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

We developed a new two-chamber system for the coculture of hepatocytes and fecal microflora under aerobic and anaerobic conditions, respectively, to i nvestigate the sequential metabolism of chemicals by the liver and microflo ra in vitro. The culture device consisted of two chambers separated by a pe rmeable polycarbonate membrane. In the aerobic compartment, hepatocytes wer e cultivated as a monolayer on the membrane and in the anaerobic compartmen t fecal microflora as a suspension. To characterize the metabolic capacity of the microflora and hepatocytes, various marker enzymes were studied. Azo reductase, nitroductase, beta-glucuronidase, beta-glucosidase and sulphatas e 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 vari ous enzymes differed only moderately in the samples from the three voluntee rs. For rat hepatocytes the activities of various cytochrome P450 forms and conjugating enzymes served as markers. The enzyme activities were tested i n the coculture system during a 4-h culture period intended for the test pr otocol. Deethylation of ethoxycoumarin and 2 alpha-, 6 beta- and 16 alpha-h ydroxylation of testosterone decreased by about 30%, 25%. 40% and 20%, resp ectively, while there was no loss of glucuronidation and sulphonation of 3- OH-benzo(a)pyrene nor of glutathione conjugation of 1-chloro-2,4-dinitroben zene during the 4-h culture period. The activities of the tested hepatic ph ase I and II enzymes were not changed after coculture of the hepatocytes wi th the microflora for 4 h. The applicability of the in vitro system for stu dying the metabolic interaction of liver and microflora was demonstrated us ing 7-ethoxycoumarin and the developmental drug EMD 57033, a thiadiazinon d erivative from Merck KGaA, as model compounds. Both compounds were oxidized and conjugated by liver cells. In the coculture of hepatocytes and fecal m icroflora the resulting glucuronides and sulphoconjugates were split by hyd rolytic enzymes of the intestinal microflora.