Ethylbenzene Exposure to Cytochrome P450: Does Ethylbenzene Produce Transient Changes in P450 2B Messenger RNA Levels?
Desai, Ketan N.
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The introduction of aromatic hydrocarbons to an organism has been shown to cause the induction of certain cytochrome P450 (P450) isozymes that function to detoxify xenobiotics from the body (Backes, 1993). A past study demonstrated the induction of P450 2B1 and 2B2 (2B1/2) proteins by ethylbenzene (Bergeron, 1995). Bergeron's study showed an unusual pattern of induction with ethylbenzene treatment. Although P450 2B1/2 mRNA and protein levels both initially increased, the protein levels remained elevated thereafter, but the mRNA returned to control levels for the rest of the treatment period. Considering that the t l/2 of 2B1/2 protein has been reported to be 20 hours (Shiraki et al., 1984), protein degradation should ordinarily be expected. To derive a possible mechanism for this pattern of induction, male Holtzman rats were treated daily with ethylbenzene for 4 days, and killed at various times thereafter. The 2B1/2-dependent catalytic activity, protein levels, and mRNA levels of liver samples from these rats were then measured. The catalytic activity, estimated by spectrophotometrically measuring the dealkylation of pentoxyresorufin by P450 2B1/2 protein, showed significantly higher levels for all ethylbenzene-treated rats relative to the control rats which were treated with corn oil. Protein levels were measured by Western blotting, using P450 2B1/2 specific antibodies to identify and quantitate the levels of these isozymes. They showed similar results as the catalytic activity, with the protein levels in all ethylbenzene treated rats being significantly higher than in the control rats. The changes in mRNA were measured using Northern blotting, where RNA samples isolated from liver were separated electrophoretically and reacted with radioactively-labeled P450 2B1 or 2B2 oligonucleotide probes. These showed significantly elevated mRNA levels between 5 and 12 hours after administration of ethylbenzene. Thus it was conclusively shown that in rats chronically treated with ethylbenzene, protein levels are maintained at elevated levels by transient increases in mRNA.