L-buthionine (S,R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, was administered to mice via drinking water for 14 days in order to establish an animal model with continuously depleted levels of GSH. No toxicity was observed at 20 mM BSO, even though a significant decrease in liver weight was observed at 30 mM BSO. GSH levels in the liver, kidney, brain, lung, heart, spleen, pancreas, small intestine, large intestine, skeletal muscle, plasma and blood cells from mice given 20 mM of BSO were all less than those from the control mice continuously throughout a 24-hr period. The ratios of the GSH levels to that of the control were 46.4% and 16.7% in the liver and kidney, respectively, suggesting a decrease in GSH conjugation activity in vivo by GSH depletion. Liver cytochrome P450 content and UDP-glucuronosyltransferase activity to p-nitrophenol were not influenced by the BSO dosing. To confirm the adequacy of this GSH-depletion model, 0.125 or 0.25% of acetaminophen (APAP) was administered via diet to this model for 14 days. Nine out of the ten mice given both 20 mM BSO and 0.25% APAP died on Day 2, and remarkable necrosis was observed in the hepatocytes and renal tubular epithelium. Moreover, focal necrosis of hepatocytes with proliferation of fibroblasts was observed on Day 15 in some mice coadministered 20 mM BSO and 0.125% APAP. However, no toxicity was observed in mice given APAP alone. Based on these results, a mouse given 20 mM of BSO via drinking water for 14 days was concluded to be an animal model with continuously depleted levels of GSH in various organs without toxicity. This model shows high susceptibility to toxicity induced by chemicals which are metabolized to electrophilic and reactive metabolite(s), such as APAP.

The ratio of urinary 6beta-hydroxycortisol (6beta-OHF) to free cortisol (F), i.e., the 6beta-OHF/F ratio, has been reported to be a specific marker for human CYP3A induction by in vivo studies of human subjects. In the development of drugs, it is quite beneficial to predict human CYP3A induction in preclinical safety studies using urine samples from experimental animals. We examined the 6beta-OHF/F ratio in urine of common marmosets administered with rifampicin, a potent inducer of CYP3A, to evaluate the usefulness of common marmosets for the prediction of CYP3A induction. Rifampicin was orally administered to three groups of four male common marmosets at doses of 0, 10, and 20 mg/kg per day for 4 days. Amounts of 6beta-OHF and F in urine samples were determined by means of high-performance liquid chromatography (HPLC) during the experimental period. One day after the 4th dosing, animals were killed, and P450 contents and P450-catalyzed. 7-alkoxycoumarin O-dealkylase (ACD) activities in the liver were measured. Western blot analysis of liver microsomes was also performed using anti-rat P450 (CYP1A1, 2B1/2, 3A, and 4A) antibodies. The results indicated elevations in the 6beta-OHF/F ratios that were dependent on both the dosing period and dose levels adopted. The ratios on day 4 reached 4.7- and 5.3-fold the pre-administration values in the 10 and 20 mg/kg per day groups, respectively. P450 contents and ACD activities were also elevated in all of the groups. Western blot analysis showed specific increases in the protein which cross-reacts with anti-rat CYP3A antibody in all of the groups. Furthermore, the 6beta-OHF/F ratio was well correlated with the CYP3A contents in liver (r = 0.906). These results indicated that increase in urinary excretion of 6beta-OHF is a specific marker of the induction of hepatic CYP3A in common marmosets just as in humans. Consequently, the present study suggested that human CYP3A induction elicited by chemical agents can be predicted in common marmosets by measuring the urinary excretion of 6beta-OHF.

No abstract

No abstract

No abstract