Improvement of short-term tests for mutagenicity: on the optimal pH for the liver microsomal assay.
The aim of this study was to optimize the pH in the liver microsomal assay (LMA) in processing short-term mutagenicity tests. pH optimization would increase the sensitivity (i.e. decrease the presence of false negatives) and increase the specificity (decrease false positives). Such optimization is a function of the relative activities and stabilities of the liver microsomal cytochrome P-450- and FAD-containing monooxygenase-dependent biotransformation enzymes present in the incubation mixtures used. The enzyme activities ethoxyresorufin O-deethylase, dinemorphan N-demethylase, aminopyrine N-demethylase, p-nitroanisole O-demethylase and thiobenzamide S-oxidase (as phase-I markers), were examined in terms of their exact incubation conditions for the LMA during a period of pre-incubation (1 h) over the pH range 6-9. As a comparison, the behaviours of glutathione S-transferase and epoxide hydrase activities (as phase-II markers) were also studied. Lipid peroxidation was also determined. Experiments were carried out on S9 fractions derived from Na-phenobarbital and beta-naphthoflavone induced mouse liver. The maximal value of the mean specific activity (Asp) was found at pH 7.8 for the phase-I drug metabolizing enzymes considered (30-45% increase). On the contrary, a lower increase of Asp for epoxide hydrase and glutathione S-transferase (approximately 14%), was observed between pH 7.4 and 7.8. Lipid peroxidation was not changed appreciably by varying pH. In vitro DNA binding of the well-known pre-mutagenic agent [14C]dimethylnitrosamine ([14C]DMNA), mediated by mouse hepatic microsomal enzymes, showed a significant increase of specific activity at pH 7.8 (2.8-fold) compared to the usual pH (7.4) employed. Additional support for the above results has come from mutagenesis experiments using DMNA on the diploid D7 strain of Saccharomyces cerevisiae as a biological test system. In fact, a significant enhancement of mitotic gene conversion (1.7-fold), mitotic cross-over (2.6-fold) and reverse point mutation (2.3-fold) frequencies were observed at pH 7.8 compared to pH 7.4. These data indicate that pH 7.8 provides a more favourable condition for in vitro mutagenesis tests resulting in greater rates of biotransformation (as measured by an increased Asp phase-I/Asp phase-II ratio), DNA binding and genotoxic response.
Paolini
M
M
Istituto di Farmacologia, Università degli Studi, Bologna, Italy.
Sapigni
E
E
Hrelia
P
P
Grilli
S
S
Lattanzi
G
G
Cantelli-Forti
G
G
D000818:Animals; D001711:Biotransformation; D004128:Dimethylnitrosamine / Q000378:metabolism; D004851:Epoxide Hydrolases / Q000032:analysis; D005982:Glutathione Transferase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015227:Lipid Peroxidation; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism*; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism
D009153:Mutagens; D010088:Oxidoreductases; D005982:Glutathione Transferase; D004851:Epoxide Hydrolases; D004128:Dimethylnitrosamine