Alternatives in the induction and preparation of phenobarbital/naphthoflavone-induced S9 and their activation profiles

With the aim of optimizing the efficiency of S9 fractions used in in vitro mutagenicity assays, different schemes for the induction of liver enzymes i n rats were tried and the amount of S9 fraction required was assessed. The activity of 2-anthramine (2AA), 2-acetylaminofluorene (2AAF), 3-methylchola nthrene (3MTCL) and benzo[a]pyrene in bacterial mutagenicity tests was comp ared with the enzymatic activity in S9 fractions obtained from rats treated with either phenobarbital (NaPB), beta-naphthoflavone (beta NF) or combina tions of both. Three pool systems prepared with different amounts of NaPB-i nduced S9 and beta NF-induced S9 were also analyzed for their activation ca pacities, Profiles of standard plate incorporation assays with Salmonella t yphimurium TA98 increased with the amount of S9 fraction added for all drug s tested, except for 2AA, which showed a maximun of activity at low protein concentrations. According to these profiles, an optimal S9 protein content of 700-1000 mu g/plate was estimated, For 2AAF and 3MTCL an S9 fraction ob tained following a simultaneous treatment with NaPB (i.p.) and beta NF (ora l gavage) (NaPB + beta NF) yielded the greatest response. This preparation was the only one which produced positive activation with 3MTCL as test drug . With the other test drugs all the S9 fractions were very active, includin g the NAPB + beta NF-induced S9, Both Phase I and Phase II cytochrome P450 enzymatic activities were enhanced in this S9 fraction, These results sugge st that the simultaneous treatment (NaPB + beta NF) would be an adequate in ducer for lit vitro activation when used at 700-1000 mu g protein/plate.