nitrobenzanthrone and Lung-Neoplasms

TP53 is one of the most commonly mutated genes in human tumours. Variations in the types and frequencies of mutations at different tumour sites suggest that they may provide clues to the identity of the causative mutagenic agent. A useful model for studying human TP53 mutagenesis is the partial human TP53 knock-in (Hupki) mouse containing exons 4-9 of human TP53 in place of the corresponding mouse exons. For an in vitro assay, embryo fibroblasts from the Hupki mouse can be examined for the generation and selection of TP53 mutations because mouse cells can be immortalized by mutation of Tp53 alone. Thus far, four environmental carcinogens have been examined using the Hupki embryo fibroblast immortalization assay: (a) UV light, which is linked to human skin cancer; (b) benzo[a]pyrene, which is associated with tobacco smoke-induced lung cancer; (c) 3-nitrobenzanthrone, a suspected human lung carcinogen linked to diesel exposure; and (d) aristolochic acid, which is linked to Balkan endemic nephropathy-associated urothelial cancer. In each case, a unique TP53 mutation pattern was generated that corresponded to the pattern found in human tumours where exposure to these agents has been documented. Therefore, the Hupki embryo fibroblast immortalization assay has sufficient specificity to make it applicable to other environmental mutagens that putatively play a role in cancer aetiology. Despite the utility of the current Hupki embryo fibroblast immortalization assay, it has several limitations that could be addressed by future developments, in order to improve its sensitivity and selectivity.

Topics: Animals; Aristolochic Acids; Benz(a)Anthracenes; Carcinogens, Environmental; Carcinoma; Environmental Exposure; Exons; Gene Knock-In Techniques; Genes, p53; Humans; Lung Neoplasms; Mice; Neoplasms; Neoplasms, Radiation-Induced; Skin Neoplasms; Smoking; Tumor Suppressor Protein p53; Ultraviolet Rays; Urothelium

Exposure to environmental and occupational contaminants leads to lung cancer. 3-Nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one, 3-NBA) is a potential carcinogen in ambient air or diesel particulate matter. Studies have revealed that short-term exposure to 3-NBA induces cell death, reactive oxygen species activation, and DNA adduct formation and damage. However, details of the mechanism by which chronic exposure to 3-NBA influences lung carcinogenesis remain largely unknown. In this study, human lung epithelial BEAS-2B cells were continuously exposed to 0-10-μM 3-NBA for 6 months. NanoString analysis was conducted to evaluate gene expression in the cells, revealing that 3-NBA-mediated transformation results in a distinct gene expression signature including carbon cancer metabolism, metastasis, and angiogenesis. Alterations in tumor-promoting genes such as EREG (epiregulin), SOX9, E-cadherin, TWIST, and IL-6 were involved in epithelial cell aggressiveness. Kaplan-Meier plotter analyses indicated that increased EREG and IL-6 expressions in early-stage lung cancer cells are correlated with poor survival. In vivo xenografts on 3-NBA-transformed cells exhibited prominent tumor formation and metastasis. EREG knockout cells exposed to 3-NBA for a short period exhibited high apoptosis and low colony formation. By contrast, overexpression of EREG in 3-NBA-transformed cells markedly activated the PI3K/AKT and MEK/ERK signaling pathways, resulting in tumorigenicity. Furthermore, elevated IL-6 and EREG expressions synergistically led to STAT3 signaling activation, resulting in clonogenic cell survival and migration. Taken together, chronic exposure of human lung epithelial cells to 3-NBA leads to malignant transformation, in which the EREG signaling pathway plays a pivotal mediating role. • Short-term exposure of lung epithelial cells to 3-NBA can lead to ROS production and cell apoptosis. • Long-term chronic exposure to 3-NBA upregulates the levels of tumor-promoting genes such as EREG and IL-6. • Increased EREG expression in 3-NBA-transformed cells markedly contributes to tumorigenesis through PI3K/AKT and MEK/ERK activation and synergistically enhances the IL-6/STAT3 signaling pathway, which promotes tumorigenicity.

Topics: Benz(a)Anthracenes; Cadherins; Carbon; Carcinogenesis; Carcinogens; Cell Transformation, Neoplastic; DNA Adducts; Epiregulin; Epithelial Cells; Humans; Interleukin-6; Lung; Lung Neoplasms; Mitogen-Activated Protein Kinase Kinases; Particulate Matter; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and potential human carcinogen identified in diesel exhaust and ambient air particulate matter. Previously, we detected the formation of 3-NBA-derived DNA adducts in rodent tissues by 32P-postlabeling, all of which are derived from reductive metabolites of 3-NBA bound to purine bases, but structural identification of these adducts has not yet been reported. We have now prepared 3-NBA-derived DNA adduct standards for 32P-postlabeling by reacting N-acetoxy-3-aminobenzanthrone (N-Aco-ABA) with purine nucleotides. Three deoxyguanosine (dG) adducts have been characterised as N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-C8-N-ABA), 2-(2'-deoxyguanosin-N2-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-N2-ABA) and 2-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-C8-C2-ABA), and a deoxyadenosine (dA) adduct was characterised as 2-(2'-deoxyadenosin-N6-yl)-3-aminobenzanthrone-3'-phosphate (dA3'p-N6-ABA). 3-NBA-derived DNA adducts formed experimentally in vivo and in vitro were compared with the chemically synthesised adducts. The major 3-NBA-derived DNA adduct formed in rat lung cochromatographed with dG3'p-N2-ABA in two independent systems (thin layer and high-performance liquid chromatography). This is also the major adduct formed in tissue of rats or mice treated with 3-aminobenzanthrone (3-ABA), the major human metabolite of 3-NBA. Similarly, dG3'p-C8-N-ABA and dA3'p-N6-ABA cochromatographed with two other adducts formed in various organs of rats or mice treated either with 3-NBA or 3-ABA, whereas dG3'p-C8-C2-ABA did not cochromatograph with any of the adducts found in vivo. Utilizing different enzymatic systems in vitro, including human hepatic microsomes and cytosols, and purified and recombinant enzymes, we found that a variety of enzymes [NAD(P)H:quinone oxidoreductase, xanthine oxidase, NADPH:cytochrome P450 oxidoreductase, cytochrome P450s 1A1 and 1A2, N,O-acetyltransferases 1 and 2, sulfotransferases 1A1 and 1A2, and myeloperoxidase] are able to catalyse the formation of 2-(2'-deoxyguanosin-N2-yl)-3-aminobenzanthrone, N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone and 2-(2'-deoxyadenosin-N6-yl)-3-aminobenzanthrone in DNA, after incubation with 3-NBA and/or 3-ABA.

Topics: Adenine; Animals; Benz(a)Anthracenes; DNA Adducts; Female; Guanine; Lung; Lung Neoplasms; Phosphorus Radioisotopes; Rats; Rats, Sprague-Dawley; Vehicle Emissions