Article

Mutagenic activation of biliary metabolites of benzo(a)pyrene by -glucuronidase-positive bacteria in human faeces

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Abstract

Human faeces hydrolysed synthetic beta-D-glucuronides of both p-nitrophenol and phenolphthalein. The origin of this activity in faeces was localised in the bacterial pellet fraction after centrifugation. Ninety-seven bacterial strains with beta-glucuronidase activity isolated from fresh human faeces were identified as species of Bacteroides, Peptostreptococcus, Fusobacterium, Propionibacterium, Clostridium, Eubacterium and Bifidobacterium. They were classified into two groups according to their activity against two synthetic beta-D-glucuronides. One group hydrolysed p-nitrophenyl glucuronide and phenolphthalein glucuronide to the same extent and the other hydrolysed p-nitrophenyl glucuronide much more strongly than phenolphthalein glucuronide. The bile of rats given benzo(a)pyrene by mouth was tested for mutagenicity in the presence and absence of cell-free extracts of human faeces and bacteria. Extracts of beta-glucuronidase-positive bacteria increased the mutagenicity of metabolites of benzo(a)pyrene, as did faecal extracts, but extracts of beta-glucuronidase-negative bacteria did not. D-Saccharic acid-1,4-lactone inhibited the increase in mutagenicity produced by the faecal extracts and extracts of beta-glucuronidase-positive bacteria except for Peptostreptococcus strains 204 and 952. These results indicate that some intestinal bacteria have beta-glucuronidases heterogenous in substrate specificity and that they may be involved in mutagenicity of benzo(a)pyrene in the intestinal tract.

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... Hydrolytic microbial enzymes, such as sulfatase and glucuronidase, are of a particular interest since they may release parent metabolites from their conjugates. These metabolites can be reabsorbed into the enterocytes, and thus circulate between the gut lumen and the epithelial cells (Nanno et al. 1986). Thus, an enormous metabolic potential of intestinal microflora is involved in the overall metabolic fate of ingested compounds (Meinl et al. 2009;Bezirtzoglou, 2012). ...
... In addition, intestinal microflora is involved in a cleavage of the xenobiotic conjugates. For instance, glucuronide conjugates of BaP are hydrolyzed and mutagenic derivatives can be reabsorbed into the enterocytes, and thus circulate between the gut lumen and the epithelial cells (Nanno et al. 1986). ...
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Flavanol dihydromyricetin (DHM) has been shown to counteract acute ethanol (EtOH) intoxication and reduce excessive EtOH consumption. Since this flavonoid is being considered for human use, the in vivo study of DHM interactions with the cytochrome P450 (CYP) multienzyme system in the respect of metabolic activation of a model food-born carcinogen, benzo[a]pyrene (BaP), is of high importance. Flavonoids of known properties, alpha-naphthoflavone (ANF) and beta-naphthoflavone (BNF) were included into the study to compare their and DHM effects on BaP-DNA adduct formation. The flavonoids were administered by oral gavage either 72 hrs prior or simultaneously with a single dose of BaP to experimental rats. The expression of CYP1A1/2 enzymes was examined based on the enzymatic activity with a marker substrate, 7-ethoxyresorufin, and on Western blots. The nuclease P1 version of the 32P-postlabeling assay was used to detect and quantify covalent DNA adducts formed by BaP. Treatment of rats with a single dose of DHM or ANF prior to or simultaneously with BaP did not produce an increase in levels of CYP1A1 and in formation of BaP-DNA adducts in liver. BNF, a known inducer of CYP1A1, showed a synergistic effect on BaP-mediated CYP1A1 induction and BaP activation in liver. Contrary to that, in small intestine the stimulatory effect of BNF on both parameters was not detected. Animal pre-treatment with DHM or ANF before BaP administration resulted in a significant elevation of BaP-DNA adducts, namely in the distal part of small intestine, while the CYP1A1 mediated 7-ethoxyresorufin-O-deethylation (EROD) was decreased markedly. It is important to note that under all regimens of animal treatment, DHM or ANF produced the higher inhibitory effect on the BaP-DNA adduct formation and BaP-induced EROD activity of CYP1A1 when administered simultaneously than sequentially with BaP. Our data show that DHM or ANF did not enhance the BaP-activation leading to BaP-mediated genotoxicity (the formation of BaP-DNA adducts) in rat liver, however, in small intestine the pre-treatment of rats with these flavonoids may enhance BaP genotoxicity. The data indicate that the intake of DHM prior to or simultaneously with the administration of BaP may increase the risk of a BaP-induced tumorigenesis in small intestine.
... The BG group, revealed by functional metagenomics, includes homologs to metagenomically identified H11G11 BG present in some strains of Firmicute and Bacteroidetes [16,17]. Numerous GUS substrates are naturally present in the diet or glucuronidated in the liver via the phase II detoxification pathway; endogenous metabolic wastes, vitamins, steroid hormones, animal-and plant-derived secondary metabolites, xenobiotics and pharmaceuticals are often conjugated with glucuronic acid [16,[18][19][20][21][22][23][24][25][26][27][28]. GUS activity increases body exposure to the deglucuronidated form and is therefore efficient for exacerbating toxicity of hormones or drugs recognized by the human MRP1/MDR1 multidrug transporters or AhR aryl hydrocarbon receptor known to be crucial in IBD [29][30][31][32]. ...
... The bacterial β-glucuronidases of the gut microbiota include harmful GUS enzymes involved in the retention of toxic compounds, and BG enzymes that are part of the 'healthy' functional core [16][17][18][19][20][21][22][23][24][25][26][27][28]. Several previous studies, including unaffected CD relatives, supported a microbiota predisposition hypothesis [12][13][14] but a functional explanation was missing. ...
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Crohn's disease, an incurable chronic inflammatory bowel disease, has been attributed to both genetic predisposition and environmental factors. A dysbiosis of the gut microbiota, observed in numerous patients but also in at least one hundred unaffected first-degree relatives, was proposed to have a causal role. Gut microbiota β-D-glucuronidases (EC 3.2.1.33) hydrolyse β-D-glucuronate from glucuronidated compounds. They include a GUS group, that is homologous to the Escherichia coli GusA, and a BG group, that is homologous to metagenomically identified H11G11 BG and has unidentified natural substrates. H11G11 BG is part of the functional core of the human gut microbiota whereas GusA, known to regenerate various toxic products, is variably found in human subjects. We investigated potential risk markers for Crohn's disease using DNA-sequence-based exploration of the β-D-glucuronidase loci (GUS or Firmicute H11G11-BG and the respective co-encoded glucuronide transporters). Crohn's disease-related microbiomes revealed a higher frequency of a C7D2 glucuronide transporter (12/13) compared to unrelated healthy subjects (8/32). This transporter was in synteny with the potential harmful GUS β-D-glucuronidase as only observed in a Eubacterium eligens plasmid. A conserved NH2-terminal sequence in the transporter (FGDFGND motif) was found in 83% of the disease-related subjects and only in 12% of controls. We propose a microbiota-pathology hypothesis in which the presence of this unique β-glucuronidase locus may contribute to an increase risk for Crohn's disease.
... In the group of PAHs, B(a)P has the highest potential to cause cancer [2] [4] [8] [9]. Furthermore, B(a)P has also mutagenic and teratogenic effects [2] [6] [10] [11] [12]. It transforms into (+)-BP-7-8-oxide by the monooxigenase enzyme, into (-)-trans-BP-7,8-dihydrodiol by the epoxide hydrolase enzyme and into (+)-anti-BP-7,8-diol- 9,10-epoxide by the monooxigenase enzyme [1] [5]. ...
... In the group of PAHs, B(a)P has the highest potential to cause cancer [2,4,8,9]. Furthermore, B(a)P has also mutagenic and teratogenic effects [2,6,101112. It transforms into (+)-BP-7-8-oxide by the monooxigenase enzyme, into (-)-trans-BP-7,8-dihydrodiol by the epoxide hydrolase enzyme and into (+)-anti-BP-7,8-diol- 9,10-epoxide by the monooxigenase enzyme [1,5]. ...
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... 2 . 1 .31; Cole et al. 1985;Gadelle et al. 1985) may result in the hydrolysis of the conjugates, and their consequent reabsorption into the circulation in their active, deconjugated form . /I-Glucuronidase activity has been implicated in the activation of benzo(a)pyrene and 1,2-dimethylhydrazine conjugates in the colon (Nanno et al. 1986;Mallett & Rowland, 1990). It has been demonstrated that the yield of colorectal tumours is lower in germ free rats exposed to 1 ,Zdimethylhydrazine than in similarly treated conventional rats (Reddy et al. 1974) and that a /I-glucuronidase inhibitor can protect rats with a conventional flora from the induction of tumours by azoxymethane, a metabolite of 1 ,Zdimethylhydrazine (Takada et al. 1982). ...
... Several well-known examples of useful enzymatic activities of probiotics are glycoside hydrolase activities, which increase bioavailability of plant polysaccharides [27]; protease activities, which result in production of numerous bioactive peptides [7]; and β-galactosidase activity, which makes dairy products well tolerated by lactose-maldigesters [28]. In addition, in order to avoid the production of potentially toxic substances, probiotics must be evaluated for the production of undesirable enzymes, such as β-glucuronidase, a carcinogenic enzyme that can produce reactive metabolites negatively affecting the liver and increasing probability of colon carcinogenesis [29,30]. ...
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... It was previously noted that diets rich in fats and red meats result in dysbiosis, favoring the growth of Proteobacteria phylum (species such as E. coli, Klebsiella, Enterobacter, and Citrobacter) and Fusobacterium nucleatum [103,104] and impacting in a negative way certain species of Firmicutes and Bacteroidetes [73]. Notably, Fusobacterium nucleatum species are able to kill maturing lymphocytes via M cells in the Peyer's patches via direct contact, lowering the number of circulating systemic lymphocytes [92,105] (Figure 2). ...
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... -Glucuronidase activity is involved in the mutagenic activation of metabolites, resulting in potential colon carcinogenesis. [31][32][33] Therefore, the presence of -glucuronidase activity is an undesirable characteristic of Bifidobacterium in the food industry, 32 and the lack of it in B. adolescentis JCS2 satisfies an essential safety feature of a probiotic in the food industry. ...
... Steroids and sorne carcinogenic compounds are metabolised in the liver and conjugated with glucuronic acid before secretion via bile into the small intestine. Bacterial -glucuronidases can hydrolyse the glucuronide releasing the parent compounds [5,7,13]. ...
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The objective of this study was to evaluate if kimchi may be related to carcinogenesis by evaluating the activities of β-glucuronidase and β-glucosidase of lactic acid bacteria isolated from kimchi, which play a role in carcinogenesis. Thirty-six lactic acid bacteria (22 Lactobacillus spp., 11 Pediococcus pentosaceus, 2 Leuconostoc spp., and 1 Weisella cibaria) isolated from various kimchi were incubated in 10 mL of lactobacilli MRS broth at 35oC for 20 h. Following incubation, 100 μL of each culture was suspended in 10 mL phosphate-buffered saline. The suspensions of 36 lactic acid bacteria strains were diluted with distilled water until OD500=1.048, and 65 μL aliquots of the suspensions were inoculated into the wells, where contained substrates to evaluate the activities of β-glucuronidase and β-glucosidase, followed by incubation at 35oC for 4 h for the enzyme reactions. Of 36 isolates, 19 strains (52.8%) produced β-glucuronidase, 3 strains (13.9%) produced β-glucosidase, and two strains (5.6%) produced both enzymes. This result indicates that consumption of kimchi may have a potential possibility for a carcinogen production in the gut by producing the pro-carcinogenic enzymes (β-glucuronidase, β-glucosidase) of the lactic acid bacteria in kimchi.
Article
This study aimed to evaluate in vivo gastrointestinal survival and safety of orally administered probiotic bacterium, Propionibacterium jensenii 702, using a male Wistar rat model. A high dose of 10(10) cfu/rat/day of P. jensenii 702 was fed to each rat for 81 days. The repeated dose toxicity and translocation of P. jensenii 702 into rat tissues were evaluated, along with the rat faecal beta-glucuronidase activities and dairy propionibacteria counts. Results showed that P. jensenii 702 had no adverse effect on general health status, body weight gain, visceral organs and faecal beta-glucuronidase activities. No viable cells of P. jensenii 702 were recovered from blood and tissue samples (mesenteric lymph nodes, liver and spleen) of rats, and no treatment-associated illness or death was observed. Faecal dairy propionibacteria counts reached 10(8) cfu/g after 36 days treatment and remained between 10(8)-10(9) cfu/g till the end of 81 days treatment. The results indicate that P. jensenii 702 was able to survive the in vivo gastrointestinal tract transit of rats, with no adverse affects on the animals. However, further human clinical trials are required before strain P. jensenii 702 could be incorporated into food for human consumption as probiotics.
Article
To investigate the modifying role of intestinal microflora in the metabolism of chemical carcinogens in vivo, we subjected bile from Fischer rats treated per os with chemical carcinogens and related compounds to a mutagenicity assay in the presence and absence of a cell-free extract from human feces. A mixture of the bile sample and potassium phosphate buffer was incubated in the presence or absence of human cell-free fecal extract and then further incubated with a bacterial suspension of Salmonella typhimurium tester strains TA98 or TA100.Bile from rats treated with 1-nitropyrene (1-NP) produced about 2700 and 400 revertants per plate in strain TA98 in the presence and absence of the fecal extract, respectively. There was a drug dose- and bile volume-related response. Treatment of 1-NP-bile with β-glucuronidase, but not aryl sulfatase, enhanced its mutagenicity. Cell-free extracts of some strains of intestinal bacteria (Bacteroides fragilis ATCC 12044, B. vulgatus ATCC 8482, B. thetaiotaomicron ATCC 12290, Bacteroides sp. strain 524, Eubacterium eligens VPI C15-48, Peptostreptococcus sp. strain 204 and Escherichia coli A-5-18) also enhanced the mutagenicity of 1-NP-bile. These bacterial cell-free extracts hydrolyzed the synthetic β-d-glucuronides of phenolphthalein and/or p-nitrophenol.These data indicate that the glucuronide(s) of 1-NP-metabolite(s) secreted into bile can be hydrolyzed in the intestine by bacterial β-glucuronides to potent mutagenic aglycone(s).
The intestinal beta-glucoronidase was studied in germfree, monocontaminated and conventional rats. The greater part of the beta-glucuronidase of the caecum and the large intestine of the contaminated animals was of bacterial origin. No bacterial beta-glucuronidase was found in the small intestine. Monocontamination with Escherichia coli gave activities corresponding to those of the conventional rats, whereas content from the caecum and the large intestine of the rats monocontaminated with Streptococcus pyogenes showed an activity approximately 10 per cent of that of the conventional rats.
Article
beta-Glucuronidase catalyzes the hydrolysis of benzo[a]pyrene-3-glucuronide to 3-hydroxybenzo[a]pyrene. During the enzymatic hydrolysis, a benzo[a]pyrene derivative is formed which binds to DNA to a far greater extent than either the 3-hydroxybenzo[a]pyrene or its glucuronide. These results suggest that conjugates of benzo(a)pyrene may be converted by beta-glucuronidase at intracellular and organ sites distal to the initial sites of oxygenation and conjugation of benzo(a)pyrene to activated intermediates that are possibly carcinogenic.
Article
The effect of high-protein (beef or soybean protein) and high-fat (beef fat, corn oil, or lard) diets on large intestinal bacterial and intestinal mucosal beta-glucuronidase was studied in female F344 rats maintained on these diets for two generations. Animals fed a 20% corn oil or 20% lard and 20% casein diet had a higher beta-glucuronidase activity in the contents of cecum and colon than did rats fed a 5% corn oil or lard and 20% casein diet. The cecal bacterial beta-glucuronidase activity was higher in animals fed diets with high levels of beef protein (40%) and beef fat (23%) or with high levels of soybean protein (39%) and corn oil (24%) than it was in rats fed diets containing 18.5% beef protein and 6.5% beef fat or 19% soybean protein and 5.4% corn oil. Animals fed diets containing high levels of beef protein and fat or high levels of soybean protein and corn oil had a higher small intestinal mucosal beta-glucuronidase activity than did the other groups. No significant difference was observed in the colonic mucosal beta-glucuronidase activity among the animals fed beef and soybean diets. It is concluded that diets high in fat and high or normal in protein are associated with elevated levels of bacterial beta-glucuronidase activity in the large intestine of rats.
Article
THE geographical variation in the incidence of large bowel cancer together with the increased incidence found in migrants from low risk to high risk areas support the view that environmental factors are involved in the aetiology of this disease1. These environmental factors are thought to be largely dietary although other influences have not been excluded. Aries et al.2 suggested that the disease is caused by carcinogens produced by gut bacteria from bile acids. Diet would influence both the bile acid levels in the bowel and also the nature of the gut flora. Faecal bile acids are more concentrated in faeces from people living in high incidence areas than in faeces from populations at low risk3. Similarly, faecal bile acids are usually more concentrated in samples from patients with large bowel cancer than in those from control patients4. Bile acids were postulated to be the substrates from which the gut flora produced carcinogens2 and indeed this suggestion has been supported by the demonstration that the faecal concentration of bacterially degraded bile salts is more highly correlated with the incidence of large bowel cancer than is the total bile acid level3. However, it has been suggested that the primary role of bile acids is as co-carcinogens and this suggestion is supported by the results of animal experimentation demonstrating the promoting effect of bile acids on the action of known carcinogens5. If it is accepted that bile acids act as co-carcinogens it becomes necessary to explain how the carcinogen reaches the large bowel without producing cancers in other parts of the gastrointestinal tract. Here we describe preliminary investigations of a mechanism by which an environmental carcinogen could be transported to the large intestine.
Article
We studied the effect of diet on the activities of four enzymes found in the intestinal flora of the male F344 rat. Animals initially fed a diet with high vegetable and grain content were shifted to a diet consisting predominantly of beef. While eating the meat diet, the rats had significantly higher levels of nitroreductase, azoreductase, and beta-glucuronidase in their fecal flora when compared to levels measured during grain feeding. However, beta-glucosidase activity was significantly lower during meat feeding, which probably reflected the lack of beta-glucosidic linkages in this diet. These findings suggested that a high-beef diet, similar in composition to that consumed by humans with a relatively high risk of colon cancer, is associated with elevated levels of specific enzymes in the colon microflora. These enzymes have been implicated in the conversion of procarcinogens into carcinogens.
Article
The effect of a mixed Western, high meat diet or a nonmeat diet on the intestinal bacterial beta-glucuronidase activity was studied in human volunteers. This enzyme was significantly higher in stools of subjects on a high meat diet as compared to the nonmeat regimen. Thus, intestinal flora of subjects on a high meat diet was more able to hydrolyze glucuronide conjugates than that of individuals on a nonmeat diet. This, in turn, may raise the amount of substances, such as carcinogens, within the colonic lumen.
Article
The effect of diet and Lactobacillus acidophilus supplements on fecal microflora enzyme activity was studied in humans. The bacterial enzymes that were investigated are known to catalyze reactions that may result in formation of proximal carcinogens. Compared to vegetarians, omnivores eating a "Western-type" diet had higher levels of beta-glucuronidase, nitroreductase, azoreductase, and steroid 7-alpha-dehydroxylase in their fecal microflora. Removal of red meat or addition of fiber in the form of bran or wheat germ to the diet of omnivores for 30 days had no effect on beta-glucuronidase, nitroreductase, or azoreductase activity. However, removal of red meat or addition of fiber reduced fecal steroid 7-alpha-dehydroxylase activity. The addition of viable Lactobacillus acidophilus supplements to the diet of omnivores significantly decreased fecal bacterial beta-glucuronidase and nitroreductase activities. Thirty days after Lactobacillus supplements were curtailed, fecal enzyme levels returned to normal base-line activities. These findings suggested that the metabolic activity of the fecal microflora was influenced by diet and could be altered by Lactobacillus supplements and to a lesser extent by dietary fiber.
Article
The effect of a high-fat, high-beef diet and of method of preparation of beef in the diet on the fecal bile acids and neutral sterols and on the activities of fecal bacterial beta-glucuronidase, cholesterol dehydrogenase and 7 alpha-dehydroxylase was studied in healthy men and women, 24-41 years old, who were consuming a customary mixed-western diet. The experimental diets were high in fat and beef, which was cooked rare, medium or well-done. The sequence of dietary regimen was selected at random and each diet phase lasted for 4 weeks. Individual 24-hour fecal specimens were collected from each volunteer for the final 3 days during each dietary period, including pre- and post-experimental periods while they were consuming a customary mixed-western diet. The mode of cooking beef in the high-fat, high-beef experimental diets had no influence on the fat and protein content of the diets but the fat content of experimental diets was high compared to customary mixed-western diet. Fecal bacterial beta-glucuronidase activity and fecal secondary bile acid and cholesterol metabolite levels were significantly higher during the experimental diet periods but the fecal bacterial activities of 7 alpha-dehydroxylase and cholesterol dehydrogenase were unaffected. The mode of cooking beef in experimental diets had no influence on the fecal bacterial enzymes and on the excretion of fecal bile acids and cholesterol metabolites.
  • E Bresnick
Bresnick E 1980 Colon carcinogenesis. Cancer 45: 1047-1 05 1.
Validity of transfer of the taxonomical position of Corynebacterium pseudopyogenes from genus Corynebacterium to genus Actinomyces
  • R Azuma
  • T Suto
Azuma R, Suto T 1970 Validity of transfer of the taxonomical position of Corynebacterium pseudopyogenes from genus Corynebacterium to genus Actinomyces. In: Iizuka H, Hasegawa T (eds) Proceedings of the first international conference on culture collections. University Park Press, Baltimore, MD, pp 493-505. 347-364.