Inducibility and functionality of rat embryonic/foetal cytochrome P-450: A study of differentiating limb-bud and mid-brain cells in vitro

Abstract

The presence of constitutive levels of cytochrome P-450 isoenzymes in cultures derived from rat embryo limb-bud (LB) and mid-brain (CNS) cells was demonstrated immunocytochemically by staining with specific monoclonal and polyclonal antibodies of cytochrome P-450. The b and e forms of cytochrome P-450 were found to be non-inducible by either in vitro co-incubation for 5 days or by transplacental maternal induction with phenobarbitone (PB), 3-methylcholanthrene (3MC) or β-naphthoflavone (βNF) in either cell type. Consistent with this lack of response was the observation that both in vitro and in vivo inducer treatment did not alter the toxicity of the teratogens diphenylhydantoin (DPH) or cyclophosphamide (CPA). In contrast, 3MC induction was achieved by both in vitro and transplacental regimens as gauged by the increased intensity of peroxidase staining using a monoclonal antibody to cytochrome t-450 c, in both cell types. There was also a concomitant increase in DPH toxicity (>20% gauged by a decrease in IC 50 values) in LB cells by both induction regimens but the CNS cells were refractory. βNF induction of cytochrome P-450 was observed following in vitro and in vivo exposures in both cell types. There was no modulation of DPH or CPA toxicity after in vitro exposure to the inducers, but in vivo induction caused a strong staining reaction in both cell types, commensurate with a 30% increase in DPH toxicity in LB cells and activation of the pro-teratogen CPA. The b and e forms of cytochrome P-450 were non-inducible but it is highly likely that the c form was both inducible (by 3MC and βNF) and functional, the latter being assessed by modulation of DPH toxicity and CPA activation. It may be possible to induce cytochrome P-450 in cells derived from embryos. The system used may be suitable for detailed investigations of the types of metabolizing systems involved in the mechanisms underlying toxicity/teratogenicity.