Improvement of short-term tests for mutagenicity: On the optimal pH for the liver microsomal assay

M. Paolini, E. Sapigni, P. Hrelia, S. Grilli, G. Lattanzi, G. Cantelli-forti

Research output: Contribution to journalArticle

Abstract

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 β-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 (∼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 in crease of specific activity at pH 7.8 (2.8-fold) compared to the usual pH (7.4) employed. Additionalsupport for the above results has come from mutagenesis experiments using DMNA on the diplold 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-IAsp phase-II ratio), DNA binding and genotoxic response.

Original languageEnglish
Pages (from-to)2265-2270
Number of pages6
JournalCarcinogenesis
Volume9
Issue number12
DOIs
Publication statusPublished - Dec 1988

ASJC Scopus subject areas

  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Physiology (medical)
  • Physiology
  • Behavioral Neuroscience
  • Cancer Research

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