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

Abstract

Objectives: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Fig. 1

Autoradiogram of ³²P-labeled DNA fragments incubated with 2-nitrotoluene (2-NO₂-T) and its metabolite -2-nitrosotoluene (2-NO-T) in the presence of Cu (II) and β-nicotinamide adenine dinucleotide disodium salt (reduced form) (NADH). a The reaction mixture contained the ³²P-5′-end-labeled 443-bp DNA fragment, 20 μM/base thymus DNA, the indicated concentrations of 2-NO₂-T or 2-NO-T, 20 μM CuCl₂, and 100 μM NADH in 10 mM sodium phosphate buffer (pH 7.8) containing 5 μM diethylenetriamine-N, N, N′, N″, N″-pentaacetic acid (DTPA). b The reaction mixture contained the ³²P-5′-end-labeled 443-bp DNA fragment, 20 μM/base thymus DNA, 0.5 μM 2-NO-T, 20 μM CuCl₂, 100 μM NADH, and a scavenger in 10 mM sodium phosphate buffer (pH 7.8) containing 5 μM DTPA. The scavenger or bathocuproine was added as follows: 5% ethanol, 0.1 M sodium formate, 0.1 M mannitol, 30 units/ml superoxide dismutase (SOD), 30 units/ml catalase, and 50 μM bathocuproine. The mixtures were incubated at 37°C for 1 h, and the DNA fragments were treated with 1 M piperidine at 90°C for 20 min, and then electrophoresed on an 8% polyacrylamide/8 M urea gel. The autoradiogram was obtained by exposing an X-ray film to the gel

Fig. 2

Site specificity of DNA damage induced by 2-NO-T in the presence of Cu (II) and NADH. The reaction mixture contained the ³²P-5′-end labeled 211-bp (HindIII*13972–ApaI 14182) DNA fragment, 20 μM/base calf thymus DNA, 0.2 μM or 0.5 μM 2-NO-T, 20 μM CuCl₂, and 100 μM NADH in 10 mM sodium phosphate buffer (pH 7.8) containing 5 μM DTPA. After incubation for 1 h at 37°C, the DNA fragments were treated with piperidine (a) or formamidopyrimidine-DNA glycosylase (Fpg) protein (b) and electrophoresed by the method described in “Materials and methods”. The relative amounts of DNA fragments were measured by scanning the autoradiogram with a laser densitometer

Fig. 3

Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG) in calf thymus DNA treated with 2-NO-T in the presence of Cu (II) and NADH. The reaction mixture contained 100 μM/base calf thymus DNA, the indicated concentrations of 2-NO-T, 20 μM CuCl₂, and no or 100 μM NADH in 4 mM sodium phosphate buffer (pH 7.8) containing 5 μM DTPA. After incubation at 37°C for 1 h, DNA was precipitated in ethanol and enzymatically digested into nucleosides. Then, the amounts of 8-oxodG were measured by an electrochemical detector coupled to an HPLC system (HPLC-ECD). Results are expressed as the means and SEM for four independent experiments. *Significant difference compared with the control by Student’s t test with Bonferroni correction (the level of significance was less than 0.05)

Fig. 4

Intracellular 8-oxodG formation and 8-oxoguanine DNA glycosylase (OGG1) gene expression induced by 2-NO₂-T and its metabolite 2-NO-T in HL-60 cells. HL-60 cells were treated with 50 μM 2-NO₂-T and 2-NO-T (a, c) or the indicated concentrations of 2-NO-T (b) in the experimental medium at 37°C for 4 h. a, b DNA was extracted and treated as described in “Materials and methods”. Amounts of 8-oxodG were determined by HPLC-ECD. c RNA was extracted and reverse-transcribed to cDNA. OGG1 gene expression was determined by real-time polymerase chain reaction (PCR) as described in “Materials and methods”. Results are expressed as the means ± SEM for four independent experiments. *Significant difference compared with the control by Student’s t test with Bonferroni correction (the level of significance was less than 0.05). GAPDH glyceraldehyde-3-phosphate-dehydrogenase

Fig. 5

Estrogenic activities of 2-NO₂-T and 2-NO-T. a An E-screen assay was performed as described in “Materials and methods”. MCF-7 cells were incubated with 10 μM 2-NO₂-T, 100 pM 17β-estradiol (E2), and 10 μM 2-NO-T + 100 pM E2 at 37°C for 6 days. b MCF-7 cells were incubated with the indicated concentrations of 2-NO₂-T and 2-NO-T at 37°C for 6 days. Then the cells were trypsinized, harvested, and counted. Results are expressed as the means ± SEM for four independent experiments. *Significant difference compared with the control by Student’s t test with Bonferroni correction (the level of significance was less than 0.05). ^#Significant difference between E2 and 2-NO-T + E2 by Student’s t test. c Estrogen receptor α and estrogen response element (ER–ERE) binding was assessed by surface plasmon resonance (SPR). Human ERα (20 nM) was liganded with 100 nM E2, 10 μM 2-NO₂-T, or 2-NO-T by incubation at 37°C for 5 min. Then, the liganded ER was introduced by a 40 μl-injection over a sensor chip immobilized with double-stranded human pS2 ERE. The binding of liganded ER to ERE was expressed as a percentage; that is, with the response to 100 nM E2 taken as 100% and that to no ligand (DMSO, 0.1%) as 0%. Results are expressed as the means for two independent experiments

Fig. 6

Possible mechanisms of a DNA damage and b estrogen-disrupting effects caused by 2-NO₂-T and 2-NO-T