DNA damage and estrogenic activity induced by the environmental pollutant 2-nitrotoluene and its metabolite

Abstract

The environmental pollutant 2-nitrotoluene (2-NO(2)-T) is carcinogenic and reproductively toxic in animals. In this study, we elucidated the mechanisms of its carcinogenicity and reproductive toxicity. We examined DNA damage induced by 2-NO(2)-T and its metabolite, 2-nitrosotoluene (2-NO-T), using (32)P-5'-end-labeled DNA. We measured 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in calf thymus DNA and cellular DNA in cultured human leukemia (HL-60) cells treated with 2-NO(2)-T and 2-NO-T. 8-Oxoguanine DNA glycosylase (OGG1) gene expression in HL-60 cells was measured by real-time polymerase chain reaction (PCR). We examined estrogenic activity using an E-screen assay and a surface plasmon resonance (SPR) sensor. In experiments with isolated DNA fragments, 2-NO-T induced oxidative DNA damage in the presence of Cu (II) and β-nicotinamide adenine dinucleotide disodium salt (reduced form) (NADH), while 2-NO(2)-T did not. 2-NO-T significantly increased levels of 8-oxodG in HL-60 cells. Real-time polymerase chain reaction (PCR) analysis revealed upregulation of OGG1 gene expression induced by 2-NO-T. An E-screen assay using the human breast cancer cell line MCF-7 revealed that 2-NO(2)-T induced estrogen-dependent cell proliferation. In contrast, 2-NO-T decreased the cell number and suppressed 17β-estradiol-induced cell proliferation. The data obtained with the SPR sensor using estrogen receptor α and the estrogen response element supported the results of the E-screen assay. Oxidative DNA damage caused by 2-NO-T and estrogen-disrupting effects caused by 2-NO(2)-T and 2-NO-T may play a role in the reproductive toxicity and carcinogenicity of these entities.