Genotoxicity of 1‐methylpyrene and 1‐hydroxymethylpyrene in chinese hamster V79‐derived cells expressing both human CYP2E1 and SULT1A1

Abstract

1-Methylpyrene (1-MP) is a widespread pollutant that is carcinogenic in animals following metabolic activation. Previous studies have shown that benzylic hydroxylation of 1-MP, catalyzed by multiple CYP isoforms, gives rise to 1-hydroxymethylpyrene (1-HMP), which becomes bioreactive following further metabolism by various sulfotransferase (SULT) isoforms. However, the mutagenic and chromosome damaging effects of 1-MP and 1-HMP in mammalian cells have not been investigated. In this study a Chinese hamster V79-derived cell line expressing both human CYP2E1 and human SULT1A1 was used to investigate the ability of 1-MP and 1-HMP to induce cytotoxicity (using the CCK-8 assay), micronuclei and Hprt gene mutations. The role of each enzyme was investigated through co-exposure in the presence of an enzyme inhibitor. We found that at concentrations of 0.5-4 μM and 5-20 μM, under conditions where no reduction in cell viability/growth occurred, 1-HMP and 1-MP induced micronuclei in V79-hCYP2E1-hSULT1A1 cells in a concentration-dependent manner; however, both compounds were inactive in V79 cells. Similarly, they both caused an increase in Hprt mutant frequency in V79-hCYP2E1-hSULT1A1 cells in these concentration ranges, with 1-MP impairing cell viability/growth at 10 μM and above in the mutagenicity assay. The compounds were again both inactive in V79 cells. The effects of 1-HMP in V79-hCYP2E1-hSULT1A1 cells were blocked or reduced by addition of pentachlorophenol (PCP), a SULT1 inhibitor; the genotoxicity of 1-MP was significantly reduced by either 1-aminobenotrazole, a CYP2E1 inhibitor, or PCP. The results suggest that human CYP2E1 and SULT1A1 cooperate to activate 1-MP and cause genotoxicity in mammalian cells.

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

“…As a chromogenic indicator for nicotinamide adenine dinucleotide (NADH) and cell viability (Ishiyama et al, 1997), CCK-8 values correspond well with cell numbers, as previously reported (Jiang et al, 2015). As a chromogenic indicator for nicotinamide adenine dinucleotide (NADH) and cell viability (Ishiyama et al, 1997), CCK-8 values correspond well with cell numbers, as previously reported (Jiang et al, 2015).…”

“…1-HMP is an intermediate metabolite of 1-MP, formed through hydroxylation of the methyl carbon of 1-MP by a CYP enzyme. 1-HMP is further activated through sulfo-conjugation by a SULT (e.g., SULT1A1) for the formation of 1-sulfooxymethylpyrene (1-SMP), which is electrophilic, capable of forming adducts with DNA and proteins (Bendadani et al, 2014b), and inducing mutagenic responses (Jiang et al, 2015). Benzo[a]pyrene is primarily activated to benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) by CYP1A1 and 1B1 (Shimada et al, 2001), with the additional involvement of microsomal epoxide hydrolase, an enzyme ubiquitously expressed in all cell lines studied, including V79 cells (Glatt et al, 1990a).…”

Impact of p53 function on the sulfotransferase‐mediated bioactivation of the alkylated polycyclic aromatic hydrocarbon 1‐hydroxymethylpyrene in vitro

“…Bioactivation of 1-HMP in humans can be catalyzed by several SULTs, of which human SULT1A1 is reported to be by far the most efficient followed by 1E1. Furthermore, SULT1A2, 1A3, 1B1, 1C2, 1C3, 2A1, and 2E1 also convert 1-HMP into a genotoxic metabolite, but to much lower extents (Meinl et al 2002;Meinl et al 2013;Jiang et al 2015). The antibody raised against SULT1A used in the present study detects SULT1A1 and 1A3 with a similar affinity.…”

“…Furthermore, SULT1A2, 1A3, 1B1, 1C2, 1C3, 2A1, and 2E1 also convert 1-HMP into a genotoxic metabolite, but to much lower extents (Meinl et al 2002;Meinl et al 2013;Jiang et al 2015). Furthermore, SULT1A2, 1A3, 1B1, 1C2, 1C3, 2A1, and 2E1 also convert 1-HMP into a genotoxic metabolite, but to much lower extents (Meinl et al 2002;Meinl et al 2013;Jiang et al 2015).…”

“…TP53(+/+), TP53(+/−), and TP53(−/−) cells were exposed to 5, 10, and 20 μM 1-HMP or DMSO alone (control) for 24 and 48 hr. These concentrations were chosen according to previous studies (Glatt et al 1994;Jiang et al 2015). TP53(+/−) cells showed the strongest sensitivity to 1-HMP exposure; at 20 μM, viability was decreased to 77% and 72% after 24 and 48 hr, respectively (Fig.…”