Martin R. Osborne

Environmental Pollutant and Potent Mutagen 3-Nitrobenzanthrone Forms DNA Adducts after Reduction by NAD(P)H:Quinone Oxidoreductase and Conjugation by Acetyltransferases and Sulfotransferases in Human Hepatic Cytosols

3-Nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one, 3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and air pollution. We compared the ability of human hepatic cytosolic samples to catalyze DNA adduct formation by 3-NBA. Using the (32)P-postlabeling method, we found that 12/12 hepatic cytosols activated 3-NBA to form multiple DNA adducts similar to those formed in vivo in rodents. By comparing 3-NBA-DNA adduct formation in the presence of cofactors of NAD(P)H:quinone oxidoreductase (NQO1) and xanthine oxidase, most of the reductive activation of 3-NBA in human hepatic cytosols was attributed to NQO1. Inhibition of adduct formation by dicoumarol, an NQO1 inhibitor, supported this finding and was confirmed with human recombinant NQO1. When cofactors of N,O-acetyltransferases (NAT) and sulfotransferases (SULT) were added to cytosolic samples, 3-NBA-DNA adduct formation increased 10- to 35-fold. Using human recombinant NQO1 and NATs or SULTs, we found that mainly NAT2, followed by SULT1A2, NAT1, and, to a lesser extent, SULT1A1 activate 3-NBA. We also evaluated the role of hepatic NADPH:cytochrome P450 oxidoreductase (POR) in the activation of 3-NBA in vivo by treating hepatic POR-null mice and wild-type littermates i.p. with 0.2 or 2 mg/kg body weight of 3-NBA. No difference in DNA binding was found in any tissue examined (liver, lung, kidney, bladder, and colon) between null and wild-type mice, indicating that 3-NBA is predominantly activated by cytosolic nitroreductases rather than microsomal POR. Collectively, these results show the role of human hepatic NQO1 to reduce 3-NBA to species being further activated by NATs and SULTs.

Identification of three major DNA adducts formed by the carcinogenic air pollutant 3-nitrobenzanthrone in rat lung at the C8 and N2 position of guanine and at the N6 position of adenine

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.

Detection of bulky DNA lesions in the liver of patients with Wilson's disease and primary haemochromatosis

In the human metal storage disorders of Wilsons disease and primary haemochromatosis, ion transport and excretion dysfunctions result in the intracellular deposition of copper and iron, respectively. These aberrant accumulations of transition metal ions lead to extensive tissue damage, especially in the liver. In order to investigate the possible role of metal ion-mediated oxygen free radical-generated DNA damage in these processes, DNA was isolated from liver of eight Wilsons disease patients and six haemochromatosis patients. Significant levels of bulky DNA damage were detected in these samples by 32P-postlabelling analysis, but were not found in liver DNA from age-matched controls. This form of novel DNA damage was detected in six out of eight Wilsons patients, varying between approximately 1 and 100 base modifications per 10(8) nucleotides, and in all of the haemochromatosis samples examined; the levels of modified species per 10(8) nucleotides varying from approximately 2 to 50. HPLC analysis of these bulky DNA lesions demonstrated that the species formed in Wilsons disease and in haemochromatosis were chromatographically identical but were not the same as putative purine dimers that can be generated in DNA by in vitro incubation with Cu+/Fe2+ and H2O2 (although the possibility that the adducts detected are closely related has not been ruled out). Analysis of the oxidative base lesion 8-hydroxydeoxyguanosine showed that levels were not elevated in liver DNA from either Wilsons disease or haemochromatosis sufferers. In fact, a statistically significantly lower level of this lesion was found in Wilsons disease patients than in controls. These data suggest that bulky DNA damage present in the liver of both wilsons disease and primary haemochromatosis patients may play a more important role in the induction of tissue damage than 8-hydroxydeoxyguanosine. The novel DNA damage detected by 32P-poslabelling may also be a significant factor in the initiation of neoplasia leading to malignant hepatoma in haemochromatosis patients.

DNA adduct formation by the ubiquitous environmental pollutant 3-nitrobenzanthrone and its metabolites in rats.

Diesel exhaust is known to induce tumours in animals and is suspected of being carcinogenic in humans. Of the compounds found in diesel exhaust, 3-nitrobenzanthrone (3-NBA) is an extremely potent mutagen and suspected human carcinogen forming multiple DNA adducts in vitro. 3-Aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA), and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) were identified as 3-NBA metabolites. In order to gain insight into the pathways of metabolic activation leading to 3-NBA-derived DNA adducts we treated Wistar rats intraperitoneally with 2mg/kg body weight of 3-NBA, 3-ABA, 3-Ac-ABA, or N-Ac-N-OH-ABA and compared DNA adducts present in different organs. With each compound either four or five DNA adduct spots were detected by TLC in all tissues examined (lung, liver, kidney, heart, pancreas, and colon) using the nuclease P1 or butanol enrichment version of the 32P-postlabelling method, respectively. Using HPLC co-chromatographic analysis we showed that all major 3-NBA-DNA adducts produced in vivo in rats are derived from reductive metabolites bound to purine bases and lack an N-acetyl group. Our results indicate that 3-NBA metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) undergo several biotransformations and that N-hydroxy-3-aminobenzanthrone (N-OH-ABA) appears to be the common intermediate in 3-NBA-derived DNA adduct formation. Therefore, 3-NBA-DNA adducts are useful biomarkers for exposure to 3-NBA and its metabolites and may help to identify enzymes involved in their metabolic activation.

Mutagenic fingerprint of ozone in human cells

Ozone is an important factor in urban pollution and represents a major concern for human health. The chemical reactivity of ozone toward biological targets and particularly its genotoxicity supports a possible link between exposure and cancer risk, but no molecular data exist on its mutagenic potential in human cells. Using a shuttle vector, we showed that ozone is indeed a potent mutagen and we characterized the mutation spectrum it produced in human cells. Almost all mutations are base substitutions, essentially located at G:Cs (75%), typical of reactive oxygen species (ROS), but occurring in a specific pattern, i.e. a similar extent of GC:TA (28%), GC:CG (23%) and GC:AT (23%). The targeted distribution of mutations and identification of hotspot sequences define the first molecular fingerprint of mutations induced by ozone in human cells. Possible applications derived from our results with respect to ozone genotoxicity should help determining quantifiable biomarkers of ozone exposure in human health, especially for carcinogenesis.

Metallothionein crypt-restricted immunopositivity indices (MTCRII) correlate with aberrant crypt foci (ACF) in mouse colon

Metallothionein (MT) crypt-restricted immunopositivity indices (MTCRII) are colonic crypt stem cell mutation markers that may be induced early and in abundance after mutagen treatment. Metallothionein is the endogenous reporter gene for MTCRII, but is not typically implicated in the classical pathway of colorectal tumorigenesis. Hence, the oncological relevance of MTCRII is unclear. This study tests the hypothesis that MTCRII induced by N-methyl-N-nitrosourea (MNU) and lambda carrageenan (λCgN) associate with aberrant crypt foci (ACF) in mouse colon. Undegraded λCgN and MNU were tested alone and in combination against MTCRII and ACF in Balb/c mice, at 20 weeks after the start of treatment. MTCRII were unaffected by λCgN alone. Combined λCgN/MNU treatments induced greater MTCRII (P<0.01) as well as greater number (P<0.001) and crypt multiplicity (P<0.01) of ACF than MNU alone. MTCRII were approximately 10-fold more numerous than ACF, although linear correlations were observed between these parameters (r=0.732; P<0.01). MTCRII are induced by λCgN/MNU interactions in sufficient numbers to provide statistical power from relatively small sample sizes and correlate with ACF formation. MTCRII could thus provide the basis for a novel medium-term murine bioassay relevant to early-stage colorectal tumorigenesis.

Synthesis and DNA reactivity of alpha-hydroxylated metabolites of nonsteroidal antiestrogens

Tamoxifen [(E)-1-(4-(2-(N,N-dimethylamino)ethoxy)phenyl)-1, 2-diphenylbut-1-ene], a nonsteroidal antiestrogen, induces liver tumors in rats by a genotoxic mechanism. The mechanism of DNA adduct formation is believed to proceed via the formation of a reactive carbocation at the alpha-position from the alpha-hydroxylated metabolite. Molecular mechanics calculations [Kuramochi, H. (1996) J. Med. Chem. 39, 2877-2886] have predicted that 4-substitution will affect the stability of the carbocation and thus will alter its reactivity toward DNA. We have synthesized the putative alpha-hydroxylated metabolites of 4-hydroxytamoxifen [(E)-1-(4-(2-(N, N-dimethylamino)ethoxy)phenyl)-1-(4-hydroxyphenyl)-3-hydroxy-2-phenyl but-1-ene] and idoxifene [(Z)-1-(4-iodophenyl)-3-hydroxy-2-phenyl-1-(4-(2-(N-pyrrolidino) ethoxy)phenyl)but-1-ene] and compared their reactivities with DNA with that of alpha-hydroxytamoxifen [(E)-1-(4-(2-(N, N-dimethylamino)ethoxy)phenyl)-3-hydroxy-1,2-diphenylbut-1-ene]. As predicted, the bis-hydroxylated compound reacted with DNA in aqueous solution at pH 5 to give 12-fold greater levels of adducts than alpha-hydroxytamoxifen, whereas alpha-hydroxyidoxifene gave one-half the number of adducts. The results demonstrate that idoxifene presents a significantly lower genotoxic hazard than tamoxifen for the treatment and prophylaxis of breast cancer.