Establishment of a novel in vitro system for studying the interaction of xenobiotic metabolism of liver and intestinal microflora

Abstract

We developed a new two-chamber system for the coculture of hepatocytes and fecal microflora under aerobic and anaerobic conditions, respectively, to investigate the sequential metabolism of chemicals by the liver and microflora in vitro. The culture device consisted of two chambers separated by a permeable polycarbonate membrane. In the aerobic compartment, hepatocytes were cultivated as a monolayer on the membrane and in the anaerobic compartment fecal microflora as a suspension. To characterize the metabolic capacity of the microflora and hepatocytes, various marker enzymes were studied. Azoreductase, nitroductase, beta-glucuronidase, beta-glucosidase and sulphatase were tested in the microflora of the feces from three volunteers who had had significantly different eating habits for years (daily meat, mixed diet, vegetarian). The microflora exhibited significant activities and the various enzymes differed only moderately in the samples from the three volunteers. For rat hepatocytes the activities of various cytochrome P450 forms and conjugating enzymes served as markers. The enzyme activities were tested in the coculture system during a 4-h culture period intended for the test protocol. Deethylation of ethoxycoumarin and 2alpha-, 6beta- and 16alpha-hydroxylation of testosterone decreased by about 30%, 25%, 40% and 20%, respectively, while there was no loss of glucuronidation and sulphonation of 3-OH-benzo(a)pyrene nor of glutathione conjugation of 1-chloro-2,4-dinitrobenzene during the 4-h culture period. The activities of the tested hepatic phase I and II enzymes were not changed after coculture of the hepatocytes with the microflora for 4 h. The applicability of the in vitro system for studying the metabolic interaction of liver and microflora was demonstrated using 7-ethoxycoumarin and the developmental drug EMD 57033, a thiadiazinon derivative from Merck KGaA, as model compounds. Both compounds were oxidized and conjugated by liver cells. In the coculture of hepatocytes and fecal microflora the resulting glucuronides and sulphoconjugates were split by hydrolytic enzymes of the intestinal microflora.

Dietary fibres as “prebiotics”: Implications for colorectal cancer

“…Certain bacteria, such as Bacteroides species, appear to enhance mutagenicity via potent carcinogens, such as HCA, through these mechanisms. Additionally, exchanges between the liver and the intestine takes place through the enterohepatic circulation [80].…”

Studying the host-microbiota interaction in the human gastrointestinal tract: basic concepts and in vitro approaches

“…This could be achieved, for instance, by means of separate compartmentalization of bacteria and cell lines, to avoid direct contact but allowing exchange of metabolites and signals between the two compartments. This appears to be a feasible approach as it is based on the typical structure of a microbial fuel cell (Pham et al 2009), with the presence of a permeable membrane, as shown by Laube et al (2000), who developed an in vitro system for studying the interaction of xenobiotic metabolism of liver and intestinal microbiota.…”

Role of intestinal microflora in xenobiotic‐induced toxicity

“…For most xenobiotics, including drugs and toxicants, the role of the intestinal microflora in metabolism has not been considered to be part of their mode of action. The interactions of hepatic drug‐metabolising enzymes and the intestinal microflora should be investigated to understand the metabolic fate of xenobiotics that undergo enterohepatic circulation. To accomplish this goal, novel in vitro systems that model the interactions of the liver with xenobiotic metabolism by the intestinal microflora and/or the interaction of the intestinal microflora with host transporters should be developed .…”