Nad(P)H:quinone oxidoreductase (DT-diaphorase) in chick embryo liver: Comparison to activity in rat and guinea pig liver and differences in co-induction with 7-ethoxyresorufin deethylase by 2,3,7,8-tetrachlorodibenzo- p-dioxin

Abstract

NAD(P)H: quinone oxidoreductase (EC 1.6.99.2; DT-diaphorase) was present in the liver of 18- and 19-day-old chick embryos as assayed both by reduction of resonifin and by the more traditional assay, reduction of 2,6-dichlorophenolindophenol (DCPIP). Both reductions had the classic characteristics of DT-diaphorase: they were equally supported by NADPH and NADH and almost entirely inhibited by dicumarol. Chick embryo liver DT-diaphorase was entirely cytosolic. It was undetectable in the microsomal and mitochondrial fractions. Chick embryo liver cytosol and mitochondrial fractions contained an enzyme oxidizer of resorufin but not of DCPIP. The k m for NADPH for resorufin reductase was an order of magnitude higher in chick embryo than in rat or guinea pig cytosol (1 mM vs 0.1 mM). Resorufin reductase activity was higher for chick embryo than for rat or guinea pig cytosols: V max (nmol resorufin reduced per mg cytosolic protein per min ± SEM) 355 ± 28 for chick embryo, 159 ± 10 for guinea pig and 68 ± 28 for rat. The V max for DCPIP reduction was also twice as high in chick embryo as rat liver cytosol. In the chick embryo, 7 days after treatment with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) at 6.4 μg/kg egg (1 nmol/egg) mortality was increased 2.4-fold, hepatic DT-diaphorase 1.3-fold, and 7-ethoxyresorufin deethylase (7-EROD) 72-fold over control levels. At 32 μg/kg, mortality was increased 4.2-fold, DT-diaphorase 2.3-fold and 7-EROD 100-fold. In the guinea pig, 5 days after treatment with TCDD at 10 μg/kg, TCDD toxicity was also evident (loss of body weight and thymus weight); there was no change in DT-diaphorase as measured by resorufin reduction, confirming by a different assay the observation of Beatty and Neal ( Biochem Pharmacol 27: 505–510, 1978) that TCDD does not induce DT-diaphorase in guinea pig liver, and 7-EROD was increased 8-fold. In contrast, in the rat, 7 days after exposure to TCDD at 10 μg/kg, there was no evidence of toxicity, DT-diaphorase was increased close to 7-fold and 7-EROD, 100-fold. The results demonstrate that avian liver contains DT-diaphorase and show that the extent to which DT-diaphorase is part of the pleiotypic response of the liver to an Ah ( aryl hydrocarbon) receptor ligand is species dependent. They also suggest that DT-diaphorase induction and TCDD toxicity may be inversely related. The possibility that DT-diaphorase protects against TCDD toxicity and participates in species differences in sensitivity to TCDD toxicity warrants further investigation.