aldrin and 7-ethoxycoumarin

The metabolism of three model substrates for the cytochrome P450 dependent mono-oxygenase enzyme system (P450-MMO) was studied in microsomes isolated from skin and liver of young adult and senescent C57B1/6J mice. The substrates chosen were aldrin (AE), 7-ethoxycoumarin (EOC), and 7-ethoxyresorufin (EOR). Both EOC and EOR activities were lower in senescent skin. By contrast, no-age related changes were seen in senescent liver. AE was similar in young and old, in both tissues. We suggest that some important age-related differences in cutaneous xenobiotic metabolism do occur, but that these are not mirrored by hepatic differences, and are substrate specific. Previous work from these laboratories would also suggest significant species differences.

Topics: Aging; Aldrin; Animals; Coumarins; Cytochrome P-450 CYP1A1; Inactivation, Metabolic; Mice; Mice, Inbred C57BL; Microsomes, Liver; Mixed Function Oxygenases; Oxazines; Skin

Hepatocytes have been isolated from samples of adult human liver by removal of extracellular calcium followed by perfusion with collagenase. The hepatocytes were isolated with a yield of up to 39 X 10(6) cells/g and with a viability of up to 74%. The cells were active in the oxidation of aldrin and 7-ethoxycoumarin. They also catalysed the conjugation of 7-hydroxycoumarin. Monooxygenase activity of the hepatocytes was linear for at least 60 min. Maintenance of the hepatocytes in suspension at 4 degrees C for 19 h resulted in a 15% loss in viability. This was accompanied by a 50% decrease in monooxygenase activity expressed per viable cell. It is concluded that human hepatocytes can be isolated in sufficient yield and with satisfactory viability for use in a range of studies on drug metabolism and toxicity.

Topics: 7-Alkoxycoumarin O-Dealkylase; Adult; Aged; Aldrin; Cell Survival; Coumarins; Culture Media; Epoxy Compounds; Female; Humans; In Vitro Techniques; Kinetics; Liver; Male; Microscopy, Electron; Middle Aged; Mixed Function Oxygenases; Oxygenases; Pharmaceutical Preparations

The multichannel perifusion system in recirculating and non-recirculating (single-pass) mode has been used to monitor the rate of oxidative metabolism of three model substrates--7-ethoxycoumarin, dichloronitroanisole and aldrin. With control hepatocytes, the rate of de-ethylation of 7-ethoxycoumarin derived from recirculating mode was essentially similar to the rate obtained with conventional flask-incubated cell suspensions. The formation of 7-hydroxycoumarin glucuronide and sulphate by hepatocytes exposed to 7-ethoxycoumarin demonstrated the retention of conjugative ability of cells in the perifusion system. The rate of demethylation of dichloronitroanisole to dichloronitrophenol was low whilst aldrin epoxidation to dieldrin was rapid using control hepatocytes in recirculating mode. The inductive effect of phenobarbitone on hepatic mixed-function oxidases was demonstrated by a marked increase in the rate of 7-ethoxycoumarin (nine-fold) and dichloronitroanisole (64-fold) dealkylation by hepatocytes from phenobarbitone-treated animals in recirculating mode. The rate of substrate oxidation by hepatocytes perifused in the recirculating and the single-pass mode were the same. With dichloronitroanisole as substrate and a single-pass mode, the rate of dichloronitrophenol formation declined rapidly on perifusion with substrate-free medium and rapidly re-attained steady state on re-introduction of the substrate; the presence of metyrapone effectively inhibited dichloronitroanisole metabolism. The perifusion system is recommended for the study of the dynamics of xenobiotic metabolism by isolated mammalian hepatocytes.

Topics: Aldrin; Animals; Biochemistry; Biotransformation; Coumarins; Equipment Design; In Vitro Techniques; Liver; Male; Metyrapone; Mixed Function Oxygenases; Phenobarbital; Rats; Rats, Inbred Strains

Topics: Aldrin; Animals; Anisoles; Benzoflavones; beta-Naphthoflavone; Butadienes; Coumarins; Cytochrome P-450 Enzyme System; Female; Kidney Tubules, Proximal; Lauric Acids; Male; Mice; Oxazines; Oxygenases; Rats

The specificity of the placental monooxygenase system to metabolize foreign compounds was studied by using different potential substrates and inhibitors and by performing electrophoresis of placental microsomes. Placental preparations from smokers catalyzed benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation and 2,5-diphenyloxazole hydroxylation, but not biphenyl hydroxylation at 2-, 3- or 4-carbon, aldrin epoxidation to dieldrin or coumarin hydroxylation or aminopyrine N-demethylation. Enzyme activities were inhibited by alpha-naphthoflavone, but to a much lesser extent by SKF 525-A or metyrapone. Correlations between the metabolism of benzo(a)pyrene, 7-ethoxycoumarin and 2,5-diphenyloxazole were highly significant. There was a clear difference in Michaelis-Menten constant of 7-ethoxycoumarin O-deethylation between placentas from smokers and nonsmokers. Gel electrophoresis revealed that protein bands of placental microsomes in the region of cytochrome P-450 enzymes were less prominent than those of rat liver microsomes, a finding that accorded with the relative amounts of cytochrome P-450. There were no consistent differences in the electrophoretic pattern between placentas of variable benzo(a)pyrene hydroxylase activities. Results show that the human placental monooxygenase system is restricted in substrate specificity, that there may be a qualitative difference between smokers and nonsmokers and that the increase in several enzyme activities by cigarette smoking cannot be detected by the standard gel electrophoresis.

Topics: Aldrin; Aminopyrine; Aryl Hydrocarbon Hydroxylases; Benzopyrenes; Biotransformation; Biphenyl Compounds; Coumarins; Electrophoresis, Polyacrylamide Gel; Female; Flavonoids; Humans; Microsomes; Placenta; Pregnancy; Proadifen; Smoking