Role of metabolism by human intestinal microflora in geniposide-induced toxicity in HepG2 cells

Possible role of metabolism by the intestinal bacteria in geniposide-induced cytotoxicity was investigated in human hepatoma HepG2 cells. Initially, toxic effects of geniposide and its metabolite genipin were compared. Genipin, a deglycosylated form of geniposide, was cytotoxic at the concentrations that geniposide was not. As metabolic activation systems for geniposide, human intestinal bacterial cultures, fecal preparation (fecalase) and intestinal microbial enzyme mix were employed in the present study. When geniposide was incubated with human intestinal bacteria, either Bifidobacterium longum HY8001 or Bacteroides fragilis, for 24 h, the cultured media caused cytotoxicity in HepG2 cells. Fecalase and intestinal enzyme mix were also effective to metabolically activate geniposide to its cytotoxic metabolite. The present results indicated that genipin, a metabolite of geniposide, might be more toxic than geniposide, and that intestinal bacteria might have a role in biotransformation of geniposide to its toxic metabolite. In addition, among three activation systems tested, intestinal microbial enzyme mix would be convenient to use in detecting toxicants requiring metabolic activation by intestinal bacteria.     

肠道菌群对药物代谢影响的研究进展

肠道菌群是寄居在肠道的种类繁多、数量庞大的微生物群落,对药物等外来化合物的代谢至关重要,近年来肠道菌群作为机体"隐形器官"被广泛关注。通过查阅国内外相关文献,该文对机体中肠道菌群的分类、功能及其对中药化学成分和化学药物代谢的影响进行了分析、归纳和总结。了解肠道菌群对药物代谢的影响,明确药物转化过程,对于指导临床合理用药、个体化用药、毒理学评估及推动药物发现和研发具有重要意义,为后期研究高原环境下肠道菌群对药物代谢的影响提供理论指导。     

The role of intestinal microflora in the metabolism of trichothecene mycotoxins

The role of faecal and intestinal microflora on the metabolism of trichothecene mycotoxins was examined in this study. Suspensions of microflora obtained from the faeces of horses, cattle, dogs, rats, swine and chickens were incubated anaerobically with the trichothecene mycotoxin, diacetoxyscirpenol (DAS). Micro-organisms from rats, cattle and swine completely biotransformed DAS, primarily to the deacylated deepoxidation products, deepoxy monoacetoxyscirpenol (DE MAS) and deepoxy scirpentriol (DE SCP). By contrast, faecal microflora from chickens, horses and dogs failed to reduce the epoxide group in DAS and yielded only the deacylation products, monoacetoxyscirpenol (MAS) and scirpentriol (SCP), in addition to unmetabolized parent compound. Intestinal microflora obtained from rats completely biotransformed DAS to DE MAS, DE SCP and SCP; and T-2 toxin to the deepoxy products, deepoxy HT-2 (DE HT-2) and deepoxy T-2 triol (DE TRIOL). Rat intestinal microflora also biotransformed the polar trichothecenes, T-2 tetraol and scirpentriol, to their corresponding deepoxy analogues. Deepoxy T-2 toxin (DE T-2) was synthesized from T-2 toxin and demonstrated to be 400 times less toxic than T-2 toxin in the rat skin irritation bioassay and non-toxic to mice given 60 mg/kg ip, demonstrating that epoxide reduction is a significant single step detoxification reaction for trichothecene mycotoxins.     

抑制肠道细菌丛和非特异性羧酸酯酶对T2毒素在原位灌流大鼠小肠袢中代谢的影响

预先用新霉素和头孢氨苄抑制肠道细菌丛后，Ｔ２毒素在灌流小肠袢内的代谢速度明显减慢，ｔ１／２β由正常的２６ｍｉｎ延长为１３１ｍｉｎ．主要代谢产物ＨＴ２的生成减少．Ｔ２毒素羟化生成３′－羟基ＨＴ２的反应被抑制．当Ｔ２毒素对预先用二异丙基氟磷酸酯抑制酯酶的小肠袢进行肠系膜一门脉血管灌流时．Ｔ２毒素的ｔ１／２β由正常的４７ｍｉｎ延长至１２０ｍｉｎ，ＨＴ２的生成部分被抑制，而３′－ＯＨＨＴ２的生成量显著增加．同时抑制肠道细菌丛和酯酶．Ｔ２毒素在小肠中的代谢转化基本被阻断．实验结果表明，Ｔ２毒素在大鼠小肠中的代谢转化反应主要在肠道细菌丛酶系统和非特异性羧酸酯酶的共同作用下进行．抑制肠道细菌丛酶系统可同时减少Ｔ２毒素在小肠中的水解和羟化反应．而抑制酯酶仅能减少水解反应．与此同时轻化产物增加．     

The role of the intestinal microflora in the reductive metabolism of acenocoumarol in man

Although the oral anticoagulant acenocoumarol (AC) is very effectively metabolized by the intestinal microflora to its amino metabolite, under clinical conditions this route of AC-disposition is of no importance because the compound is rapidly absorbed from its pharmaceutical application form. Only when the gastro-intestinal absorption is retarded, for instance by using a capsule as vehiculum, are appreciable amounts of reduced metabolites recovered in urine.     

Influence of the intestinal microflora on the elimination of warfarin in the rat

The effect of the intestinal microflora on the half-life and elimination of warfarin in rats was examined. When the intestinal microflora was reduced with neomycin, bacitracin, and tetracycline, or was nonexistent as in germ-free animals, more radioactivity was found in feces and less in the urine after ip administration of 14C-warfarin. The ratio of conjugated to free metabolites in the feces was higher in germ-free rats compared to conventional or ex-germ-free animals. In addition, fecal beta-glucuronidase levels were markedly decreased in antibiotic-treated rats and in germ-free rats when compared to conventional and ex-germ-free rats. In a crossover study, a 30% decrease in warfarin half-life was observed in germ-free and antibiotic-treated animals compared to the same rats in an ex-germ-free state. The antibiotic treatment, however, had effects other than reduction of the microflora. A significant decrease in the volume of distribution of warfarin was noted in antibiotic-treated animals which may invalidate the use of this widely used mixture as a model for the study of intestinal microflora-drug interactions. To confirm enterohepatic recycling of warfarin, bile from donor rats administered 14C-warfarin ip was infused into the upper duodenum of recipient rats. Bile from the recipient rats was shown to contain 0.1-0.9% of the radioactivity administered to the donor rats. At 6-8 hr after injection, serum of the recipient rats contained 2-10% of the radioactivity present in the serum from donor rats, and contained mainly free warfarin. These data are consistent with an important role for the intestinal microflora in facilitating enterohepatic recycling of warfarin in the rat.     

Role of the intestinal flora in the metabolism of misonidazole

The radiation sensitizer misonidazole is metabolized to its amino derivative [1-(2-aminoimidazol-1-yl)-3-methoxypropan-2-o1] in pure or mixed cultures of the intestinal microflora. This metabolite appears in the excreta of conventional rats but is not detectable in the excreta of germfree rats. Thus. its formation appears to be due to the activity of the intestinal flora in vivo as well as in vitro. CO₂ is liberated from 1(2aminoimidazol1yl)3methoxypropan2o1 by pure and mixed cultures of the In cultures of Clostridium perfringens that lack urease, the release of CO₂ depends on added urease, suggesting that urea is an intermediate in this pathway.     

Role of prostaglandins in the antimyoclonic action of clonazepam

Possible involvement of prostaglandins (PG) in the antimyoclonic action of clonazepam was examined in the p,p′-DDT-animal model of myoclonus. PG synthesis inhibitors and the PG antagonist polyphloretin phosphate (PPP) counteracted the antimyoclonic action of clonazepam in mice. PGE₂ reduced DDT-induced myoclonus; this effect was blocked by PPP. Another antimyoclonic drug combination, L-5-hydroxytryptophan plus chlorimipramine, was not blocked by PPP or indomethacin. The antimyoclonic action of clonazepam may be mediated by enhancement of PG synthesis.     

Review article: Role of the enteric microflora in the pathogenesis of intestinal inflammation and arthritis

Strong associations exist between intestinal inflammation and arthritis, ranging from infections with enteric pathogens to idiopathic inflammatory bowel disease. Increased exposure of the lamina propia and systemic circulation to enteric microflora and their products are a result of increased proliferation of the luminal bacteria, pathogenic invasion or enhanced mucosal permeability. Data suggest that anaerobic bacteria and other constituents of the normal luminal microbial flora induce and sustain chronic intestinal inflammation and arthritis. However, the normal host develops a tolerance to such bacteria and maintains homeostasis through a controlled inflammatory response and an almost impermeable mucosal barrier.     

Oxidative metabolism of estrogens in rat intestinal mitochondria.

Intestinal epithelial cells are capable of metabolizing a wide variety of exogenous substrates. To determine how this metabolic capacity may affect endogenous substances such as steroid hormones, we examined the ability of rat gut epithelial preparations to hydroxylate estradiol at the C-2 position. Utilizing a site-specific tritium exchange assay, an active estrogen 2-hydroxylase system was shown to be localized to gut mitochondria throughout the intestine, with enzymatic activities comparable to the activity in crude hepatic homogenates of non-induced animals (0.2 nmol/min/mg protein). Gas chromatography-mass spectrometry confirmed the formation of C-2 hydroxylated estrogens by these mitochondrial preparations. The enzyme system was shown to involve a saturable monooxygenase, utilizing NADH (preferably) or NADPH in a protein- and time-dependent fashion. The Michaelis-Menten constant for this pathway was approximately 150 microM. Enzyme activity decreased by 20% in the presence of carbon monoxide, and was largely unaffected by organic P450 inhibitors such as alpha-naphthoflavone, metyrapone, and SKF-525A. These studies suggest that intestinal mitochondria are able to contribute to the oxidative metabolism of endogenous estrogens circulating within the enterohepatic pool.     

Presystemic and systemic intestinal metabolism of fenoterol in the conscious rat

The intestinal and liver first pass metabolism of fenoterol.HBr (Berotec, Partusisten) was investigated in the conscious rat. Fenoterol plasma concentrations (2-1000 ng/ml) were measured with a new HPLC determination using electrochemical detection. After intraduodenal administration, fenoterol was incompletely absorbed (47-66% not absorbed). Presystemic intestinal (EGpre) and liver (EH) extraction ratios, EGpre = 0.93 +/- 0.01, EH = 0.67 +/- 0.04, were calculated from AUC values after intraduodenal, intraportal, and iv administration. Saturation of intestinal and/or liver metabolism was checked by using three dose levels at different administration routes. Total systemic availability after intraduodenal administration ranged from 0.8 (10 mg/kg) to 1.2% (40 mg/kg). The contribution of the splanchnic region to the systemic clearance of fenoterol was assessed by measuring fenoterol and fenoterol-glucuronide concentrations in arterial and portal venous blood under steady state conditions. During iv infusion (30 micrograms fenoterol/min X kg), an intestinal extraction ratio of EG = 0.26 was observed. After iv administration of fenoterol (1 and 2 mg/kg), dose-dependent pharmacokinetics were observed. Doubling of the dose resulted in an increase of systemic clearance (Cl = 53.8 +/- 2.7 and 74.4 +/- 1.8 ml/min X kg) and distribution volume (Vss = 0.95 +/- 0.13 and 1.21 +/- 0.11 liters/kg); the mean residence time (17.9 +/- 2.4 and 16.3 +/- 1.4 min) and terminal half-life (45.8 +/- 5.5 and 46.8 +/- 2.8 min) were not changed.     

Protective role of metabolism by intestinal microflora in butyl paraben-induced toxicity in HepG2 cell cultures

Parabens are alkyl esters of p-hydroxybenzoic acid (BA), including methyl paraben (MP), ethyl paraben, propyl paraben (PP), and butyl paraben (BP). In the present study, possible role of metabolism by fecalase in BP-induced cytotoxicity was investigated in HepG2 cell cultures. As an intestinal bacterial metabolic system, a human fecalase prepared from human fecal specimen was employed. Among the parabens tested, cytotoxicity of BP was most severe. BA, the de-esterified metabolite, did not induce cytotoxicity when compared to other parabens. When BP was incubated with fecalase, it rapidly disappeared, in association with reduced cytotoxicity in HepG2 cells. In addition, BP incubated with fecalase significantly caused an increase in Bcl-2 expression together with a decrease in Bax expression and cleaved caspase-3. Moreover, anti-apoptotic effect by the incubation of BP with fecalase was also confirmed by the TUNEL assay. Furthermore, BP induced a sustained activation of the phosphorylation of JNK only when it was treated alone. Meanwhile, BP-induced cell death was reversed by the pre-incubation of BP with either fecalase or SP600125. Taken together, the findings suggested that metabolism of BP by human fecalase might have protective effects against BP-induced toxicity in HepG2 cells.     

Role of metabolism by intestinal bacteria in arbutin-induced toxicity in vitro

A possible role of metabolism by intestinal bacteria in arbutin-induced toxicity was investigated in mammalian cell cultures. Following an incubation of arbutin with intestinal bacteria, either Bifidobacterium longum HY81 or Bifidobacterium adolescentis, for 24 h, its aglycone hydroquinone could be produced and detected in the bacterial culture media. The bacterial growth was not affected up to 10 mM arbutin in the culture medium. When the toxicity of bacteria cultured medium with arbutin was tested in the HepG2 cell lines, the medium with arbutin was more toxic than either parent arbutin only or bacteria cultured medium without arbutin, indicating that metabolic activation might be required in arbutin-induced toxicity. In addition, bacteria cultured medium with arbutin could suppress LPS and ConA mitogenicity in splenocyte cultures prepared from normal mice. The results indicate that the present toxicity testing system might be applied for assessing the possible role of metabolism by intestinal bacteria in certain chemical-induced toxicity in mammalian cell cultures.