Role of exposure/recovery schedule in micronuclei induction by several promutagens in V79-derived cells expressing human CYP2E1 and SULT1A1

Abstract

The standard procedure for the micronucleus test in cell lines requires a short exposure (≤0.5 cell cycle) to the test compounds followed by a long recovery (≥1.5 cell cycle), and in case of negative or equivocal results, a second test with extended exposure (≥2 cell cycles) without or with a recovery time. In general the two procedures are advantageous for detecting clastogens and aneugens, respectively. However, whether the recommended procedures apply to micronucleus tests with promutagens in cell lines genetically engineered for expressing biotransformation enzymes has not been identified. In this study, several promutagens dependent on cytochrome P450 (CYP) 2E1 and/or sulfotransferase (SULT) 1A1 were used in the micronucleus test in a Chinese hamster V79-derived cell line expressing human CYP2E1 and SULT1A1 (V79-hCYP2E1-hSULT1A1), with varying exposure/recovery schedules: 3h/21h, 6h/18h, 12h/12h, 18h/6h, and 24h/0h, in comparison with known clastogens and aneugens in V79 control cells. The results showed peaked micronuclei induction by mitomycin C and bleomycin (clastogens) at the 12h/12h schedule, while colchicine and vinblastine (aneugens) showed the strongest effect at 24h/0h. Catechol and trihydroxybenzene (activated by CYP2E1) induced micronuclei most strongly at 6h/18h, whereas somewhat longer exposures were optimal for hydroquinone, another compound activated by CYP2E1. 1-Hydroxymethylpyrene (activated by SULT1A1) and 1-methylpyrene (activated sequentially by CYP2E1 and SULT1A1) produced the highest response with the 18h/6h treatment regimen. Moreover, mitotic arrest by 1-hydroxymethylpyrene was observed in V79-hCYP2E1-hSULT1A1 cells but not in V79 cells, and 1-methylpyrene arrested mitosis in V79-hCYP2E1-hSULT1A1 more strongly than in V79 cells. Our study suggests that intracellular bioactivation of promutagens may not delay the induction of micronuclei in the present model, and 1-methylpyrene and 1-hydroxymethylpyrene may be activated to mitosis-arresting metabolites.

Following the adverse outcome pathway from micronucleus to cancer using H2B-eGFP transgenic healthy stem cells

“…Further, it could be confirmed for colchicine and 4-NQO that prolonged recovery time leads to higher MNi frequencies (Fig. 4a) as already shown by Pfeiffer et al for V79 and for colchicine and vinblastine treatment by Jia et al (Jia et al 2016;Pfeiffer et al 1998). For two test compounds DES and EMS it could be shown that MNi frequencies decrease (Fig.…”

Induction of micronuclei and cell cycle arrest by some tri‐ and tetrachlorobiphenyls in mammalian cells deficient in xenobiotic‐metabolizing enzymes

“…Under the 12 h/12 h treatment schedule, a compromised treatment condition for valid detection of micronuclei formation by both clastogenic and aneugenic mechanisms under a single treatment regimen [Jia et al, ], the induction of micronuclei by six trichlorobiphenyls and four tetrachlorobiphenyls with subtle structural differences were determined in V79‐Mz and V79 cells. As shown in Figure A, PCB 66 which has an ortho ‐substituted pattern induced micronuclei in V79‐Mz cells, while PCB 77 whose structure is very similar to PCB 66 except for having a coplanar configuration (without ortho‐ substitution) was inactive, up to 40 μM (a proximate limit of solubility).…”

“…The mitotic index was measured as recently described [Jia et al, ]. A 4.5 cm 2 round glass slide was placed onto each well (9.6 cm 2 , 3 ml medium) of the 6‐well culture boards.…”

Micronuclei Formation by Promutagens in Metabolism‐Incompetent V79 Cells Interacting With Activation‐Proficient Cells in Various Experimental Settings

“…The micronucleus test was conducted under a 3 hr/21 hr (exposure/recovery) regime, which is favorable for the detection of clastogens, as described recently (Jia et al, 2016), whereas a potential microtubuledisturbing effect (relevant to aneugenesis) in V79-Mz cells could be minimized (Matsuoka et al, 1997). Generally, cells were inoculated at the density of 3 × 10 5 per 12.5 cm 2 flask.…”

“…V79 cells, instead of V79-Mz cells, were used in the micronucleus tests in the presence of S9 mix, as the latter subclone of cells is more susceptible to metabolism-independent microtubule damage (aneugenesis) and cell cycle disturbance (Jia et al, 2016;Wang et al, 2017), thus limiting the concentration of a test compound applied to avoid an unspecific microtubule or cell cycle change. The micronucleus tests in V79 cells with rat liver S9 mix was performed as described recently (Jin et al, 2019).…”