Metabolic differences in colon mucosal cells

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1 Metabolic differences in colon mucosal cells Ross A. McKinnon a Wendy M. Burgess a, Frank J. Gonzalez b and Michael E. McManus c a Department of Clinical Pharmacology, Flinders University, South Australia 5042, Australia, b Laboratory of Molecular Carcinogenesis, National Institutes of Health, Bethesda, MD 20892, USA and c Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Queensland 4067, Australia Summary The colonic expression of cytochromes P450 from the CYPIA, CYP3A and CYP4B subfamilies has been characterized in rabbit and human tissues using RNA blotting, immunoblotting, immunohistochemistry and hybridization histochemistry. These studies demonstrate negligible expression of the CYP1A subfamily in either rabbit or human colon. The CYP3A6 gene is expressed in rabbit colon although at markedly reduced levels relative to liver and small intestine. Whilst at least two CYP3A genes are expressed at the mrna level in human colon tissue from some individuals, no expression was demonstrated in others. Where expression was observed, this expression was continuous throughout the length of the colon. In rabbits, CYP4B1 represents a major colon P450 enzyme, expressed at levels in colon comparable to liver and small intestine. In contrast, the human CYP4B1 gene is expressed at low levels in some individuals. These studies highlight individual differences in the expression of cytochrome P450 enzymes of importance in procarcinogen metabolism. Keywords: Colon mucosal cells; Cytochromes P450; CYP genes Abbreviations: MelQ, 2-amino-3,4-dimethylimidazo[4,5-f]- quinoline; PhIP, 2-amino-l-methyl- 6-phenylimidazo[4,5-b]- pyridine. The nomenclature used to define each of the P450s discussed in this report has been published (Nelson et al., 1993). The gastrointestinal tract represents a major portal of entry for many toxic chemicals and carcinogens. Indeed, Ames et al. have estimated that the vast majority of chemicals to which humans are exposed derive from dietary sources and must therefore traverse the gastrointestinal tract (Ames et al., 1987, Ames, 1989). Given that the vast majority of chemical carcinogens require metabolism before being mutagenic/carcinogenic, it is essential to characterize the xenobiotic metabolizing enzyme systems of all gastrointestinal tissues. Further stimulus for studies of xenobiotic metabolizing enzyme expression in the col- orectal region is provided by the high incidence of malignancies in this region Cytochromes P450 comprise a superfamily of structurally and functionally related heme proteins involved in the phase I metabolism of numerous xenobiotics and endogenous substrates (Nelson et al., 1993). Individual cytochrome P450 isoforms are characterized by diversity in developmental regulation, substrate specificity and tissue distribution. Whilst the majority of cytochrome P450 catalysed oxidations result in diminished toxicity of the parent compound, on occasion this process may generate a more toxic intermediate. Many such metabolites have been implicated in carcinogenesis Substrates for P450 enzymes include a variety of procarcinogens including a group of compounds termed food-derived heterocyclic amines, isolated from cooked meat (Sugimura et al., 1988; Felton et al., 1990) The activation of these compounds to genotoxins involves an obligatory P450 catalysed N- hydroxylation reaction prior to further metabolism by phase II enzymes such as sulfotransferases, Nacetyltransferases and UDP-glucuronosyltransferases. Studies in this laboratory and others have demonstrated that in liver, this obligatory process is primarily catalysed by members of the CYP1A subfamily (McManus et al., 1988a,b, 1989, 1990; Aoyama et al., 1990). In addition, we have recently demonstrated a possible role for CYP3A

2 enzymes in this process in gastrointestinal tissues in the presence of dietary flavonoids (McKinnon et al., 1992). If the colorectal mucosa possesses a significant CYPIA or CYP3A complement, it is possible that activation of foodderived heterocyclic amines to ultimate carcinogens may occur in close proximity to their putative target tissue. Alternatively, the N-hydroxylated metabolites may be formed in liver and transported to the gastrointestinal tract where they may undergo further metabolism by the various phase II enzyme systems. The possible pathways of food-derived heterocyclic amine activation in gastrointestinal tissues are represented schematically in Fig. 1. Given the high incidence of malignancies in the colorectal region, it is prudent to fully characterize the colonic expression of cytochrome P450 enzymes implicated in the activation of procarcinogens such as the food-derived heterocyclic amines and other aromatic amines. The current understanding of the human cytochrome P450 system is based primarily on hepatic studies given its role as the major drug metabolism tissue. In contrast, colorectal expression of individual cytochrome P450 enzymes is poorly characterized. Several studies have reported the expression of a CYP3A protein in human colon using immunoblotting techniques (Peters et al., 1989, 1992; de Waziers et al., 1991). It is unclear from these studies however which CYP3A enzyme(s) is being expressed. In addition, several other studies failed to demonstrate CYP3A protein in human colon (Murray et al., 1988; McKinnon et al., 1992). White et al. (1992) have recently demonstrated the presence of CYP1A protein in human colon microsomes, although several other studies have failed to demonstrate CYP1A expression in colon (de Waziers et al., 1990; McKinnon et al., 1992; Massaad et al., 1992). Given this limited and conflicting data, we have utilized a variety of techniques to further characterize cytochrome P450 expression in colorectal tissues from both rabbits and humans. These studies have focussed on several forms of P450 known to activate procarcinogens. The results demonstrate that whilst the expression of these cytochrome P450 genes in colon is generally minimal, the colon of both humans and rabbits possesses a distinct P450 profile relative to both liver and small intestine. In addition, considerable interindividual heterogeneity is observed in both the level of expression and the individual P450 genes expressed.

3 Expression of cytochromes P450 in rabbit and human colon CYPIA subfamily The CYP1A subfamily comprises two members in both humans and rabbits, designated CYP1A1 and CYP1A2 (Nelson et al., 1993). Using a variety of approaches including purified forms of cytochrome P450, cdna expression systems, hybridization histochemistry, immunochemical techniques and metabolic inhibition studies, we have demonstrated that both members of the CYP1A subfamily are expressed in rabbit and human liver and are primarily responsible for the activation of the food-derived heterocyclic amines (McManus et al., 1988a, 1989, 1990; McKinnon et al., 1991). Utilising a range of techniques including RNA blotting, immunoblotting, immunohistochemistry and hybridization histochemistry, we have been unable to demonstrate CYP1A1 or CYP1A2 expression in any of 18 specimens of human colon. Whilst RNA blotting of human colon mrna samples with CYP1A1 and CYP1A2 RNA probes did not demonstrate the presence of either CYP1A1 and CYP1A2 transcripts, a smaller 2.3- kb transcript was observed in a number of mrna samples. The identity of this transcript is presently unclear although it is unlikely to encode a highly related P450 protein given the lack of protein on immunoblots probed with anti-cyp1a IgG or antisera. In an attempt to gain an insight into the identity of this unknown transcript, a colon cdna library was screened using a CYP1A2 RNA probe which recognizes both CYP1A1 and CYP1A2 sequences. This screening resulted in the isolation of several clones which demonstrated complete sequence similarity with human liver CYPIA1, other than a single conservative base change. As a further indication of CYP1A expression, microsomes were prepared from 4 human colon specimens and used as the activating source for a series of Ames Salmonella tests assessing the activation of the model food-derived heterocyclic amine MelQ. Only one of the four samples of colon microsomes tested exhibited activity towards MelQ. This activity was exceedingly low relative to liver and was completely inhibited by a-naphthoflavone suggesting that in this colon, low level CYP1A expression may be present. Using a similar range of techniques to study the rabbit gastrointestinal tract, no CYP1A expression was observed in colon although CYP1A1 protein was detected on immunoblots of small intestine microsomes. This finding contrasts with that in humans where no CYP1A expression was observed in any gastrointestinal tissue. Collectively the results in both humans and rabbits indicate that the expression of CYP1A enzymes is 30 minimal in colorectal tissue. Based on this data, it therefore appears unlikely that gastrointestinal tissues contribute significantly to the activation of procarcinogens such as the food-derived heterocyclic amines whose activation is primarily dependent on CYP1A enzymes. CYP3A subfamily Stimulus for characterizing the expression of the CYP3A subfamily was generated by the observation that CYP3A enzymes may play a role in the activation of the food-derived heterocyclic amines in the presence of dietary flavonoids (Mc- Kinnon et al., 1992). The adult human CYP3A subfamily comprises at least three genes encoding highly related enzymes designated CYP3A3, CYP3A4 and CYP3A5 (Molowa et al., 1986; Beaune et al., 1986; Gonzalez et al., 1988; Aoyama et al., 1989; Schuetz et al., 1989). Immunoblot and RNA blot analysis of liver samples demonstrates that the CYP3A3 and CYP3A4 isoforms are not distinguishable electrophoretically. One or both of these enzymes represents the major liver species and is expressed in all individuals. A third form designated CYP3A5 has been observed in liver at the protein level in 19 of 66 (29%) subjects (Wrighton et al., 1990). Immunoblot studies in our laboratory have demonstrated the CYP3A5 enzyme in 5 of 22 liver microsomal samples (R.A. McKinnon, W.M. Burgess and M.E. McManus, unpublished results). As outlined earlier, several studies have documented that CYP3A protein is present in human colon although the precise isoforms present remain unresolved (Peters et al., 1989; de Waziers et al., 1991; White et al., 1991). In an attempt to address this problem we have employed a CYP3A4 RNA probe which recognizes all three CYP3A mrna sequences for RNA blotting and hybridization histochemistry analyses of CYP3A mrna in human colon specimens. RNA blot analysis of mrna samples from 11 human colons revealed marked heterogeneity in CYP3A mrna with at least two genes expressed in some colons and none in others (Table 1). A 2.2-kb band representing CYP3A3/3A4 was observed in 5 of the 11 samples. The level of this mrna species was considerably lower in colon specimens rela- tive to both small intestine and liver. A smaller 1.9-kb transcript of variable intensity was observed in 6 of 11 individuals. The lower molecular weight of this mrna is consistent with the bp CYP3A5 cdna as compared with the larger CYP3A3 and CYP3A4 cdnas (Aoyama et al., 1989). This smaller mrna is also observed in liver mrna samples from individuals demonstrating the presence of the CYP3A5 enzyme on immunoblot analysis. It therefore appears that the

4 CYP3A3/3A4 and CYP3A5 genes are both expressed at highly variable levels in human colon. The observation of CYP3A5 mrna in more than half of the colon specimens studied is of particular interest given the low incidence of CYP3A5 expression in liver (approximately 25-30%). In addition, we have been unable to demonstrate CYP3A5 mrna in the small intestine where CYP3A3/3A4 mrna and protein is abundant. The marked heterogeneity in CYP3A mrna patterns between colons does not appear to correlate with gender or the region of colon studied (Table 1). In addition, none of the subjects were being treated with known inducers of CYP3A enzymes. Hybridization histochemistry analysis of multiple segments of colon from single individuals confirmed that in individuals in whom CYP3A genes were expressed, expression was observed throughout the colon, although mrna levels in rectal specimens were generally very low. CYP3A mrna was highest in colon mucosal cells in the non-proliferative compartment. It remains presently unclear if the CYP3A3/3A4 and CYP3A5 mrnas are translated into proteins as we have been unable to demonstrate any CYP3A enzyme on immunoblots of colon microsomes treated with an anti-cyp3a3 IgG known to recognize these enzymes in liver microsomes. The possibility exists that this discrepancy may simply reflect the different sensitivity of the RNA blot and immunoblot techniques, particularly when RNA probes are utilized for RNA blotting. The rabbit CYP3A subfamily presently comprises only a single member designated CYP3A6. Immunoblot analysis of rabbit gastrointestinal microsomes demonstrates the presence of this enzyme in both the small and large intestine (Mc- Kinnon et al., 1992). The level of CYP3A6 decreases from the small to the large intestine, consistent with a decline in total cytochrome P450 levels. CYP4B1 The CYP4B subfamily comprises only a single gene product in rabbits, rats and humans designated CYP4B1 (Nelson et al., 1993). The CYP4B1 enzyme has created particular interest amongst researchers due to its species- and tissuedependent regulation (Robertson et al., 1983; Parandoosh et al., 1987), and capacity to N-hydroxylate the procarcinogen aromatic amine, 2-aminofluorene (Vanderslice et al., 1987). The gastrointestinal expression of CYP4B1 has not been characterized in human or rabbit tissues although analysis of rat mrna samples with a human CYP4B1 cdna demonstrated the presence of the putative CYP4B1 mrna in intestine (Nhamburo et al., 1989). This study did not however specify the segment of intestine analyzed. We therefore employed both blotting and histological analyses to characterize CYP4B1 expression in rabbit and human gastrointestinal tissues. Using both immunoblotting and RNA blotting, we were unable to demonstrate the presence of CYP4B1 expression in any human gastrointestinal tissue. However, employing a human CYP4B1 RNA probe for hybridization histochemistry analyses of routine formalin-fixed, paraffin-embedded biopsy specimens, we were able to detect low levels of CYP4B1 mrna in approximately 50% of human colons. Similar to the findings with the CYP3A subfamily, CYP4B1 mrna was present throughout the length of the colon and was present at highest levels in the non-proliferative cells. This finding demonstrates the enhanced sensitivity of histological techniques which preserve cellular integrity relative to blotting techniques. In contrast to the human situation, CYP4B1 expression was demonstrated in all rabbit gastrointestinal tissues other than stomach. This expression was demonstrated at both the protein and mrna level using both

5 blotting and histological techniques. Of particular interest was the observation that CYP4B1 levels in rabbit colon were comparable to those observed in both the small intestine and liver. This contrasts with the decline seen in total cytochrome P450 levels from small to large intestine and also the parallel decline in CYP1A and CYP3A expression between these tissues. Thus, this particular cytochrome P450 isoform demonstrates a novel pattern of expression in the gastrointestinal tract of both rabbits and humans. Discussion Characterization of cytochrome P450 expression in all gastrointestinal tissues is of considerable importance in understanding the role that metabolism of xenobiotics by the intestinal mucosa plays in pharmacological and toxicological processes. Expression of cytochromes P450 in the colorectal region is of particular interest given the possible role of xenobiotics in the etiology of cancers of this region. The possibility exists that heterogeneity in the expression of cytochromes P450 in colonic mucosa, and the subsequent capacity to activate dietary procarcinogens in close proximity to the putative target tissue, may be a contributing factor to variations in colon cancer incidence. Considering the present data, both in this laboratory and elsewhere, it is clear that cytochromes P450 are expressed in both the rabbit and human gastrointestinal tract. The expression of P450 enzymes in colon is however vastly reduced relative to both hepatic and small intestinal expression, particularly in humans. It appears unlikely therefore that the low level expression of the cytochrome P450 isoforms discussed here plays a significant role in the metabolism of procarcinogens such as the food-derived heterocyclic amines in human colon as represented in Fig. 1. If these compounds do represent human colon carcinogens, it is probable that they are N-hydroxylated by CYP1A enzymes in the liver prior to transport to the colon via the mesenteric artery (Fig. 1). In colon mucosal cells, the N- hydroxylated metabolite may then be further metabolized by phase II enzymes with the resultant formation of covalent adducts. Whilst the studies discussed here suggest a minimal role for members of the CYP1A, CYP3A and CYP4B subfamilies in colonic metabolism, they do demonstrate interesting heterogeneity in the expression of cytochromes P450, both between organs and between individuals. The colon clearly possesses a unique cytochrome P450 complement which differs markedly from the liver and small intestine, both in terms of the isoforms expressed and the level of expression. Further studies involving larger numbers of individuals are clearly required to assess possible mechanisms underlying such heterogeneity. Molecular analysis of the regulation of the CYP3A subfamily in human colon would represent one particularly interesting research area. This subfamily represents an example of clear differences in the regulation of cytochrome P450 genes between the small and large intestines. It may be envisaged that such studies may also provide insights into the mechanisms underlying the polymorphic expression of the CYP3A5 gene observed in human liver. The demonstration of CYP4B1 expression in rabbit colon at levels comparable to liver and small intestine represents a particularly interesting finding. In humans, CYP4B1 was demonstrated in colon by hybridization histochemistry but not in liver or the small intestine. Prior to these studies, assessments of P450 expression in colon have been based primarily on those isoforms of P450 expressed at appreciable levels in liver including members of the CYP1A and CYP3A subfamilies. CYP4B1 represents a constitutive lung enzyme rather than a predominantly hepatic enzyme and thus is a useful model for a "non-hepatic" P450. Given the expression of CYP4B1 in human colon in the absence of hepatic expression, the possibility exists that colonspecific forms of cytochrome P450 also exist. Such isoforms would not necessarily be detected by probes designed purely on the basis of hepatic expression. Clearly, further insights into the role of colon mucosal cells in chemical carcinogenesis will be derived from further studies aimed at characterizing the expression of all identified phase I and phase II xenobiotic metabolizing enzymes in these cells. Further insights into the possible role of colon metabolism in chemical carcinogenesis will also necessitate closer attention to the influence of dietary and environmentally derived chemicals on both the expression and function of these enzymes. The possible importance of such interactions is illustrated by the enhanced ability of CYP3A enzymes to activate food-derived heterocyclic amines in the presence of dietary flavonoids. Clearly the possibility exists for further such interactions given the myriad of chemicals ingested by humans. The role of such interactions in the mucosal metabolism of xenobiotics may play a significant role in the marked susceptibility of this tissue to cancer.

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