Previous studies in the Swiss Webster
Previous studies in the Swiss Webster mouse (Rosengren et al., 1995) and the Sprague–Dawley rat (El Sisi et al., 1993) have shown that retinol pretreatment potentiates the hepatotoxicity of paracetamol. The current study is designed to investigate further this interaction in mice. To accomplish this, we decided to probe the interaction between retinol and paracetamol in the liver and kidney, since both these organs are susceptible to paracetamol-induced toxicity. We will determine whether retinol induces CYP450 isoforms important in the bioactivation of paracetamol, as has been reported in other species. The CYP450 isoforms will be profiled at a time-point pertinent to N-acetyl p-benzoquinoneimine (NAPQI) formation. An increase in CYP1A2, CYP2E1 or CYP3A at the time of paracetamol administration (24 h after retinol treatment) would increase the production of NAPQI and thereby increase the hepatotoxicity of paracetamol. Additionally, we will also determine whether retinol has an organ-specific effect on CYP450 activity in the mouse. The involvement of CYP450 isoforms in the gpr120 agonist of retinol and the ability of retinol to induce various CYP450 isoforms in a variety of different species validates our investigation of an interaction between retinoids and CYP450 isoforms in the mouse.
As paracetamol's nephrotoxicity (McMurtry et al., 1978, Richie et al., 1992) and hepatotoxicity (Mitchell et al., 1973, James et al., 1993) have been shown to correlate with glutathione levels, we will also examine the effect of retinol on glutathione in both the liver and kidney. Retinol interacts with the glutathione system both as an antioxidant and as a xenobiotic. Through its action as an antioxidant, retinol is able to reduce glutathione depletion following a toxicant challenge, and therefore protect from injury (Colin et al., 1991, Savoure et al., 1996, Montilla et al., 1998). Such protection does not fit with our previous findings of retinol's potentiation of paracetamol-induced injury. However, retinol itself has been reported to modulate glutathione stores. For example, in rats retinol deficiency caused a decrease in the hepatic (Gupta et al., 1983) and pulmonary (Tom et al., 1985) glutathione stores. However, retinol supplementation also had the effect of decreasing glutathione stores in the lung (Tom et al., 1985). This finding supports the possibility of retinol decreasing the conjugation of NAPQI and therefore increasing the hepatotoxicity of paracetamol. At the conclusion of these experiments we will have used a murine model to demonstrate the effect of retinol on CYP450 activity and glutathione conjugation in two organs susceptible to xenobiotic-induced damage.
Materials and methods
Results We have previously demonstrated that 4 days of retinol pretreatment (75 mg/kg/day) potentiates paracetamol-induced hepatotoxicity in the male Swiss Webster mouse (Rosengren et al., 1995). We have replicated this finding in another strain, the BALB/c mouse (Fig. 1). To determine whether this modulation of toxicity would occur in other organs susceptible to paracetamol-induced toxicity, we investigated the response of the kidney in the same paradigm. We found no significant increase in BUN between the retinol+paracetamol and the vehicle+paracetamol treated animals (Fig. 2), therefore this potentiation is organ-specific. In order to establish that the retinol treatment was not causing any damage in the organs investigated, plasma ALT, BUN and creatinine clearance were measured. No elevation above normal was found in either ALT (Fig. 1) or BUN (Fig. 2) following retinol treatment. Additionally, plasma creatinine showed no statistical increase following retinol treatment (41.9±7.1 vs 34.5±3.7 mg/dl, retinol vs untreated, respectively). To further investigate the mechanism underlying the hepatic potentiation, retinol's effect on the three major CYP450 isoforms involved in the bioactivation of paracetamol were analysed in both the kidney and liver. It was postulated that the hepatic potentiation effect could be due to an induction of CYP1A2 (ethoxyresorufin O-deethylation), CYP2E1 (p-nitrophenol hydroxylation) or CYP3A (erythromycin N-demethylation). Our results demonstrate no change in the catalytic activity of either hepatic CYP1A2 or CYP2E1 following 4 days of retinol treatment (Table 1). These results were supported by Western immunoblotting, which showed no significant difference in the polypeptide levels of either hepatic CYP1A2 (92.8±13.6% of control) or CYP2E1 (100±6.8% of control). CYP1A2 catalytic activity and polypeptide levels were undetectable in renal microsomes, while renal CYP2E1 catalytic activity (Table 1) and polypeptide levels were not different from control (101±12.2% of control) following retinol treatment. However, 4 days of retinol treatment did reduce both the catalytic activity (Table 1) and polypeptide levels (58±0.6% of control) (Fig. 3) of hepatic CYP3A. This was an organ-specific response since kidney microsomal CYP3A catalytic activity (Table 1) was not altered following retinol treatment and polypeptide levels were below detectable levels in all treatment groups.