Abstract
The potential role of embryonic cells in the IN VITRO bioactivation of teratogens was investigated, with limb bud mesenchyme cells, (LB), and mid brain cells,(CNS), derived from 13 day old rat embryos. The cells were cultured for 5 days and during this period differentiated into foci of chondrocytes (LB) or neurones (CNS). The development of the cells in culture mirrored the IN VIVO development and therefore can be used as a teratogen screen. The presence of constitutive forms of cytochrome P450 isoenzyme forms b, c and d (Levin et. al. 1978 nomenclature) were detected in both cell types after the 5 day culture period by immunocytochemistry. Isoenzyme cytochrome P450 b was found to be non-inducible, whereas isoenzymes c and d were found to be inducible by both transplacental administration of 3-methylcholanthrene (3MC) and B-naphthoflavone (BNF) and by IN VITRO coincubation with 3MC and BNF. There was a difference in the developmental profile of the appearance of isoenzyme forms b and c over the 5 day culture period. Having established the presence of embryonic cytochrome P450's in the culture system the role of metabolism in the bioactivation of diphenylhydantoin (DPH) was investigated. Five approaches were used: 1. Modulation of cytochrome P450 activity IN VITRO by coincubation with a variety of inhibitors caused an increase in DPH toxicity to the extent of 13-82% in LB and 3-52% in CNS cells. 2. Modulation of cytochrome P450 activity IN VITRO by coincubation with inducers, only LB cells coincubated with 3MC caused a 21% increase in DPH toxicity. 3. Modulation of cytochrome P450 activity IN VITRO by transplacentally administered inducers; only LB cells derived from 3MC or BNF pretreated dams increased DPH toxicity, by 20 and 30% respectively. In addition, only LB cells from BNF pretreated dams had the ability to activate the pro-teratogen cyclophosphamide (CPA), (CPA is only toxic to LB cells in the presence of an external metabolising source). 4. Modulation of the formation of the potentially reactive arene oxide intermediate of DPH showed that although the degree of covalent binding could be modulated this did not correlate with the modulation in toxicity and it was therefore concluded that the arene oxide intermediate did not play an important role in DPH teratogenesis. 5. Identification by HPLC analysis of DPH metabolites formed by the cells IN VITRO. Cells (especially LB) were capable of hydroxylation and hydantoin ring cleavage.