Effect of beta-carotene on hepatic cytochrome P-450 in ethanol-fed rats.

BACKGROUND: Hepatotoxicity of ethanol is increased by beta-carotene in both rodents and nonhuman primates. Furthermore, in smokers who are also drinkers, beta-carotene increases the incidence of pulmonary cancer. The hepatotoxicity was associated with proliferation of the membranes of the smooth endoplasmic reticulum, suggesting the involvement of cytochromes P-450. Therefore, the aim of the present study was to assess the effect of beta-carotene and ethanol treatment on rodent hepatic cytochromes P-450. METHODS AND RESULTS: Weanling male Sprague-Dawley rats were pair-fed beta-carotene (56.5 mg/l of diet) for 8 weeks, with and without ethanol (Lieber-DeCarli, 1994 liquid diet). As expected, ethanol increased CYP2E1 (measured by Western blots) from 67 +/- 8 to 317 +/- 27 densitometric units (p < 0.001). Furthermore, beta-carotene potentiated the ethanol induction to 442 +/- 38 densitometric units (p < 0.01) with a significant interaction (p = 0.012). The rise was confirmed by a corresponding increase in the hydroxylation of p-nitrophenol, a specific substrate for CYP2E1, and by the inhibition with diethyl dithiocarbamate (50 microM). Beta-carotene alone also significantly induced CYP4A1 protein (328 +/- 49 vs. 158 +/- 17 densitometric units, p < 0.05). The corresponding CYP4A1 mRNA (measured by Northern blots) was also increased (p < 0.05) and there was a significant interaction of the two treatments (p = 0.015). The combination of ethanol and beta-carotene had no significant effect on either total cytochrome P-450 or CYP1A1/2, CYP2B, CYP3A, and CYP4A2/3 contents. CONCLUSIONS: Beta-carotene potentiates the CYP2E1 induction by ethanol in rat liver and also increases CYP4A1, which may, at least in part, explain the associated hepatotoxicity.

Inducibility of cytochromes P-4502E1 and P-4501A1 in the rat pancreas.

Cytochrome P-450 (CYP) isoenzymes have been incriminated in the toxicity and carcinogenicity of various xenobiotics in different tissues, but prior measurements of their activity in pancreatic microsomes have been disappointing. We now applied new isolation methods and a highly sensitive procedure to assay for the metabolism of p-nitrophenol and 7-ethoxyresorufin, specific substrates for CYP2E1 (2E1) and CYP1A1 (1A1), respectively. 2E1 and 1A1 content was estimated with high-resolution chemiluminescent Western blots using recombinant 2E1 and 1A1 as standards. We found that p-nitrophenol hydroxylase activity was 5.07 +/- 0.66 and 1.50 +/- 0.26 pmol/ min/mg of protein in pancreatic microsomes of ethanol-fed and control rats, respectively. Chronic ethanol treatment increased 2E1 content in pancreatic microsomes 3.6-fold. Activity and content of 2E1 were also assessed in hepatic microsomes: specific activity (expressed per 2E1 content) was similar in pancreatic and hepatic microsomes. There was also an inductive effect of 3-methylcholanthrene (MC) on 1A1 in pancreatic microsomes. Pancreatic microsomal 7-ethoxyresorufin-O-dealkylation activity in MC-treated rats was 19.6 +/- 1.7 pmol/min/mg of protein, 61-fold higher than in controls. MC treatment increased the 1A1 content in pancreatic microsomes 42-fold. These results demonstrate that, in pancreatic microsomes, ethanol and MC exert striking inductive effects on 2E1 and 1A1 activities, which could play a role in the pathogenesis of pancreatitis and/or pancreatic cancer.

Respective roles of human cytochrome P-4502E1, 1A2, and 3A4 in the hepatic microsomal ethanol oxidizing system.

The microsomal ethanol oxidizing system comprises an ethanol-inducible cytochrome P-4502E1, but the involvement of other P-450s has also been suggested. In our study, human CYP2E1, CYP1A2, and CYP3A4 were heterologously expressed in HepG2 cells, and their ethanol oxidation was assessed using a corresponding selective inhibitor: all three P-450 isoenzymes metabolized ethanol. Selective inhibitors-4-methylpyrazole (CYP2E1), furafylline (CYP1A2), and troleandomycin (CYP3A4)-also decreased microsomal ethanol oxidation in the livers of 18 organ donors. The P-450-dependent ethanol oxidizing activities correlated significantly with those of the specific monooxygenases and the immunochemically determined microsomal content of the respective P-450. The mean CYP2E1-dependent ethanol oxidation in human liver microsomes [1.41+/-0.11 nmol min(-1) (mg protein)(-1)] was twice that of CYP1A2 (0.61+/-0.07) or CYP3A4 (0.73+/-0.11) (p < 0.05). Furthermore, CYP2E1 had the highest (p < 0.05) specific activity [28+/-2 nmol min(-1) (nmol CYP2E1)(-1) versus 17+/-3 nmol min(-1) (nmol CYP1A2)(-1), and 12+/-2 nmol min(-1) (CYP3A4)(-1), respectively]. Thus, in human liver microsomes, CYP2E1 plays the major role. However, CYP1A2 and CYP3A4 contribute significantly to microsomal ethanol oxidation and may, therefore, also be involved in the pathogenesis of alcoholic liver disease.