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

11 Citations (Scopus)

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

Fingerprint

Mutagenicity Tests
Liver
Assays
Enzymes
Fold
Mutagenesis
Lipid Peroxidation
Glutathione
Lipids
DNA
Mouse
Viperidae
Enzyme activity
Decrease
Epoxide Hydrolases
Yeast
Optimization
Test System
Saccharomyces Cerevisiae
False Positive

ASJC Scopus subject areas

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

Cite this

Improvement of short-term tests for mutagenicity : On the optimal pH for the liver microsomal assay. / Paolini, M.; Sapigni, E.; Hrelia, P.; Grilli, S.; Lattanzi, G.; Cantelli-forti, G.

In: Carcinogenesis, Vol. 9, No. 12, 12.1988, p. 2265-2270.

Research output: Contribution to journalArticle

Paolini, M. ; Sapigni, E. ; Hrelia, P. ; Grilli, S. ; Lattanzi, G. ; Cantelli-forti, G. / Improvement of short-term tests for mutagenicity : On the optimal pH for the liver microsomal assay. In: Carcinogenesis. 1988 ; Vol. 9, No. 12. pp. 2265-2270.
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AU - Lattanzi, G.

AU - Cantelli-forti, G.

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N2 - 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.

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