Garret B. Nelson

Comparison of the genotoxic activities of the K-region dihydrodiol of benzo[a]pyrene with benzo[a]pyrene in mammalian cells: morphological cell transformation; DNA damage; and stable covalent DNA adducts.

Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides evidence for the DNA damaging and morphological cell transforming activities of the K-region dihydrodiol of B[a]P, in the absence of covalent stable DNA adducts. While trans-B[a]P-4,5-diol and B[a]P both induce morphological cell transformation, their activities as DNA damaging agents differ, both qualitatively and quantitatively. In concert with the morphological cell transformation activities of other K-region dihydrodiols of PAHs, these data suggest a new mechanism/pathway for the morphological cell transforming activities of B[a]P and its metabolites.

Lack of contribution of covalent benzo[a]pyrene-7,8-quinone–DNA adducts in benzo[a]pyrene-induced mouse lung tumorigenesis☆

Benzo[a]pyrene (B[a]P) is a potent human and rodent lung carcinogen. This activity has been ascribed in part to the formation of anti-trans-7,8-dihydroxy-7,8-dihydroB[a]P-9,10-epoxide (BPDE)-DNA adducts. Other carcinogenic mechanisms have been proposed: (1) the induction of apurinic sites from radical cation processes, and (2) the metabolic formation of B[a]P-7,8-quinone (BPQ) that can form covalent DNA adducts or reactive oxygen species which can damage DNA. The studies presented here sought to examine the role of stable BPQ-DNA adducts in B[a]P-induced mouse lung tumorigenesis. Male strain A/J mice were injected intraperitoneally once with BPQ or trans-7,8-dihydroxy-7,8-dihydroB[a]P (BP-7,8-diol) at 30, 10, 3, or 0mg/kg. Lungs and livers were harvested after 24h, the DNA extracted and subjected to (32)P-postlabeling analysis. Additional groups of mice were dosed once with BPQ or BP-7,8-diol each at 30 mg/kg and tissues harvested 48 and 72 h later, or with B[a]P (50mg/kg, a tumorigenic dose) and tissues harvested 72 h later. No BPQ or any other DNA adducts were observed in lung or liver tissues 24, 48, or 72 h after the treatment with 30 mg/kg BPQ. BP-7,8-diol gave BPDE-DNA adducts at all time points in both tissues and B[a]P treatment gave BPDE-DNA adducts in the lung. In each case, no BPQ-DNA adducts were detected. Mouse body weights significantly decreased over time after BPQ or BP-7,8-diol treatments suggesting that systemic toxicity was induced by both agents. Model studies with BPQ and N-acetylcysteine suggested that BPQ is rapidly inactivated by sulfhydryl-containing compounds and not available for DNA adduction. We conclude that under these treatment conditions BPQ does not form stable covalent DNA adducts in the lungs or livers of strain A/J mice, suggesting that stable BPQ-covalent adducts are not a part of the complex of mechanisms involved in B[a]P-induced mouse lung tumorigenesis.

Interaction Analyses of Binary Mixtures of Carcinogenic PAHs Using Morphological Cell Transformation of C3H10T1/2CL8 Mouse Embryo Fibroblasts in Culture

Abstract Studies of defined mixtures of carcinogenic polycyclic aromatic hydrocarbons (PAH) have identified three major categories of interactions: antagonism; synergism; and additivity depending on the biological model, tissue, route of exposure, and specific PAH. To understand the bases of these interactions we studied binary mixtures of benzo[a]pyrene (B[a]P) and dibenz[a,h]anthracene (DBA) in transformable C3H10T1/2C18 (C3H10T1/2) mouse embryo fibroblast cells in culture. C3H10T1/2 cells treated with binary mixtures of B[a]P and DBA gave less than additive morphological cell transformation based on response additivity. These results were consistent with those reported in mice and rats on the antagonistic effects of B[a]P and DBA on tumorigenesis. 32P-Postlabeling DNA adduct studies revealed that DBA reduced B[a]P-DNA adduct levels by 47% with no effect on DBA-DNA adduct levels. This suggests that one mechanism for the inhibition of morphological cell transformation of binary mixtures of B[a]P and DBA is due to alterations in the metabolic activation of B[a]P.

Quantitative changes in endogenous DNA adducts correlate with conazole in vivo mutagenicity and tumorigenicity

The mouse liver tumorigenic conazole fungicides triadimefon and propiconazole have previously been shown to be in vivo mouse liver mutagens in the Big Blue™ transgenic mutation assay when administered in feed at tumorigenic doses, whereas the nontumorigenic conazole myclobutanil was not mutagenic. DNA sequencing of the mutants recovered from each treatment group as well as from animals receiving control diet revealed that propiconazole- and triadimefon-induced mutations do not represent general clonal expansion of background mutations, and support the hypothesis that they arise from the accumulation of endogenous reactive metabolic intermediates within the liver in vivo. We therefore measured the spectra of endogenous DNA adducts in the livers of mice from these studies to determine if there were quantitative or qualitative differences between mice receiving tumorigenic or nontumorigenic conazoles compared to concurrent control animals. We resolved and quantitated 16 individual adduct spots by (32)P postlabelling and thin layer chromatography using three solvent systems. Qualitatively, we observed the same DNA adducts in control mice as in mice receiving conazoles. However, the 13 adducts with the highest chromatographic mobility were, as a group, present at significantly higher amounts in the livers of mice treated with propiconazole and triadimefon than in their concurrent controls, whereas this same group of DNA adducts in the myclobutanil-treated mice was not different from controls. This same group of endogenous adducts were significantly correlated with mutant frequency across all treatment groups (P = 0.002), as were total endogenous DNA adduct levels (P = 0.005). We hypothesise that this treatment-related increase in endogenous DNA adducts, together with concomitant increases in cell proliferation previously reported to be induced by conazoles, explain the observed increased in vivo mutation frequencies previously reported to be induced by treatment with propiconazole and triadimefon.

Abstract 2814: Comparative DNA damage and transcriptomic effects of engineered nanoparticles in human lung cells in vitro

A series of six titanium dioxide and two cerium oxide engineered nanomaterials were assessed for their ability to induce cytotoxicity, reactive oxygen species (ROS), various types of DNA damage, and transcriptional changes in human respiratory BEAS-2B cells exposed in vitro at several concentrations for 72 hours. Only limited cytotoxicity was observed at concentrations up to 300 μg/ml for all of the nanomaterials. Small increases in 8-oxo-deoxyguanosine were induced by some of the nanomaterials, but did not achieve statistical significance. No increases in ethenoadenosine or ethenocytidine were detected by ELISA assays for any of the tested nanomaterials. Several of the nanomaterials exhibited concentration related increases in levels of apurinic/apyrimidinic sites, endogenous DNA adducts measured by 32 P-postlabeling, lipid peroxidation, and ROS. Consistent with these findings, several of the nanomaterials also affected expression of genes involved in p53, ATM, and mismatch repair pathways. Integrin signaling pathways were also altered by a majority of the nanomaterials tested. There was general agreement between activity in DNA damage assays and extent of pathway transcriptional alteration. One out of the cerium oxide nanomaterials tested did not induce a high enough incidence of differentially expressed genes relative to controls to allow analysis at the pathway level, and also elicited the lowest response in multiple DNA damage assays. Taken together, these data are consistent with the contribution of DNA damage induced by reactive oxygen species as mediators of potentially adverse biological effects following exposure to engineered titanium and cerium oxide nanomaterials, and suggests the utility of short term in vitro tests to predict relative potencies of these particles. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy. Citation Format: Garret B. Nelson, Sheau-Fung Y. Thai, Carlton P. Jones, Audrey Barbee, Micaela Killius, Jeffrey A. Ross. Comparative DNA damage and transcriptomic effects of engineered nanoparticles in human lung cells in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2814.

Morphological Cell Transformation and DNA Adduction by Benz(J)Aceanthrylene and its Presumptive Reactive Metabolites in C3H10T1/2CL8 Cells

Benz(j)aceanthrylene [B(j)A], a cyclopenta-fused polycyclic aromatic hydrocarbon found in coal combustion emissions [Grimmer et al., 1985; Schmidt et al., 1986], is a strong inducer of morphological transforming activity in C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts [Mohapatra et al., 1987]. The metabolism of B(j)A by these cells has been studied and the results differ from those found with Aroclor-1254 induced rat liver S9. In C3H10T1/2 cells the major metabolite is 9,10dihydro-9,10-dihydroxy-B(j)A with minor amounts of 1,2-dihydro-1,2-dihydroxy-B(j)A [Mohapatra et al., 1987], while in rat liver S9 the major metabolite is 1,2-dihydro-1,2-dihydroxy-B(j)A [Nesnow et al., 1988]. These results suggest that while the major route of metabolic activation by Aroclor-1254 induced rat liver S9 is through B(j)A-1,2-oxide, the arene oxide at the cyclopenta-ring, metabolic activation of B(j)A in C3H10T1/2 cells might be via two routes: botharene oxide [B(j)A-1,2-oxide] formation and bay-region diol-epoxide formation [9,10-dihydro-9,10-dihydroxy-B(j)A-7,8-oxide]. This study was undertaken to clarify the role of the putative reactive intermediates, 9,10-dihydro-9,10-dihydroxy-B(j)A-7,8-oxide [B(j)A-diol-epoxide], and B(j)A-1,2-oxide in the metabolic activation of B(j)A in C3H10T1/2 cells.