Metabolic activation of 7,12-dimethylbenz(a)anthracene: role of cytochrome P-450 isoenzymes in the formation of DNA and protein adducts in vitro.

el nl. [ 5 ] on to nylon (Hybond-N, Amersham). Dot blots using a BRL apparatus were on to nitrocellulose (BRL) for RNA and nylon (Amersham) for DNA. Gene probes isolated from rat cDNA libraries corresponding to two members of cytochrome 1’-450 superfamily, namely a 1.2 kb fragment of P-450 lVAl (1’-450 LAW, 1’452) and a 1.1 kb fragment of P-450 RLM6 (probably the same as the alcohol inducible 1’-450 IIEl/P-450j; T. H. Richardson, personal communication) were used to detect homologous sequences. The results shown in Fig. I( a) demonstrate that sequences homologous to rat 1’-450 RLM6 cannot be detected in mussel, even under low stringency washing conditions (42”C, 2 x SSC [ S ] ) . In contrast, the P 4 5 0 lVAl probe does bind to mussel RNA (Fig. lh), even under more stringent washing conditions (55°C. 0.5 x SSC). These observations are in agreement with the proposal that the 1’-450 I I family is of comparatively recent origin and might not be present in lower organisms, whereas the P 4 5 0 IV family is of more ancient origin [6, 71. It might be expected therefore that sequences homologous to 1’-450 lVAl would be expressed in lower organisms and indeed preliminary work in this laboratory has detected homology to RNA from a range of marine invertebrates. We have also detected sequences homologous to the 1’450 lVAl probe in mussel genomic DNA; however, much less stringent washing conditions were required (37”C, 2 x SSC) owing to the presence of intron sequences. BIOCHEMICAL SOCIETY TRANSACTIONS