The hepatic microsomal catalytic activity and polypeptide

The hepatic microsomal catalytic activity and polypeptide levels for CYP3A were decreased in male BALB/c mice following 4 days of retinol treatment. However, retinol did not alter renal microsomal CYP3A catalytic activity. Hepatic constitutive CYP3A catalytic activity was 100-fold greater than renal CYP3A and a minimal amount of constitutive CYP3A in the kidney has been documented by other groups (Maurel, 1996, Seree et al., 1996). Additionally, our studies demonstrate that retinol does not induce renal CYP3A. However, induction of renal CYP3A is reported to occur following treatment with metalaxyl (Paolini et al., 1997). Our observation regarding a decrease in hepatic CYP3A is contrary to findings in other species. Various groups have documented an induction of CYP3A following retinol treatment. Murray et al. (1991) reported a 158% increase in the polypeptide levels of CYP3A in rats fed a diet supplemented with 25 IU/g of retinol for 15 weeks. Rabbits fed a diet containing 500 IU retinyl palmitate for 7 weeks demonstrated an increase in 7-ethoxycoumarin deethylase activity, a non-specific measure of CYP3A activity (Miranda and Chhabra, 1981). Furthermore, Badger et al. (1998) reported a 132% increase in u73122 6β-hydroxylation following 1 day of retinol treatment in rats. However, these values returned to within normal limits 48 h following retinol and by 96 h CYP3A catalytic activity had dropped to below control levels (Badger et al., 1998). It appears from these three studies that mice respond distinctly compared to other species following retinol administration. Therefore, the differences seen between our work and that of other laboratories, is likely a species-specific response. In rats it would appear that retinoids induce CYP2E1 or CYP3A and in guinea pigs retinol induces CYP2E1, but in mice retinol has no significant effect on the catalytic activity or polypeptide levels of CYP2E1 or CYP1A2. Retinol supplementation did however result in a significant decrease of both the catalytic activity and polypeptide levels of hepatic CYP3A, a result not seen in previously published rat studies. No changes were observed in the catalytic activity or polypeptide levels of renal CYP2E1 following retinol supplementation, a finding not previously documented in the literature. These results show that the organ-specific response in potentiation of paracetamol-induced toxicity is not due to differential CYP450 induction and, therefore, potentiation of paracetamol hepatotoxicity must be produced through another mechanism. This conclusion is based on the fact that CYP450 activities were measured 24 h following retinol pretreatment, which would detect changes in CYP450 at a time-point pertinent to paracetamol bioactivation. As retinol was not inducing any of the isoforms of CYP450 responsible for the bioactivation of paracetamol, we decided to examine retinol\'s effect on renal and hepatic glutathione to determine whether retinol was potentiating hepatotoxicity through a mechanism of glutathione depletion. Studies, utilising the classical glutathione depletor diethyl maleate and a glutathione synthesis inhibitor buthionine sulfoximine, have demonstrated that under conditions of hepatic glutathione depletion potentiation of paracetamol-induced hepatotoxicity occurs (James et al., 1993). The authors were also able to conclude that phenylpropanolamine potentiated paracetamol-induced hepatotoxicity in mice through a mechanism of moderate (30–50%) glutathione depletion. Similarly, a decrease in renal glutathione content is associated with paracetamol-induced nephrotoxicity (McMurtry et al., 1978, Richie et al., 1992). As retinol potentiated the hepatotoxicity but not the nephrotoxicity of paracetamol, our glutathione investigation would further characterise this organ-specific response. Our results showed that hepatic and renal total reduced glutathione levels were not different from control following retinol treatment. Therefore, retinol is acting through a mechanism independent of both glutathione depletion and CYP450 induction. We can conclude that the mechanism of retinol\'s potentiation of paracetamol-induced hepatotoxicity does not involve an increased bioactivation of paracetamol through an induction of CYP450 and it also does not involve a decrease in NAPQI conjugation through glutathione depletion. Furthermore, neither of these systems is responsible for the differential response observed between the liver and the kidney. We propose that retinol may be causing potentiation through a mechanism of glucuronide saturation, which would limit the capacity of this major conjugation pathway, and are currently investigating this aspect. However, it is conceivable that retinol treatment impairs glutathione S-transferase activity which, if decreased, could lead to higher levels of NAPQI in the presence of sufficient reduced glutathione. Alternatively, since retinol has been shown to activate Kupffer cells in rats (Badger et al., 1997, Hoglen et al., 1997), retinol may increase the production of reactive oxygen species in mice which would lead to a subsequent increase in the progression of paracetamol-induced hepatotoxicity. This type of mechanism may also explain the organ-specific response between the liver and the kidney and should also be investigated.