Mechanisms linked to differences in the mutagenic potential of 1,3-dinitropyrene and 1,8-dinitropyrene

J A Holme; H E Nyvold; V Tat; V M Arlt; A Bhargava; K B Gutzkow; A Solhaug; M Låg; R Becher; P E Schwarze; K Ask; L Ekeren; J Øvrevik

doi:10.1016/j.toxrep.2014.07.009

Mechanisms linked to differences in the mutagenic potential of 1,3-dinitropyrene and 1,8-dinitropyrene

Toxicol Rep. 2014 Jul 27:1:459-473. doi: 10.1016/j.toxrep.2014.07.009. eCollection 2014.

Authors

J A Holme¹, H E Nyvold¹, V Tat², V M Arlt³, A Bhargava², K B Gutzkow¹, A Solhaug⁴, M Låg¹, R Becher¹, P E Schwarze¹, K Ask², L Ekeren¹, J Øvrevik¹

Affiliations

¹ Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo, Norway.
² Department of Medicine, McMaster University, Hamilton, ON, Canada.
³ Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health, King's College London, London, United Kingdom.
⁴ Norwegian Veterinary Institute, Oslo, Norway.

Abstract

This study explores and characterizes the toxicity of two closely related carcinogenic dinitro-pyrenes (DNPs), 1,3-DNP and 1,8-DNP, in human bronchial epithelial BEAS-2B cells and mouse hepatoma Hepa1c1c7 cells. Neither 1,3-DNP nor 1,8-DNP (3-30 μM) induced cell death in BEAS-2B cells. In Hepa1c1c7 cells only 1,3-DNP (10-30 μM) induced a mixture of apoptotic and necrotic cell death after 24 h. Both compounds increased the level of reactive oxygen species (ROS) in BEAS-2B as measured by CM-H₂DCFDA-fluorescence. A corresponding increase in oxidative damage to DNA was revealed by the formamidopyrimidine-DNA glycosylase (fpg)-modified comet assay. Without fpg, DNP-induced DNA damage detected by the comet assay was only found in Hepa1c1c7 cells. Only 1,8-DNP formed DNA adduct measured by ³²P-postlabelling. In Hepa1c1c cells, 1,8-DNP induced phosphorylation of H2AX (γH2AX) and p53 at a lower concentration than 1,3-DNP and there was no direct correlation between DNA damage/DNA damage response (DR) and induced cytotoxicity. On the other hand, 1,3-DNP-induced apoptosis was inhibited by pifithrin-α, an inhibitor of p53 transcriptional activity. Furthermore, 1,3-DNP triggered an unfolded protein response (UPR), as measured by an increased expression of CHOP, ATF4 and XBP1. Thus, other types of damage possibly linked to endoplasmic reticulum (ER)-stress and/or UPR could be involved in the induced apoptosis. Our results suggest that the stronger carcinogenic potency of 1,8-DNP compared to 1,3-DNP is linked to its higher genotoxic effects. This in combination with its lower potency to induce cell death may increase the probability of causing mutations.

Keywords: 1,3-DNP, 1,3-dinitropyrene; 1,3-Dinitropyrene; 1,8-DNP, 1,8-dinitropyrene; 1,8-Dinitropyrene; 1-NP, 1-nitropyrene; 3-NBA, 3-nitrobenzanthrone; AhR, aromatic hydrocarbon receptor; Apoptosis; B[a]P, benzo[a]pyrene; CM-H2DCFDA or H2DCFDA, 5-(and 6-)chloromethyl-2,7-dichlorodihydrofluorescein diacetate; CYP, cytochrome P450; Chk, checkpoint kinases; DDR, DNA damage response; DHE, dihydroethidium; DMSO, dimethyl sulfoxide; DNA damage; ER, endoplasmic reticulum; Hoechst 33258, 2(2-(4-hydroxyphenyl)-6-benzimidazole-6-(1-methyl-4-piperazyl)benzimidazole hydrochloride); Hoechst 33342, 2′-(4-ethoxyphenyl)-2′,5′-bis-1H-benzimidazole hydrochloride); NR, nitro-reductasesnitro-PAHnitro substituted-polycyclic aromatic hydrocarbon; Nitro-PAHs; PAH, polycyclic aromatic hydrocarbon; PARP, poly(ADP-ribose) polymerase; PFT, pifithrin; PI, propidium iodide; PM, particular matter; RNS, reactive nitrogen species; ROS, reactive oxygen species; SSB, single strand breaks; UPR, unfolded protein response; fpg, formamidopyrimidine-DNA glycosylase; zVAD-FMK, benzyolcarbonayl-Val-Ala-Asp-fluoromethyl ketone; γH2AX, phosphorylated H2AX.