1984) - - a 12-carbon form, fecapentaene-12 and a 14-carbon form, fecapentaene-14. These novel compounds are unstable in air, mild acid and light, hence they are very difficult to work with. Nevertheless, fecapentaene12 has been synthesized in quantity by several research groups (Gunatilaka et al., 1983; deWit et al., 1984; Pflaendler et al., 1986; Govindan et al., 1987) and it has been tested in a wide variety of short-term genotoxicity testing systems. The fecapentaenes are directly active in most short-term systems in which they have been tested. The fecapentaenes are potent mutagens in the Ames test on both TA98 and TA100 (Wilkins et al., 1980; Goggleman et al., 1986; Govindan et al., 1987; Curren et al., 1987; Peters et al., 1988). In mammalian cells they induce unscheduled D N A synthesis and cellular transformations (Curren et al., 1987), mutations and sister-chromosome exchanges (Plummer et al., 1986), single-stranded breaks in D N A (Hinzman et al., 1987; Plummer et al., 1986) and nuclear aberrations and mitotic figures (Vaughn et al., 1987). These compounds are the most prevalent mutagens found in the colon (Dion and Bruce, 1983), accounting for a majority of the direct-acting fecal mutagenicity (Schiffman et al., 1989). In recent years, most of the research on the fecapentaenes has been on their epidemiology, mechanism of action, carcinogenic potential and origin(s). Epidemiological studies have shown that the fecapentaenes are excreted in the feces of most ( > 75%) North Americans (Schiffman et al., 1989a, b). Although the relative amounts of endogenous F12 and F14 vary considerably among individuals (Van Tassell et al., 1986), their ratios within any one individual can remain constant for years (Wilkins and Van Tassell, 1983). In a collaborative effort coordinated by Dr. Mark Schiffman of NCI we showed that both the fecapentaenes and their precursors are excreted in significantly lower concentrations in individuals who have colorectal cancer than in normal individuals (Schiffman et al., 1989b). Although the diagnostic work-up or the diseased state itself might be expected to alter the fecal concentrations of these compounds, control experiments examining the effects of cleansing regimens, colonoscopy

213 and colorectal bleeding showed no significant reasons why lower fecapentaene levels were observed in colorectal cancer cases. Although these lower levels suggest that these compounds somehow may be involved in protection - - as will be discussed later - - the overall implications of these recent observations are unclear and can only be determined by further studies on the metabolism, tissue-binding, carcinogenicity and origin(s) of these compounds. What is clear, however, is that excretion of the fecapentaenes, or their precursors, is not necessarily a good marker for risk for colorectal cancer. If any association exists for these genotoxic lipids and colorectal cancer it most likely occurs early in the natural history of the disease, i.e. when the tumors are initiated, and not years later at the time of diagnosis. The studies on the chemistry and mechanism of action of the fecapentaenes primarily have involved synthesizing stereoisomers and analogs and comparing their genotoxicities in a variety of short-term tests. These studies are revealing the existence of reactive electrophilic intermediates which are likely the actual genotoxic forms which react with DNA. For example, a recent report showed that both F12 and F14 can hydroxylate the C-8 position of guanine in D N A in vitro (Shioya et al., 1989). For details of the chemistry and postulated mechanisms of action of these compounds see the excellent reviews of Venitt (1988) and Krepinsky (1988). There are currently several studies underway to determine the carcinogenic potential of the fecapentaenes in experimental animals. The first completed study, a collaborative NCI effort (Ward et al., 1988), found no evidence that these mutagens induce tumors in the colon or in other sites of rat or mice. However, as has been observed over the years in our laboratory and has been recently described in detail by the NCI group (Streeter et al., 1989), the fecapentaenes are notoriously unstable compounds which require special precautionary considerations when designing and conducting carcinogenicity or other biological assays. And, even when the utmost care is taken to control for "decomposition" of the fecapentaenes before and after administration to animals, there is currently no way to determine if these unstable compounds reach D N A targets in a biologically

active form. Nevertheless, a recent collaborative effort between our research group and that of Dr. John Weisburger of the American Health Foundation indicated that fecapentaene-12, although not active when intrarectally administered to rats, does have weak carcinogenic activity in newborn mice (Weisburger et al., 1989). When newborn mice were injected with fecapentaene-12 intraperitoneally, low but significant numbers of lung, liver and stomach tumors and subcutaneous sarcomas were observed. This observation - - perhaps reflecting a decreased level of cellular protective mechanisms in the newborn rodent - - indicates that although the fecapentaenes are not potent carcinogens, they may be weak ones which cannot be detected in most standard animal bioassays. The inability of standard bioassays to demonstrate the carcinogenic potential of the fecapentaenes may reflect (i) the forementioned methodological problems concerning the instability of these compounds, (ii) the involvement of efficient protection mechanisms in adult animals or (iii) both of these possibilities. If mechanisms at the cellular level in the colon protect adult animals from the genotoxic effects of the fecapentaenes, then contributing factors such as polyps (known "predisposers" of colon cancer), bacterially-induced colitis (e.g.C. difficile toxicity) or other "conditions" in which the natural cellular defenses are compromised and cell growth is stimulated may play a major role in the in vivo "expression" of fecapentaene carcinogenicity. In addition, if the fecapentaenes are human carcinogens, that they may be weak ones, as opposed to potent ones, would not be surprising. In fact, it would be expected since they are so prevalent within individuals in Western populations ( > 75%) and often occur in relatively high concentrations in vivo (up to 50 g g / g ) yet the lifetime probability of developing colorectal cancer is only about 3-5%.

In vitro production of fecapentaenes We showed several years ago that the fecapentaenes are produced by several species of the common intestinal anaerobes Bacteroides (Van Tassell et al., 1982a) and that their production in vitro is greatly enhanced by bile (Van Tassell et al., 1982b). These observations were key factors in the early isolation work; in vitro anaerobic in-

214 cubation of feces provided researchers with a means of increasing their purification yields by 50-100-fold. Although colonic bacteria and bile are required for production of the fecapentaenes, precursor molecules must also be present (Van Tassell et al., 1986; Schiffman et al., 1989b). However, the origins of these precursors are unknown. They are found in uniform concentrations throughout the colon of an individual, yet like the fecapentaenes, their concentrations vary greatly among a group of individuals (Schiffman et al., 1988). In addition, like the fecapentaenes, the plasmalopentaenes also are excreted in significantly lower amounts by colon cancer patients than normal individuals. It is the nature of these precursors, particularly their origin and structures, which has become the focus of our work over the past few years.

Isolation of precursors of fecapentaenes Purification and structural elucidation of the precursors has been confounded by the same obstacles encountered during purification and structural elucidation of the fecapentaenes themselves: their instability to oxygen, light and mild acids, their low concentrations in feces and their molecular heterogeneity. Overcoming these obstacles, as described in a preliminary report (Van Tassell et al., 1986), we observed that the precursors (i) have the same UV absorbance spectrum as the fecapentaenes, (ii) contain functional groups covalently linked to the sn-2 and sn-3 positions of the glycerol backbone and (iii) behave like phospholipids. In a recent report (Van Tassell et al., 1989) we described the methods for isolation and purification of a major form of precursor from feces using a series of extractions, precipitation and two normal phase HPLC techniques. In addition, we characterized the purified precursor as to its chromatographic properties by comparison to the fecapentaenes and a synthetic " m o d e l " ether phospholipid. Although our results supported our beliefs that the precursors are phospholipids, the evidence remained circumstantial and indirect. However, this changed when we recently examined the effects of various lipolytic enzymes on the precursor molecules.

Enzymology of fecapentaene production Purified precursor is converted to fecapentaene when incubated with whole cells or membranes of certain Bacteroides species. Recently, using "micellar enzymology", we used commercial lipolytic enzymes to model what is likely occurring in the bacteria (Van Tassell et al., 1989). We incorporated purified precursor into artificial membranes using a combination of standard techniques for producing liposomes and the anaerobic techniques required for handling pentaenyl compounds. When the "precursor" liposomes or micelles were incubated with combinations of commercial enzymes, hydrolysis to fecapentaene occurred with a mixture of lipase and phospholipase C. None of the individual classes of phospholipases (A, B, C or D), sphingomyelinase or any of several lipases alone converted the purified precursor to fecapentaenes. Although the bacterial membranes converted precursor to fecapentaene whether or not the precursor was associated with liposomes or micelles, the "free" commercial enzymes hydrolysed the precursor only when it was incorporated into the artificial membranes. In all cases, the form of fecapentaene produced by bacterial membranes or commercial enzymes from the purified precursor was fecapentaene-12. That two different lipolytic enzymes are required for hydrolysis of precursor to fecapentaenes fits well with our observations on the bacterial membranes. The bacterial lipolytic enzymes which produce the fecapentaenes are intrinsically associated with the membranes of the Bacteroides. When purified bacterial membranes are dissociated with detergents and assayed under non-micellar conditions using "free" precursor, no fecapentaenes are produced. It appears that under dissociated conditions a complex of two or more bacterial enzymes is disrupted and rendered nonfunctional. However, under micellar conditions, using precursor micelles, membranes disrupted by the mild non-ionic detergent octyl-glucoside retain hydrolytic activity and produce fecapentaenes too. From these observations we predict that the Bacteroides have a lipase and phospholipase-C complexed within their membranes - - whose normal role is most likely turnover of bacterial membrane phospholipids - - but which fortuitously

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hydrolyse precursors to the fecapentaenes within the colon contents.

The structure of precursors of fecapentaenes The hydrolysis of precursors of fecapentaenes by Candida lipase and Clostridium phospholipaseC confirms their general structures as phospholipids. In addition, based on these observations we can deduce the mechanism by which the hydrolysis occurs. The phospholipase C removes the phosphate group from the sn-3 carbon; the lipase then removes the ester-linked functional group from the sn-2 carbon yielding the fecapentaene (see Fig. 1). The pentaenyl group remains because of the inability of the enzymes to hydrolyse ether linkages. We have now obtained the structure of the purified precursor by direct chemical analysis using specific degradation and GC-MS. The sn-3 polar unit is a phosphoryl ethanolamine and the sn-2 acid portion is a mixture of 16-18-carbon fatty acids (Kingston et al. 1989). The sn-1 group is the ether-linked 12-carbon pentaenyl chain as was suggested by the studies using the lipolytic enzymes. Thus the purified precursor is a heterologous group of polyunsaturated ether-linked phosphatidylethanolamines. Being 1-O-(1-pentaenyl) ether-linked glycerophosphatides, the precursors are members of the class of lipids known as plasmalogens (1-O-(1-alkenyl)phospholipids). We

Plasmalopentaene Upose

~

Phosphollpase C

C-O-C O ~ C I .H C-O--PO--C--C--N--I'~" / I_ "H tO

~

C-O-C I C-OH I O-OH Fecapentaene Fig. 1. Hydrolysis of plasmalopentaenes to direct-acting fecapentaenes by phospholipase-C and lipase, the presumed mechanism by which several species of Bacteroides produce these novel genotoxins in vivo.

have proposed referring to the precursors of fecapentaenes (as a general class) by the common name of "plasmalopentaenes". To our knowledge, they are the first examples of such highly conjugated polyunsaturated phospholipids to be isolated from biological materials.

The origin of the plasmalopentaenes Determining the origin of the plasmalopentaenes has proven to be the most difficult aspect of our work to date. For years we have considered the 3 probable origins to be the diet, the colonic microflora or the host itself. Using autopsy samples from humans and pigs (the only other animal shown to excrete fecapentaenes) we showed that plasmalopentaenes were distributed in uniform concentrations throughout the colons of both species (Schiffman et al., 1988). Since we found no plasmalopentaenes in the small intestines, we concluded that they probably do not originate in the diet. If they entered the colon as components of digested food, a dramatic decrease in their levels - - with a corresponding increase in fecapentaene levels - - from proximal to distal regions would be expected. This was not observed. During our early work, we felt that the bacterial microflora itself might produce the plasmalopentaenes. Ether lipids are common membrane components of anaerobic bacteria and should be present in relatively high amounts in the colon contents. However, after years of trying to isolate a precursor-producing bacterium, we recently obtained results which appear to rule out the possibility of a bacterial origin. Using standard HPLC screening techniques, we analysed the feces of 12 neonatal germ-free pigs. In 11 of the 12 pigs we detected plasmalopentaenes; we did not detect any fecapentaenes (Van Tassell and Wilkins, 1989). Since the plasmalopentaenes were present in the feces in the absence of a bacterial flora and neither fecapentaenes nor plasmalopentaenes were detected in the diets, the precursors likely are made by the host. The polyunsaturation and conjugation of the plasmalopentaenes make them unique 1-O(1,3,5,7,9-alkenyl) plasmalogens which resemble the plasmalogens found in most animal tissues. Although few specific cellular or physiological

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functions have been proven for plasmalogens in biomembranes it is felt that they may play an important role as cellular defense barriers as well as maintaining membrane stability, permeability and conductivity. Recent observations suggest that membrane plasmalogens may protect cells by scavenging toxic oxidative radicals from cellular environments (Zoellar et al., 1988), thus they may also act as "sinks" for certain genotoxins. Considering that a highly conjugated polyene chain may enhance a plasmalogen's ability to trap free radicals, the plasmalopentaenes may be better suited to this task than normal plasmalogens. If the plasmalopentaenes do have such a protective function at the cellular level in the host, thereby acting as antigenotoxins, then an interesting scenario may exist. That is, the plasmalopentaenes may be protecting cells from their own bacterial metabolites, the fecapentaenes. This would once again demonstrate the "fine line" which cancer researchers often encounter when trying to distinguish between potentially harmful and potentially beneficial compounds. For a discussion on the structures and functions of natural and "unnatural" plasmalogens, as well as other ether lipids, the reader is directed to the excellent reviews of Mangold and Weber (1987) and Mangold and Paltauf (1983). Because it is felt that the majority of the plasmalogens found in humans are synthesized by the human tissues themselves, we are now concentrating on screening human and porcine tissues for these mutagen precursors. Once the origin of the plasmalopentaenes is determined, we may finally be able to address the questions: How are these unique phospholipids synthesized in vivo?, How do they reach the colon where they are converted to potent mutagens?, What is their role in normal cellular physiology and, could this role be antimutagenesis, anticarcinogenesis or both?

The heterocyclic amines, historical perspectives The formation of potent carcinogens in proteinaceous food during cooking has been known for many years (Commoner et al., 1978; Sugimura and Sato, 1986). The mechanisms by which these "pyrolysis" carcinogens are formed (Jagerstad et al., 1986), or by which their formation is inhibited

(Weisburger, 1989b), have been extensively studied and the structures of many of these heterocyclic amines have been determined. IQ is one of the most potent of this broad class of compounds which also includes MelQ, MelQx, Try-P-2, GluP-l, Orn-P-1 and Phi-P (Kasal et al., 1980; Hargraves and Pariza, 1983). IQ, like most of these heterocyclic amines is highly mutagenic in the Salmonella/microsomal mutagenicity assay on TA98 when activated by hepatic microsomal enzymes (Kasai et al., 1980; Grivas and Jagerstad, 1984; Carman et al., 1988). IQ is also active in a variety of other bacterial and mammalian shortterm mutagenicity tests. It induces forward mutations in two other bacterial systems and in cultured mammalian cells it induces sister-chromatid exchanges, unscheduled DNA synthesis, in vitro transformations and DNA-adduct formation (Wild et al., 1986; Holme et al., 1987). Although IQ is a potent mutagen, its carcinogenicity is the major reason for concern. In laboratory mice, high levels of IQ in the diet induce tumors of the liver, stomach and lungs. In orally challenged rats, it induces tumors in organ sites which include the liver, small intestines, colon, and skin as well as the oral cavity (Barnes et al., 1983; Weisburger et al., 1986). Consequently, it is believed that human consumption of IQ and the other heterocyclic amines may play a role in the etiology of colorectal and other cancers (Weisburger, 1988). The pharmacokinetics of the heterocyclic amines have been studied in experimental animals. The distribution of these compounds in humans has also been studied, albeit to a lesser and indirect extent, using subjects who consumed various levels of fried meats. Although each compound has its own distribution and excretion pattern, it generally can be found in both the urine and feces in parental and conjugated forms, including the carcinogenic N-acetylated form (Sjodin and Jagerstad, 1984; Sato et al., 1986; Hayatsu et al., 1987; Stormer et al., 1987). In 1984, we began to study the fate of the heterocyclic amines once they reach the colonic flora. We decided to (i) synthesize radiolabeled compounds, (ii) screen them for the ability to be metabolized in feces and bacterial cultures, (iii) purify and characterize any observed metabolites,

217 NH2

NH=

IQ

7-hydroxy-IQ

Fig. 2. Hydroxylation (hydration-dehydrogenation) of IQ to direct-acting 7-hydroxy-IQ by the colonic microflora - specifically, species of Eubacterium and Clostridium.

(iv) synthesize these metabolites and (v) determine their genotoxic potential. After screening radiolabeled representatives of what were then the major classes of the heterocyclic amines, IQ, Try-P-2 and GIu-P-1, we observed that [14C]IQ was converted to a major bacterial metabolite in whole feces and anaerobic cultures of diluted human feces (Bashir et al., 1987a, b). The reaction was an anaerobic "hydroxylation" (hydration-dehydrogenation) at the C-2 position of the quinoline ring (position 7 of IQ) yielding the metabolite 2-amino-3,6-dihydro-3-methyl-7H-imidazo[4,5-f]quinoline-7-one shown in Fig. 2. Metabolism of IQ by colonic bacteria

Recently we described the in vitro production of this metabolite, 7-hydroxy-IQ, by a mixed fecal consortium and by pure cultures of Eubacterium and Clostridium spp. (Carmen et al., 1988). We showed that 4 species of Eubacterium produce 7-hydroxy-IQ from IQ in vitro; E. moniliforme VPI strain 13480 was the most active strain and became our reference strain for further studies. Using analytical HPLC, we determined that VPI 13480 reversibly converted IQ to 7-hydroxy-IQ during all growth phases. Using selective and enrichment techniques, we isolated a bacterial strain from feces which could hydroxylate IQ and which closely resembled E. moniliforme biochemically. The reversible hydroxylation of IQ seemed similar to the reversible hydroxylation of nicotinic acid reported for Clostridium barkeri (Harary, 1957; Tsai et al., 1966). We found that pure cultures and cell-free extracts of C. barkeri produced 7-hydroxy-IQ as efficiently as many of the strains of Eubacterium.

Genotoxicity of 7-hydroxy-IQ

We characterized IQ and 7-hydroxy-IQ for mutagenicity in the Ames assay using preincubation modifications on tester strains TA98 and TA100 with and without microsomal activation. As reported by others, IQ was highly mutagenic on TA98 and less mutagenic on TA100 when activated by the liver microsomes. Conversely, 7hydroxy-IQ was directly mutagenic on TA98; no activity was observed on TA100 (Carman et al., 1988). Since this first report on the bacterial conversion of IQ and the direct mutagenicity of 7-hydroxy-IQ on TA98, we have observed considerable variability of 7-hydroxy-IQ in its mutagenicity toward this tester strain. Some of the variability - - as is often the case in "Ames" testing (Arimoto et al., 1982; Booth et al., 1980) - - is a function of the solvent used to dissolve the compound, the duration of the preincubation step and the age of the "overnight" tester strain broth culture. However, preliminary data from our laboratory indicates that 7-hydroxy-IQ may not be as stable as IQ, thus we are currently performing long-term stability studies to determine what, if any, degradative products are formed. The effect of liver-microsome preparations on the mutagenicity of 7-hydroxy-IQ also varied, depending on the source and lot of the $9 used. While one commercial $9 reduced the TA98 mutagenicity of 7-OH-IQ by 50-75%, another $9 (from a different supplier) enhanced its TA98 mutagenicity 2-3-fold over that observed without $9 (unpublished data). This most likely reflects the variability in P-450 microsomal enzyme induction that occurs during $9 preparation due to subtle differences in, (i) the compounds used to induce the liver, (ii) the strains of rodents induced and (iii) the induction protocols used by the various manufacturers. In addition to short-term testing in Salmonella we have recently adopted the SOS Chromotest as an adjunct rapid screening method for directacting genotoxins. Although in our hands, the fecapentaenes are "dose-response" positive in the SOS system, 7-hydroxy-IQ is negative up to 100 /~g/ml (unpublished data). This is not, however, unexpected in that it has been shown that compounds which are "frameshift" mutagens, i.e. active only on TA98, are often negative in the SOS

218 system (vonder Hude et al., 1989). In addition to IQ, we have studied the fecal metabolism of other "quinoline" pyrolysis mutagens including the highly mutagenic MeIQ. Like IQ, MeIQ also was hydroxylated to a direct-acting form in feces and cultures of E. moniliforme. We have synthesized 7-hydroxy-MeIQ (Bashir et al., 1989) and have determined the mutagenicity of the synthetic compound to be the same as the bacterially derived form. Like 7-hydroxy-IQ, 7-hydroxy-MeIQ was directly mutagenic only on TA98. Production of 7-hydroxy-IQ in humans In order to determine if 7-hydroxy-IQ is produced in vivo, two individuals volunteered to eat fried meats prepared in ways which have been reported to maximize the formation of IQ (Van Tassell et al., 1989). In addition, since the binding of heterocyclic amines to colonic contents such as bacterial cell walls and dietary fibers has been demonstrated (Barnes et al., 1983; Howes et al., 1989), the fried meats were consumed during periods of fasting in order to minimize such binding of IQ or its metabolites in vivo. From the feces of both subjects, both IQ and 7-hydroxy-IQ were isolated. Of these two compounds, only IQ was detected and isolated from the unconsumed fried meat controls. Admittedly, the relatively high amounts of fried meats used in our in vivo study resulted in fecal levels of IQ and 7-hydroxy-IQ which were far above that present in "normal" feces. Yet, our relative recoveries of IQ and 7-hydroxy-IQ from feces generally agreed with the levels which would be expected in feces based on previously published levels of IQ in cooked foods. Thus, the direct activity of 7-hydroxy-IQ in Salmonella, combined with the likelihood of a continued low level of exposure in vivo in persons consuming "normal" western diets, continues to raise concerns over its possible role in colon carcinogenesis. We are currently beginning genotoxicity testing of 7-hydroxy-IQ in mammalian cell systems and carcinogenicity testing in experimental animals. Until its genotoxic and carcinogenic potential is thoroughly examined, the importance of our current findings remains indeterminate. If it is a carcinogen, there are serious implications to its

production by the colonic microflora. Formed by the colonic bacteria in vivo, 7-hydroxy-IQ would not have to undergo enterohepatic circulation before exerting its effect on DNA. As with the fecapentaenes, the possibility exists that 7-hydroxy-IQ could directly target the DNA of mucosal cells of the colon and initiate tumor formation. Promotion, that all too often disregarded second stage of carcinogenesis, would then be the deciding factor as to whether a carcinoma develops. Conclusions The relationship among the diet, the host and carcinogenesis has proven to be one of the most complex problems in human health and disease. Unfortunately, the human colonic microflora has often been disregarded when considering the "host" metabolism of potentially genotoxic compounds in vivo. Fortunately this situation is rapidly changing. An increasing amount of evidence, particularly that concerning the fecapentaenes, clearly shows that the colonic bacteria are involved in the generation of active, potentially harmful colonic genotoxins as well as being involved in tumor promotion. What is not resolved is the importance of the bacterially-derived genotoxins in colon carcinogenesis. If they are involved in this disease, they are obviously only one of many contributing factors which include genotoxic hepatic metabolites, chemical and physiological promoters and host-related factors such as age, genetic predisposition and organ site specificity (Weisburger, 1987). However, our synergistic life with the coIonic microflora - - and its potential to metabolically activate compounds - - may be one risk factor, particularly in the case of colorectal cancer, which may be amenable to measures of prevention such as dietary intervention, i.e. preventing the formation of the heterocyclic amines during cooking, or alteration of the flora itself, i.e. modulating the Bacteroides species which produce the fecapentaenes. Whereas the former approach is beginning to be realized (Weisburger, 1989b), the latter is well beyond current microbiological capabilities. The days of changing ones' " b a d microflora" for a "good microflora", which of course are relative terms and still undefined, remain far in the future - - if possible at all!

219

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