Garret Nelson

A quantitative comparison of dibenzo[a,l]pyrene-DNA adduct formation by recombinant human cytochrome P450 microsomes†

Dibenzo[a,l]pyrene (DB[a,l]P), an extremely potent environmental carcinogen, is metabolically activated in mammalian cells and microsomes through the fjord‐region dihydrodiol, trans‐DB[a,l]P‐11,12‐diol, to syn‐ and anti‐DB[a,l]P‐11,12‐diol‐13,14‐epoxides (syn‐ and anti‐DB[a,l]PDEs). The role of seven individual recombinant human cytochrome P450s (1A1, 1A2, 1B1, 2B6, 2C9, 2E1, and 3A4) in the metabolic activation of DB[a,l]P and formation of DNA adducts was examined by using 32P postlabeling, thin‐layer chromatography, and high‐pressure liquid chromatography. We found that, in the presence of epoxide hydrolase, only P450 1A1 and P450 1B1 catalyzed the formation of DB[a,l]PDE‐DNA adducts and several unidentified polar adducts. Human P450 1A1 catalyzed the formation of DB[a,l]PDE‐DNA adducts and unidentified polar adducts at rates threefold and 17‐fold greater than did human P450 1B1 (256 fmol/h/nmol P450 versus 90 fmol/h/nmol P450 and 132 fmol/h/nmol P450 versus 8 fmol/h/nmol P450, respectively). P450 1A1 DNA adducts were derived from both anti‐ and syn‐DB[a,l]PDE at rates of 73 fmol/h/nmol P450 and 51 fmol/h/nmol P450, respectively. P450 1B1 produced adducts derived from anti‐DB[a,l]PDE at a rate of 82 fmol/h/nmol, whereas only a small number of adducts were derived from syn‐DB[a,l]PDE (0.4 fmol/h/nmol). These results demonstrated the potential of human P450 1A1 and P450 1B1 to contribute to the metabolic activation and carcinogenicity of DB[a,l]P and provided additional evidence that human P450 1A1 and 1B1 differ in their stereospecific activation of DB[a,l]P. Mol. Carcinog. 26:74–82, 1999. Published 1999 Wiley‐Liss, Inc.

Abstract 826: DNA adducts induced by in vitro activation of diesel and biodiesel exhaust extracts

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Petroleum diesel exhaust is a complex mixture containing many probable and known carcinogens. The development of biodiesel and biodiesel blends offers a renewable alternative to petroleum diesel, but few data are available concerning the carcinogenic potential of biodiesel exhausts. We compared the formation of covalent DNA adducts by the in vitro metabolic activation of organic extracts of diesel exhaust particles (DEP)from petroleum diesel and biodiesel. Two different petroleum diesel DEPs were examined (C-DEP and B0), as well as one biodiesel DEP (B100), and DEP resulting from combustion of a blend of 20% B100 and 80% B0 (B20), which is representative of commercially available biodiesel. Oxidative activation was performed in the presence of calf thymus DNA (ctDNA) using rat liver microsomal fractions with required cofactors (S9), and nitroreductive activation was performed using xanthine oxidase (XO) and hypoxanthine in the presence of ctDNA. The modified DNA was hydrolyzed and analyzed by 32P-postlabeling using either butanol extraction or nuclease P1 pre-enrichment. Multiple DNA adducts were produced with chromatographic mobilities consistent with polycyclic aromatic hydrocarbon (PAH) and nitro-PAH adducts. The types and quantities of DNA adducts produced from two independent petroleum diesel DEPs were similar, with evidence of formation of both PAH- and nitroPAH- derived adducts. In contrast, the biodiesel DEP B100 induced higher total levels of DNA adducts in both activation systems. The lowest levels of DNA adduct formation were observed with B20 DEP. These results suggest that the DEP from available biodiesel blends (B20) pose lower risk for induction of DNA damage than petroleum diesel. This is an abstract or a proposed presentation and does not necessarily reflect EPA policy. Citation Format: Jeffrey A. Ross, Garret B. Nelson, Esrta Mutlu, Sarah H. Warren, Matthews P. Peggy, M. Ian Gilmour, David M. DeMarini. DNA adducts induced by in vitro activation of diesel and biodiesel exhaust extracts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 826. doi:10.1158/1538-7445.AM2015-826

Abstract 3594: Mutations and DNA adducts induced by diesel exhaust particles.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC We performed bioassay-directed fractionation and analyzed the polycyclic aromatic hydrocarbon (PAH) levels of a composite sample of diesel-exhaust particles (C-DEP) generated on site from petroleum diesel with a 30-kW 4-cylinder Deutz BF4M1008 diesel engine connected to an air compressor. C-DEP was generated to be representative of contemporary DEP. We extracted particles with dichloromethane (DCM) and determined the percentage of extractable organic material (EOM), solvent-exchanged extracts into dimethyl sulfoxide, and evaluated them for mutagenicity in Salmonella strains TA100 and TA98 +/- S9. More than 50% of the C-DEP EOM mass eluted in fraction 1, but this fraction was not mutagenic. Fraction 2 contained 6% of the mass of OEM, and had 60% of the TA100+S9 activity, suggestive of PAHs. The 3rd fraction contained 14% of the OEM mass and contributed 60% of the TA98-S9 activity, suggestive of nitroarenes. S9 mediated binding of C-DEP extracts was assessed by 32P-postlabeling analysis. Unfractionated C-DEP EOM was incubated with S9 in the presence of calf thymus DNA. The modified DNA was enzymatically hydrolyzed and then subjected to postlabeling analysis using either butanol extraction or nuclease P1 pre-enrichment. Multiple DNA adducts were produced with chromatographic mobilities consistent with PAH and nitro-PAH adducts. Although previous studies of the mutagenicity and adduct-forming potential of diesel exhaust have been reported, most of those utilized exhaust particles that were generated by older diesel engines that were not representative of contemporary diesel exhaust. Data derived from the C-DEP particles are more relevant to understanding the risks posed by contemporary diesel engines. This study provides further characterization of the biochemical potential for these particles to induce DNA damage and gene mutations, key events in the process of chemical carcinogenesis by diesel exhausts. [This is an abstract of a proposed presentation and does not necessarily reflect the views of the U.S. EPA.] Citation Format: Jeffrey A. Ross, Esra Mutlu, Charly King, Sarah H. Warren, David M. DeMarini, M. Ian Gilmour, William P. Linak, Garret B. Nelson. Mutations and DNA adducts induced by diesel exhaust particles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3594. doi:10.1158/1538-7445.AM2013-3594

Abstract 2548: Quantitative changes in endogenous DNA adducts correlate with conazole mutagenicity and tumorigenicity in mouse liver

We have previously shown that the conazole fungicides triadimefon and propiconazole, which are tumorigenic in mouse liver, are in vivo mouse liver mutagens in the Big Blue™ transgenic mutation assay when administered in feed at tumorigenic doses. The nontumorigenic conazole myclobutanil was not mutagenic under the same conditions. DNA sequencing of the mutants recovered from each treatment group and from untreated control animals revealed that the mutations induced by propiconazole and triadimefon do not result from clonal expansion of background mutations. We hypothesized that these mutations arise from the accumulation of reactive electrophilic metabolic byproducts within the liver in vivo. We therefore measured the spectra of endogenous DNA adducts in the livers of mice from these studies in order to determine if quantitative or qualitative differences in DNA adducts correlated with mutagenicity and tumorigenicity. We resolved and quantitated 16 individual adduct spots by 32 P postlabeling and thin layer chromatography using 3 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. We hypothesize that this treatment-related increase in endogenous DNA adducts may explain the observed increased in vivo mutation frequencies previously reported to be induced by treatment with propiconazole and triadimefon. This abstract does not necessarily reflect EPA policy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2548. doi:1538-7445.AM2012-2548

Effect of the Route of Administration on the Induction of Cytogenetic Damage and DNA Adducts in Peripheral Blood Lymphocytes of Rats and Mice by Polycyclic Aromatic Hydrocarbons

Experiments were designed to investigate how the route of exposure to polycyclic aromatic hydrocarbons (PAHs) in mice and rats affects the induction of cytogenetic end points and DNA adduction. Both mice and rats were exposed to 100 mg/kg of benz[ a ]anthracene (B[ a ]A), benzo[ b ]fluoranthene (B[ b ]F), benzo[ a ]pyrene (B[ a ]P), or chrysene (Chr) by gavage or by intraperitoneal injection (i.p.). Peripheral blood was removed by cardiac puncture 7 days after PAH administration. Blood samples were analyzed in parallel for sister chromatid exchange (SCE) frequency, the frequency of micronuclei in cytochalasin B-induced binucleate cells (MN bn ), and DNA adduction using 32P-postlabeling. The i.p. route of exposure produced both the highest levels of cytogenetic damage and DNA adducts for each PAH. The mouse was more sensitive than the rat to PAH exposure as measured by SCE induction and the total amount of DNA adducts/ w g DNA.

Benzo[A]Pyrene and Dibenzo[A, L]Pyrene do not Alter gap Junction Communication in Rat Liver Epithelial Cells

Abstract Dibenzo[a, l]pyrene (DB[a, l]P) is more potent than benzo[a]pyrene (B[a]P) in causing tumors in rodents. We investigated the possibility that this higher DB[a, l]P potency involves alterations in gap junction communication (GJC), a cell activity associated with cancer promotion processes. Rat liver epithelial cells, treated with various concentrations up to 50 μM (0.0126 mg/ml) B[a]P or 40 μM (0.0121 mg/ml) DB[a, l]P for 24 hours, were examined for changes in GJC; no changes were observed. However, cells treated with the maximum soluble concentrations of these chemicals had between 213 and 378 attomoles of DNA adduct per μg of DNA. Thus the observed lack of GJC alteration is not attributable to an inability of these cells to metabolize PAHs, and indicates that the different potency exhibited by the two chemicals is not associated with alterations in GJC.

Relationship Between Tumorigenic Potency, Ki-ras Codon 12 Mutations, and DNA Adducts Induced by Cyclopenta[cd]pyrene

Abstract Cyclopenta[cd]pyrene (CPP) was examined for its lung tumorigenic activity in strain A/J mice, for the formation and persistence of CPP-induced DNA adducts in lung tissue, and for its induction of mutations in the Ki−ras oncogene from CPP-induced tumors. CPP displayed high tumorigenic activity, inducing 97.7 lung adenomas/mouse at 200 mg/kg. Ki−ras codon 12 mutations in the DNA of induced tumors were: GGT→CGT (50%); GGT→KTT (15%); GGT→TGT (25%); GGT→GAT (10%). All DNA adducts in the lungs of CPP-treated mice were CPP−3,4−oxide derived and most were CPP−3,4−oxide−2′−deoxyguanosine adducts. CPP is highly tumorigenic in the strain A/J mouse lung adenoma model, being 5 times more active than benzo[a]pyrene. The increased activity of CPP may be related to the unique induction of the GGT→CGT, Ki−ras codon 12 mutation.