ATLA::Alternatives to Laboratory Animals

Volume 24, Number 4

Role of Phase II enzymes in the bioactivation of 1,4-dichlorobenzene and 1,4-dibromobenzene.

ATLA 24, 603-608, July/August 1996

Moreno Paolini,^1-3 Laura Pozzetti,¹ Renata Mesirca,¹ Andrea Sapone,¹ Paola Silingardi,² Sandro Grilli,² Glara Dalla Croce,⁴ Giorgio Bronzetti,⁴ and Giorgio Cantelli-Forti^1-5

¹Department of Pharmacology, Biochemical Toxicology Unit and ²Institute of Cancerology, University of Bologna, 40126 Bologna, Italy; ³Department of Pharmacology/Biology, Pharmacology Unit, University of Bard, via Orabona 4, 70125 Bard, Italy; ⁴Institute of Mutagenesis and Differentiation, CNR, via Suezia 10, 56100 Pisa, Italy; ⁵Department of Preventative Medicine and Community Health, University of Texas Medical Branch, Galveston, TX 77555, USA.

SUMMARY

The use of sodium phenobarbital (PB, CYP2B1 inducer) combined with β-naphthoflavone (β-NF, 1A1) to induce certain Phase I reactions in S9 liver fractions is a standard method for conducting short-term bioassays for genotoxicity. However, because post-oxidative enzymes are also able to activate many precarcinogens, we tested the possibility of adapting S9 liver fractions derived from Phase Il-induced rodents to the field of genetic toxicology. In this study, S9 liver fractions derived from Swiss albino CD1 mice fed 7.5g/kg 2-(3)-tert-butyl4hydroxyanisole (BHA; a monofunctional Phase Il-inducer) for 3 weeks, show a clear pattern of induction with an approximately 3.5-9.5-fold increase in glutathione S-transferase activity. In vitro DNA binding of the promutagenic agents, [¹⁴C]-1,4-dichlorobenzene (DCB) and [¹⁴C]1,4dibromobenzene (DBB), is mediated by such metabolic liver preparations and showed a signfficant increase in covalent binding capability. In some instances, enzyme activity was more elevated when compared to that obtained with traditional (Phase l-induced) S9. Together with DNA binding, the genetic response of these chemicals in the diploid D₇ strain of Saccharomyces cerevisiae used as a biological test system, revealed the ability of the BHA-derived preparations to activate the promutagenic agents, as exemplified by the significant enhancement of mitotic gene-conversion (up to 5.2-fold for DCB and 3.4-fold for DBB) and reverse point mutation (up to 3.6-fold for DCB and 2.5-fold for DBB) at a 4 mM concentration. This novel metabolising biosystem, with enhanced Phase II activity, is recommended together with a traditional S9, for detecting unknown promutagens in genotoxicity studies. The routine use of either oxidative or post-oxidative S9 increases the responsiveness of the test and can contribute to the identification of promutagens not detected when using traditional protocols.

Keywords: monooxygenase, glutathione S-transferase, enzyme induction, 1,4-dichlorobenzene, 1,4-dibromobenzene