Immunochemical detection of different isoenzymes of cytochrome P-450 induced in chick hepatocyte cultures

Abstract

This study investigated whether the same cytochrome P-450 (P-450) isoenzymes were inducible in cultures of chick-embryo hepatocytes as in the liver of chicken embryos. We purified two isoenzymes of cytochrome P-450 from the livers of 17-day-old-chick embryos: one of molecular mass approx. 50 kDa induced in vivo by the phenobarbital-like inducer glutethimide, and the second of approx. 57 kDa induced by 3-methylcholanthrene. Rabbit antiserum against the 50 kDa protein inhibited benzphetamine demethylase activity in hepatic microsomes (microsomal fractions) from glutethimide-treated chick embryo. Antiserum to the 57 kDa protein inhibited ethoxyresorufin de-ethylase activity in hepatic microsomes from methylcholanthrene-treated chick embryo. Cultured chick hepatocytes were treated with chemicals known to induce isoenzymes of P-450 in rodent liver. The induced P-450s were quantified spectrophotometrically and characterized by immunoblotting and enzyme assays. From these studies, chemical inducers were classified into three groups: (i) chemicals that induced a P-450 isoenzyme of 50 kDa and increased benzphetamine demethylase activity: glutethimide, phenobarbital, metyrapone, mephenytoin, ethanol, isopentanol, isobutanol, lindane, lysodren; (ii) chemicals that induced a P-450 isoenzyme of 57 kDa and increased ethoxyresorufin de-ethylase activity: 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl; and (iii) the mono-alpha-substituted 2,3',4,4',5-pentabromobiphenyl, which induced both proteins and both activities. The immunochemical data showed that chick-embryo hepatocytes in culture retain the inducibility of glutethimide- and methylcholanthrene-induced isoenzymes of P-450 that are inducible in the liver of the chicken embryo.

Purification and characterization of heme oxygenase from chick liver

“…Fractions containing NADPH -cytochrome reductase activity were pooled and stored at -80°C for further purification of the reductase. Cytochrome P-450 eluted in two distinct peaks, probably representing at least two distinct isoforms of this enzyme, in agreement with earlier observations [53].…”

Involvement of Cytochrome P450 1A in the toxicity of aryl hydrocarbon receptor agonists : alteration arachidonic acid metabolism and production of reactive oxygen species

“…While CYP1A mRNA remains elevated in rainbow trout exposed to high doses of CB 77 or 126 and in scup exposed to high doses of CB 77, CYP1A activity and protein both decrease at high doses (Gooch et al, 1989;Newsted et al, 1995;White et al, 1997a). Similar results have been described in studies of cultured chick embryo hepatocytes and porcine aorta endothelial cells exposed to high doses of CB 77 (Lambrecht et al, 1988;Sinclair et al, 1989;Stegeman et al, 1995;. In scup, the CB 77-dependent decrease in CYP1A was shown to be posttranscriptional and hypothesized to result from oxidative modification of the protein (White et al, 1997a).…”

“…We suggest that the attacking species is OH•, formed via Fenton chemistry between H202 and the heme iron. The oxidation of CYPlA couid target it for degradation (Davies, 1987), which could explain the decrease in CYP1A content seen in animals and cells in culture exposed to higher concentrations of pHAH (Sinclair et al, 1989;Stegeman et al, 1995;White et al, 1997a). Such an in vivo effect has not been described for oxidative inactivation of other CYP.…”

“…3,3',4, and other planar PCBs not only induce CYP1A but also can elicit sharp declines in CYP1A activity and content following exposure to high doses (Gooch et al, 1989;Sinclair et al, 1989;Stegeman et al, 1995).…”

“…Loss of catalytic activity following treatment with high doses of coplanar chlorobiphenyl (CB) congeners occurs without the loss of protein in vivo (Melancon and Lech, 1983;Voorman and Aust, 1988;Miranda et al, 1990;Monosson and Stegeman, 1991;Lindstrom-Seppa et al, 1994) and in cultured cells (Sawyer and Safe, 1982;Rodman et al, 1989;Kennedy et al, 1993;, suggesting that coplanar CB congeners could inhibit CYP1A in vivo. Last, TCB, at high doses, suppresses CYP1A protein induction in several species (Lambrecht et al, 1988;Sinclair et al, 1989;Newsted et al, 1995;Stegeman et al, 1995;White et al, 1997a).…”