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The apoptotic effects of docosahexaenoic acid (DHA) and other omega-3 polyunsaturated fatty acids (PUFAs) have been documented in cell and animal studies. The molecular mechanism by which DHA induces apoptosis is unclear. Although there is no direct evidence, some studies have suggested that DNA damage generated through lipid peroxidation may be involved. Our previous studies showed that DHA, because it has a high degree of unsaturation, can give rise to the acrolein-derived 1,N(2)-propanodeoxyguanosine (Acr-dG) as a major class of DNA adducts via lipid oxidation. As a first step to investigate the possible role of oxidative DNA damage in apoptosis induced by DHA, we examined the relationships between oxidative DNA damage and apoptosis caused by DHA in human colon cancer HT-29 cells. Apoptosis and oxidative DNA damage, including Acr-dG and 8-oxo-deoxyguanosine (8-oxo-dG) formation, in cells treated with DHA and omega-6 PUFAs, including arachidonic acid (AA) and linoleic acid (LA), were measured. DHA induced apoptosis in a dose- and time-dependent manner with a concentration range from 0 to 300 microM as indicated by increased caspase-3 activity and PARP cleavage. In contrast, AA and LA had little or no effect at these concentrations. The Acr-dG levels were increased in HT-29 cells treated with DHA at 240 and 300 microM, and the increases were correlated with the induction of apoptosis at these concentrations, while no significant changes were observed for 8-oxo-dG. Because proteins may compete with DNA to react with acrolein, we then examined the effects of BSA on DHA-induced apoptosis and oxidative DNA damage. The addition of BSA to HT-29 cell culture media significantly decreases Acr-dG levels with a concomitant decrease in the apoptosis induced by DHA. The reduced Acr-dG formation is attributed to the reaction of BSA with acrolein as indicated by increased levels of total protein carbonyls. Similar correlations between Acr-dG formation and apoptosis were observed in HT-29 cells directly incubated with 0-200 microM acrolein. Additionally, DHA treatment increased the level of DNA strand breaks and caused cell cycle arrested at G1 phase. Taken together, these results demonstrate the parallel relationships between Acr-dG level and apoptosis in HT-29 cells, suggesting that the formation of Acr-dG in cellular DNA may contribute to apoptosis induced by DHA.

2-amino-9H-pyrido[2,3-b]indole (AalphaC) is a carcinogenic heterocyclic aromatic amine (HAA) that is produced in high quantities in tobacco smoke and that also forms in charred meats. The bioactivation of AalphaC occurs by cytochrome P450-mediated (P450 1A2) N-oxidation of the exocyclic amine group, to form 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AalphaC). The HONH-AalphaC metabolite can then undergo further activation by phase II enzymes to form the penultimate ester species, which bind to DNA. Some epidemiological studies suggest a role for NAT2 genetic polymorphisms in human susceptibilities to various cancers from tobacco smoke and from consumption of well-done meats, where the exposures to AalphaC can be substantial. In this investigation, we have measured the genotoxicity of AalphaC in nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human P450 1A2 and either the NAT2*4 (rapid, wild-type acetylator) or the NAT2*5B (the most common slow acetylator) allele, to determine the role of NAT2 phenotype in the biological effects of AalphaC. Mutations at the hypoxanthine phosphoribosyl transferase (hprt) locus were induced in a dose-dependent manner by AalphaC and were found to be highest in cells transfected with P450 1A2 and NAT2*4, followed by cells transfected with P450 1A2 and NAT2*5B. The level of formation of the deoxyguanosine (dG) adduct N-(deoxyguanosin-8-yl)-2-amino-9H-pyrido[2,3-b]indole (dG-C8-AalphaC) paralleled the mutagenic potency in these cell lines. However, AalphaC did not form DNA adducts or induce mutations in untransfected CHO cells or in cells only expressing P450 1A2. These findings clearly demonstrate that NAT2 genetic polymorphism plays a major role in the genotoxic potency of AalphaC.

Dibenzo[a,l]pyrene (DBP) is the most potent tumor initiating polycyclic aromatic hydrocarbon tested to date in rodent tumor models. To investigate how DBP adduct formation and removal might influence carcinogenesis, we have examined the effects of treatment of several nucleotide excision repair (NER)-proficient (NER(+)) and -deficient (NER(-)) cell lines with the carcinogenic metabolite (+/-)-anti-DBP-11,12-diol-13,14-epoxide (DBPDE). The treatment of NER(-) cells with (+/-)-anti-DBPDE for 0.5, 1, or 2 h yielded similar total adduct levels, indicating that adduct formation was essentially complete during a 2 h treatment period with no additional adducts produced after replacement of media. In all cell lines, treatment with (+/-)-anti-DBPDE generated five major and at least two minor adducts that were chromatographically identical to those formed by direct treatment of 3'-GMP and 3'-AMP with (+/-)-anti-DBPDE. When adduct levels were assessed in NER(-) cells, the number of adducts/10(9) nucleotides decreased over time, suggesting that DNA replication was ongoing, so we incorporated a normalization strategy based on DNA synthesis. This strategy indicated that DBPDE-DNA adduct levels in NER(-) cells are stable over time. After normalization for DNA synthesis in the NER(+) cells, our data indicated that three adducts showed biphasic repair kinetics. A faster rate of removal was observed during the first 6 h following DBPDE removal followed by a slower rate for up to 34 h. Importantly, two of the major guanine adducts were particularly refractory to removal in the NER(+) cells. Our results suggest that the extreme carcinogenicity of DBPDE may result from the ability of a substantial percentage of two structurally distinct DBPDE-DNA adducts to escape repair.

The (6S,8R,11S) 1,N(2)-HNE-dGuo adduct of trans-4-hydroxynonenal (HNE) was incorporated into the duplex 5'-d(GCTAGCXAGTCC)-3'.5'-d(GGACTAGCTAGC)-3' [X = (6S,8R,11S) HNE-dG], in which the lesion was mismatched opposite dAdo. The (6S,8R,11S) adduct maintained the ring-closed 1,N(2)-HNE-dG structure. This was in contrast to when this adduct was correctly paired with dCyd, conditions under which it underwent ring opening and rearrangement to diastereomeric minor groove cyclic hemiacetals [ Huang , H. , Wang , H. , Qi , N. , Lloyd , R. S. , Harris , T. M. , Rizzo , C. J. , and Stone , M. P. ( 2008 ) J. Am. Chem. Soc. 130 , 10898 - 10906 ]. The (6S,8R,11S) adduct exhibited a syn/anti conformational equilibrium about the glycosyl bond. The syn conformation was predominant in acidic solution. Structural analysis of the syn conformation revealed that X(7) formed a distorted base pair with the complementary protonated A(18). The HNE moiety was located in the major groove. Structural perturbations were observed at the neighbor C(6).G(19) and A(8).T(17) base pairs. At basic pH, the anti conformation of X(7) was the major species. The 1,N(2)-HNE-dG intercalated and displaced the complementary A(18) in the 5'-direction, resulting in a bulge at the X(7).A(18) base pair. The HNE aliphatic chain was oriented toward the minor groove. The Watson-Crick hydrogen bonding of the neighboring A(8).T(17) base pair was also disrupted.

The inhibitory effects of four chlorophyll derivatives (chlorophyllide [Chlide] a and b and pheophorbide [Pho] a and b) on aflatoxin B1 (AFB1)-DNA adduct formation, and on the modulation of hepatic glutathione S-transferase (GST) were evaluated in murine hepatoma (Hepa-1) cells. Enzyme-linked immunosorbent assay showed that pretreatment with Chlide or Pho significantly reduced the formation of AFB1-DNA adducts, and that Pho was the most potent inhibitor. However, wash-out prior to adding AFB1 totally eliminated inhibition by Childe and partially eliminated inhibition by Pho, indicating that the inhibitory effect of Chlide, and to some extent Pho, was mediated through direct trapping of AFB1. Furthermore, spectrophotometric analysis showed that Pho treatment could increase GST activity in Hepa-1 cells. These observations indicate that the chlorophyll derivatives studied may attenuate AFB1-induced DNA damage in the Hepa-1 cell by direct trapping of AFB1. Pho provided additional protection not only by direct trapping, but also by increasing GST activity against hepatic AFB1 metabolites.

Purpose: We present work that demonstrates that cisplatin reacts rapidly with dimethyl sulfoxide (DMSO) in solution and identify the structure and reactivity of the resulting compound., Methods: Electrospray ionization-mass spectrometry (ESI-MS) and NMR were used to identify the chemical structure of compounds formed when DMSO reacts with cisplatin. We studied the reactivity of the identified compound with DNA. In vitro toxicity studies in neurons and cancer cells and in vivo toxicity studies in rats were used to determine both the cancer chemotherapeutic and toxic effects of the identified compound., Results: Cisplatin binds rapidly with DMSO to form a DMSO adduct. The resulting compound has reduced ability to bind to double-stranded DNA both in vitro and in cells. This compound has reduced toxicity for cancer cells and neurons in vitro. In vivo nephrotoxicity studies show that the adducted compound has different nephrotoxicity and elimination characteristics than cisplatin., Conclusions: From this work, we conclude that dissolving cisplatin in DMSO results in formation of an adducted compound with different therapeutic and biological characteristics. Furthermore, future studies which propose using DMSO in combination with cisplatin for chemotherapeutic treatment in patients must be reconsidered. Due to the rapidity and nature of the reaction, DMSO and cisplatin should not be combined for patient treatment.

Heterocyclic amines (HCAs), such as Trp-P-1, PhIP, and IQ, are notorious food-born carcinogens. This study examined the inhibitory effects of HCAs on the expression of interleukin-8 (IL-8), which is an important chemokine for the initiation of innate immune responses that function by recruiting immune cells to inflamed sites. Among HCA types tested, only Trp-P-1 showed a robust inhibition of the lipopolysaccharide (LPS)-induced IL-8 expression at both mRNA and protein levels in a human monocytic cell-line, THP-1. However, Trp-P-1 did not inhibit the DNA-binding abilities of the transcription factors NF-kappaB, AP-1, and NF-IL6, all of which are known to regulate IL-8 transcription. Meanwhile, treatment with actinomycin D facilitated the Trp-P-1-induced down-regulation of IL-8 expression in LPS-stimulated THP-1 cells, implying that the inhibition might be due to a decrease in the stability of IL-8 mRNA rather than an attenuation of IL-8 transcription. Furthermore, Trp-P-1 also inhibited phosphorylation of p38 MAP kinase, which is involved in the regulation of IL-8 mRNA stability. Exogenous addition of ionomycin rescued both the IL-8 protein levels and phosphorylation of p38 MAP kinase inhibited by Trp-P-1. Collectively, these results suggest that Trp-P-1 suppresses LPS-induced IL-8 production in human monocytic cells through down-regulation of an intracellular calcium/p38 MAP kinase-dependent pathway, leading to the reduction of IL-8 mRNA stability.

Polycyclic aromatic hydrocarbons (PAH) are widespread pollutants commonly found in air, food, and drinking water. Benzo[a]pyrene is a well-studied representative PAH found in air from fossil fuel combustion and a transplacental carcinogen experimentally. PAHs bind covalently to DNA to form DNA adducts, an indicator of DNA damage, and an informative biomarker of potential cancer risk. Associations between PAH-DNA adduct levels and both cancer risk and developmental deficits have been seen in previous experimental and epidemiologic studies. Several genes have been shown to play an important role in the metabolic activation or detoxification of PAHs, including the cytochrome P450 genes CYP1A1 and CYP1B1 and the glutathione S-transferase (GST) genes GSTM1, and GSTT2. Genetic variation in these genes could influence susceptibility to adverse effects of PAHs in polluted air. Here, we have explored interactions between prenatal PAH exposure and 17 polymorphisms in these genes (rs2198843, rs1456432, rs4646903, rs4646421, rs2606345, rs7495708, rs2472299, rs162549, rs1056837, rs1056836, rs162560, rs10012, rs2617266, rs2719, rs1622002, rs140194, and gene deletion GSTM1-02) and haplotypes on PAH-DNA adducts in cord blood of 547 newborns and in maternal blood of 806 mothers from three different self-described ethnic groups: African Americans, Dominicans, and Caucasians. PAHs were measured by personal air monitoring of mothers during pregnancy. Significant interactions (p < 0.05) were observed between certain genetic polymorphisms and CYP1A1 haplotype and PAHs in mothers and their newborns in the three ethnic groups. However, with our limited sample size, the current findings are suggestive only, warranting further study.

The mutagenesis of the major DNA adduct N-(deoxyguanosin-8-yl)-1-aminopyrene (C8-AP-dG) formed by 1-nitropyrene was compared with the analogous C8-dG adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) in simian kidney (COS-7) cells. The DNA sequence chosen for this comparison contained 5'-CCATC GCTACC-3' that has been used for solution NMR investigations. The structural and conformational differences among these lesions are well-established [Patel, D. J., Mao, B., Gu, Z., Hingerty, B. E., Gorin, A., Basu, A. K., and Broyde,S. (1998) NMR solution structures of covalent aromatic amine-DNA adducts and their mutagenic relevance. Chem. Res. Toxicol. 11, 391- 407.]. Accordingly, we found a notable difference in the viability of the progeny, which showed that the AAF adduct was most toxic and that the AF adduct was least toxic, with the AP adduct exhibiting intermediate toxicity. However, analysis of the progeny showed that translesion synthesis was predominantly error-free. Only low-level mutations (<3%) were detected with G-->T as the dominant type of mutation by all three DNA adducts. When C8-AP-dG was evaluated in a repetitive 5'-CGC GCG-3' sequence, higher mutational frequency ( approximately 8%) was observed. Again, G-->T was the major type of mutations in simian kidney cells, even though in bacteria CpG deletions predominate in this sequence [Hilario, P., Yan, S., Hingerty, B. E., Broyde, S., and Basu, A. K. (2002) Comparative mutagenesis of the C8-guanine adducts of 1-nitropyrene,and 1,6- and 1,8-dinitropyrene in a CpG repeat sequence: A slipped frameshift intermediate model for dinucleotide deletion. J. Biol. Chem. 277, 45068- 45074.]. Mutagenesis of C8-AP-dG in a 12-mer containing the local DNA sequence around codon 273 of the p53 tumor suppressor gene, where the adduct was located at the second base of this codon, was also investigated. In this 5'-GTGC GTGTTTGT-3' site, the mutations were slightly lower but not very different from the progeny derived from the 5'-CGC GCG-3' sequence. However, the mutational frequency increased by more than 50% when the 5'-C to the adduct was replaced with a 5-methylcytosine (5-MeC). With a 5-MeC, the most notable change in mutation was the enhancement of G-->A, which occurred 2.5 times relative to a 5'-C. The C8-AP-dG adduct in codon 273 dodecamer sequence with a 5'-C or 5-MeC was also evaluated in human embryonic kidney (293T) cells. Similar to COS cells, targeted mutations doubled with a 5-MeC 5' to the adduct. Except for an increase in G-->C transversions, the results in 293T were similar to that in COS cells. We conclude that C8-AP-dG mutagenesis depends on the type of cell in which it is replicated, the neighboring DNA sequence, and the methylation status of the 5'-C.

Recent studies from our laboratory have shown that the transactivation of nuclear factor kappa B (NF kappa B) and activator protein-1 (AP-1) plays an important mechanistic role in ultraviolet (UV)-induced skin carcinogenesis in mice. We also demonstrated that a methanol extract (ME) fraction from black raspberries (Rubus occidentalis) (RO; RO-ME) inhibits benzo[a]pyrene-7,8-diol-9,10-epoxide [B(a)PDE]-induced activation of NF kappa B and AP-1 in cultured mouse epidermal cells. In the present study, we determined if RO-ME might also inhibit the induction of NF kappa B and AP-1 in mouse epidermal cells exposed to mid UV radiation (UVB) and short UV radiation (UVC) and whether methanol fractions from strawberries and blueberries would also be effective. Our results showed that RO-ME inhibited UVB-induced activation of NF kappa B in mouse epidermal cells in a time- and dose-dependent manner; however, the methanol fractions from strawberries and blueberries were ineffective. Interestingly, none of the fractions from all 3 berry types inhibited UVB- or UVC-induced activation of AP-1, suggesting that inhibition of UV-induced signaling pathways is specific for black raspberries and NF kappa B. Cyanidin-3-rutinoside, an anthocyanin found in abundance in black raspberries and not in strawberries or high-bush blueberries, was found to contribute to the inhibition of UVB-induced activation of NF kappa B. These results suggest that berries differ in their ability to influence signaling pathways leading to activation of NF kappa B and AP-1 when using UV light as the inducer.

Cytotoxicity of two fluorescent acridine derivatives - 9-isothiocyanatoacridine (AcITC) and N-(9-acridinylthiocarbamoyl) cytosine (AcTCC) - a novel acridine compound, were investigated. Both substances have cytotoxic activity against the L1210 cellular line, IC50 values were in the micromolar range. Despite the high reactivity of AcITC towards thiols, its effects on leukemia cells were similar to naturally occurring isothiocyanates. AcITC changed the intracellular level of glutathione (GSH), and induced apoptosis. Arrest of cell cycle (G2/M-phase) was also observed. AcITC primarily reacted with -SH groups on cellular surface, and the study of the interaction of the isotiocyanate with human erythrocyte ghosts confirmed that the plasma membrane was the first place where AcITC bound. AcTCC does not react with cellular thiols; images obtained with fluorescent microscopy confirmed interaction of AcTCC with chromatine. Although AcTCC induced cellular arrest in the G2/M phase, apoptosis was not confirmed.

Reaction of crotonaldehyde or two molecules of acetaldehyde with DNA generates 3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxy-6-methylpyrimido[1,2-a]purine-10(3H)one (2, Scheme 1), which occurs in (6R, 8R) and (6S, 8S) configurations (Fig. 1). These diastereomers were site-specifically incorporated into oligonucleotides, which were then inserted into a double-stranded DNA vector for genotoxicity studies. Modified DNA was introduced into human xeroderma pigmentosum A (XPA) cells to allow replication. Analysis of progeny plasmid revealed that these DNA adducts inhibit DNA synthesis to similar degrees. (6S, 8S)-2 miscodes more frequently than (6R, 8R)-2: 10% versus 5%. For both adducts, major miscoding events were G-->T transversions, but G-->A transitions were also observed at a comparable level for (6R, 8R)-2. G-->C transversions were the second most common events for (6S, 8S)-2. Comparison of these results with those of other 1,N2-propanodeoxyguanosine (PdG) adducts, which were evaluated by the same system, indicates that (i) their synthesis inhibiting potencies are stronger than that of the unsubstituted analog, 3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)one (1, Scheme 1), but weaker than that of 3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (3, Scheme 1); (ii) both isomers of 2 are more miscoding than 1; (iii) the miscoding potency of (6S, 8S)-2 is comparable to those of 3 and a model PdG 4 lacking a hydroxyl and a methyl group (Fig. 1). Therefore, considering the fact that 2 are formed endogenously as well as exogenously, they may play a significant role in aging and cancer in humans.

Carcinogen-DNA adducts are thought to be a useful biomarker in epidemiologic studies seeking to show that environmental exposures to xenobiotics cause cancer. This paper reviews the literature in this field from an epidemiologic perspective and identifies several common problems in the epidemiologic design and analysis of these studies. Carcinogen-DNA adducts have been used in studies attempting to link xenobiotic exposures to hepatocellular carcinoma, smoking related cancers and breast cancer. Adduct measurements have been useful in further implicating aflatoxin exposure in the etiology of hepatocellular carcinoma. For smoking related cancers, associations with carcinogen-DNA adducts are commonly seen in current smokers but less so in ex- or non-smokers. In breast cancer the associations have been inconsistent and weak and there is little evidence that carcinogen-DNA adducts implicate xenobiotic exposures in the etiology of breast cancer. Methodological issues common to these studies are the use of target versus surrogate tissues and how this choice impacts control selection, disease effects on adduct levels, the time period reflected by adduct levels, the use of inappropriate statistical analyses and small sample sizes. It is unclear whether the lack of association between carcinogen-DNA adducts and cancer reflects a lack of association between the xenobiotic exposure of interest and cancer or the effects of these methodological issues. A greater focus needs to be placed on designs that allow measurements of adduct levels in tissues collected years prior to cancer diagnosis, there is little need for further hospital based case-control studies in which adducts are measured at the time of or after diagnosis. New designs that address these issues are suggested in the paper.

Exocyclic alkylamino purine adducts, including N(2)-ethyldeoxyguanosine, N(2)-isopropyldeoxyguanosine, and N(6)-isopropyldeoxyadenosine, occur as a consequence of reactions of DNA with toxins such as the ethanol metabolite acetaldehyde, diisopropylnitrosamine, and diisopropyltriazene. However, there are few data addressing the biological consequences of these adducts when present in DNA. Therefore, we assessed the mutagenicities of these single, chemically synthesized exocyclic amino adducts when placed site-specifically in the supF gene in the reporter plasmid pLSX and replicated in Escherichia coli, comparing the mutagenic potential of these exocyclic amino adducts to that of O(6)-ethyldeoxyguanosine. Inclusion of deoxyuridines on the strand complementary to the adducts at 5' and 3' flanking positions resulted in mutant fractions of N(2)-ethyldeoxyguanosine and N(2)-isopropyldeoxyguanosine-containing plasmid of 1.4+/-0.5% and 5.7+/-2.5%, respectively, both of which were significantly greater than control plasmid containing deoxyuridines but no adduct (p=0.04 and 0.003, respectively). The mutagenicities of the three exocyclic alkylamino purine adducts tested were of smaller magnitude than O(6)-ethyldeoxyguanosine (mutant fraction=21.2+/-1.2%, p=0.00001) with the N(6)-isopropyldeoxyadenosine being the least mutagenic (mutant fraction=1.2+/-0.5%, p=0.13). The mutation spectrum generated by the N(2)-ethyl and -isopropyldeoxyguanosine adducts included adduct site-targeted G:C-->T:A transversions, adduct site single base deletions, and single base deletions three bases downstream from the adduct, which contrasted sharply with the mutation spectrum generated by the O(6)-ethyldeoxyguanosine lesion of 95% adduct site-targeted transitions. We conclude that N(2)-ethyl and -isopropyldeoxyguanosine are mutagenic adducts in E. coli whose mutation spectra differ markedly from that of O(6)-ethyldeoxyguanosine.

S(N)1-type alkylating agents that produce cytotoxic O(6)-methyl-G (O(6)-meG) DNA adducts induce cell cycle arrest and apoptosis in a manner requiring the DNA mismatch repair (MMR) proteins MutSalpha and MutLalpha. Here, we show that checkpoint signaling in response to DNA methylation occurs during S phase and requires DNA replication that gives rise to O(6)-meG/T mispairs. DNA binding studies reveal that MutSalpha specifically recognizes O(6)-meG/T mispairs, but not O(6)-meG/C. In an in vitro assay, ATR-ATRIP, but not RPA, is preferentially recruited to O(6)-meG/T mismatches in a MutSalpha- and MutLalpha-dependent manner. Furthermore, ATR kinase is activated to phosphorylate Chk1 in the presence of O(6)-meG/T mispairs and MMR proteins. These results suggest that MMR proteins can act as direct sensors of methylation damage and help recruit ATR-ATRIP to sites of cytotoxic O(6)-meG adducts to initiate ATR checkpoint signaling.

Tamoxifen, a breast cancer drug, has recently been approved for the chemoprevention of this disease. However, tamoxifen causes hepatic carcinomas in rats through a genotoxic mechanism and increases the risk of endometrial tumors in women. DNA adducts have been detected at low levels in human endometrium, and there is much interest in determining whether DNA damage plays a role in tamoxifen-induced endometrial carcinogenesis. This study investigates the mutagenicity of tamoxifen DNA adducts formed by alpha-acetoxytamoxifen, a reactive ester producing the major DNA adduct formed in livers of tamoxifen-treated rats. pSP189 plasmid DNA containing the supF gene was treated with alpha-acetoxytamoxifen and adduct levels (0.5-8.0 adducts per plasmid) determined by (32)P-postlabeling. Adducted plasmids were transfected into nucleotide excision repair proficient (GM00637) or deficient (GM04429, XPA) human fibroblasts. After replication, plasmids were recovered and screened in indicator bacteria. Relative mutation frequencies increased with the adduct level, with 1.3-3.6-fold higher numbers of mutations in the XP cells compared to the GM00637 cells, indicating that NER plays a significant role in the removal of these particular tamoxifen DNA adducts. The majority of sequence alterations (91-96%) occurred at GC base pairs, as did mutation hotspots, although the type and position of mutations was cell-specific. In both cell lines, as the adduct level increased, the proportion of GC --> AT transitions decreased and GC --> TA transversions, mutations known to arise from the major tamoxifen adducts, increased. Given the high mutagenicity of dG-N(2)-tamoxifen adducts, if not excised, they may potentially contribute to the initiation of endometrial cancer in women.

Vinyl chloride and crotonaldehyde are known mutagens and carcinogens that, through their reaction with DNA, form specific deoxyguanosine adducts. To investigate the mutagenic potential of a subset of the possible deoxyguanosine lesions, site-specific adducts of vinyl chloride and crotonaldehyde were synthesized, inserted into a shuttle vector, and replicated in mammalian cells. Mutation yields of the DNA adducts of vinyl chloride and crotonaldehyde were found to be 2% and 5-6%, respectively, thus suggesting that these adducts could contribute to the overall genotoxicity and carcinogenicity associated with exposure to these chemicals.

DNA adducts are mutagenic and clastogenic. Because of their harmful nature, lesions are recognized by many proteins involved in DNA repair. However, mounting evidence suggests that lesions also are recognized by proteins with no obvious role in repair processes. One such protein is topoisomerase II, an essential enzyme that removes knots and tangles from the DNA. Because topoisomerase II generates a protein-linked double-stranded DNA break during its catalytic cycle, it has the potential to fragment the genome. Previous studies indicate that abasic sites and other lesions that distort the double helix stimulate topoisomerase II-mediated DNA cleavage. Therefore, to further explore interactions between DNA lesions and the enzyme, the effects of exocyclic adducts on DNA cleavage mediated by human topoisomerase IIalpha were determined. When located within the four-base overhang of a topoisomerase II cleavage site (at the +2 or +3 position 3' relative to the scissile bond), 3,N(4)-ethenodeoxycytidine, 3,N(4)-etheno-2'-ribocytidine, 1,N(2)-ethenodeoxyguanosine, pyrimido[1,2-a]purin-10(3H)-one deoxyribose (M(1)dG), and 1,N(2)-propanodeoxyguanosine increased DNA scission approximately 5-17-fold. Enhanced cleavage did not result from an increased affinity of topoisomerase IIalpha for adducted DNA or a decreased rate of religation. Therefore, it is concluded that these exocyclic lesions act by accelerating the forward rate of enzyme-mediated DNA scission. Finally, treatment of cultured human cells with 2-chloroacetaldehyde, a reactive metabolite of vinyl chloride that generates etheno adducts, increased cellular levels of DNA cleavage by topoisomerase IIalpha. This finding suggests that type II topoisomerases interact with exocyclic DNA lesions in physiological systems.

Mechanisms of toxicity continue to be important in developing rational strategies to deal with chemicals present in the environment. Understanding and predicting toxicity have also become a critical step in the process of drug development. Covalent binding of chemicals to macromolecules is one aspect of toxicity, and the principles and outcomes of the process are considered. Two examples of chemicals for which several aspects of metabolism and reactions are understood are aflatoxin B(1) and polyhalogenated olefins. Ethylene dibromide is a compound that is activated to genotoxic half-mustards by conjugation with glutathione or the DNA repair protein O(6)-alkylguanine DNA alkyltransferase (AGT). The AGT reaction is unusual, in that crosslinking of the protein to DNA increases mutagenicity. One of the involved mechanisms is formation of N(7)-guanyl crosslinks and depurination to produce G-->T transversions; other reactions appear to yield the additional mutagenic events. The phenomenon of thiol conjugation to increase mutagenicity is widespread among bis-electrophiles.

Furan is an environmental chemical that induces liver toxicity and tumor formation in rodents, leading to its classification as a probable human carcinogen. cis-2-Butene-1,4-dial, the metabolite considered responsible for furan's toxicological effects, is mutagenic in the Ames assay and reacts with 2'-deoxycytidine (dCyd), 2'-deoxyadenosine (dAdo), and 2'-deoxyguanosine (dGuo) to form previously characterized diastereomeric adducts. The initially formed dCyd adducts are stable to rearrangement, while the dAdo and dGuo adducts are unstable and rearrange to form secondary products. On the basis of UV absorbance, fluorescence, 1H NMR, and mass spectral data, the rearrangement product of the dAdo adduct was identified as the substituted etheno-dAdo adduct, 1''-[3-(2'-deoxy-beta-D-erythropentafuranosyl)-3H-imidazo[2,1-i]purin-8-yl]ethane-2''-al. The NMR characterization of the O-methyloxime derivative of the secondary dGuo adduct, along with mass spectral and UV data on the underivatized adduct, allowed for its structural assignment as the substituted etheno-dGuo compound, 3-(2'-deoxy-beta-D-erythropentafuranosyl)imidazo-7-(ethane-2''-al)[1,2-alpha]purine-9-one. The characterization of the primary and secondary products formed in the reaction of cis-2-butene-1,4-dial with nucleosides is important for understanding the mechanism of furan-induced carcinogenesis. These secondary adducts retain a reactive aldehyde with the potential to form cross-links and are likely to contribute significantly to furan's toxic and carcinogenic effects.

The carcinogen 2-acetylaminofluorene is metabolically activated in cells and reacts with DNA to form N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxyguanosin-N(2)()-yl)-2-acetylaminofluorene (dG-N(2)-AAF) DNA adducts. The dG-N(2)-AAF adduct is the least abundant of the three isomers, but it persists in the tissues of animals treated with this carcinogen. The miscoding and mutagenic properties of dG-C8-AAF and dG-C8-AF have been established; these adducts are readily excised by DNA repair enzymes engaged in nucleotide excision repair. In the present study, oligodeoxynucleotides modified site-specifically with dG-N(2)-AAF were used as DNA templates in primer extension reactions catalyzed by mammalian DNA polymerases. Reactions catalyzed by pol alpha were strongly blocked at a position one base before dG-N(2)-AAF and also opposite this lesion. In contrast, during translesion synthesis catalyzed by pol eta or pol kappa nucleotides were incorporated opposite the lesion. Both pol eta and pol kappa incorporated dCMP, the correct base, opposite dG-N(2)-AAF. In reactions catalyzed by pol eta, small amounts of dAMP misincorporation and one-base deletions were detected at the lesion site. With pol kappa, significant dTMP misincorporation was observed opposite the lesion. Steady-state kinetic analysis confirmed the results obtained from primer extension studies. Single-stranded shuttle vectors containing (5)(')TCCTCCTCXCCTCTC (X = dG-N(2)-AAF, dG-C8-AAF, or dG) were used to establish the frequency and specificity of dG-N(2)-AAF-induced mutations in simian kidney (COS-7) cells. Both lesions promote G --> T transversions overall, with dG-N(2)-AAF being less mutagenic than dG-C8-AAF (3.4% vs 12.5%). We conclude from this study that dG-N(2)-AAF, by virtue of its persistence in tissues, contributes significantly to the mutational spectra observed in AAF-induced mutagenesis and that pol eta, but not pol kappa, may play a role in this process.

Chromium(VI) [Cr(VI)], a ubiquitous environmental contaminant, is a well-known carcinogen to both humans and experimental animals, although it is a weak mutagen by itself. Occupational exposure to Cr(VI) is strongly associated with a high incidence of lung cancer, but the underlying mechanisms remain unclear. Tobacco smoking is the major cause of lung cancer, and polycyclic aromatic hydrocarbons (PAHs) in tobacco smoke are the major etiological agents. Since humans are frequently exposed to both Cr(VI) and PAHs, it is possible that Cr(VI) and PAHs have a synergistic effect on mutagenecity and cytotoxicity that contributes to the high incidence of lung cancer associated with exposure to both agents. In this study, we tested this possibility by determining the effect of Cr(VI) exposure on (+/-)-anti-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE, an active metabolite of PAHs) induced cytotoxicity, mutagenicity, and DNA adduct formation in Chinese hamster ovary (CHO) cells. Using the adenine phosphoribosyltransferase (APRT(+)) --> APRT(-) forward mutation assay, we found that while Cr(VI) alone induced low mutation frequency, it greatly enhanced BPDE-induced mutations in nucleotide excision repair (NER)-proficient CHO cells. Cr(VI) exposure also greatly enhanced BPDE-induced killing in NER-proficient cells. It is known that the cytotoxicity and mutagenicity of BPDE are mainly caused by the formation of DNA adduct, which are removed by NER. To test the possibility that the enhancement of cytotoxicity and mutagenicity by Cr(VI) is caused by the inhibition of NER, NER-deficient cells were used, and the enhancement effects of Cr(VI) were not observed in those cells. We further found that while Cr(VI) exposure does not change the total BPDE-DNA adduct formation, it significantly inhibited the repair of BPDE-DNA adducts from genomic DNA in NER-proficient cells. Using a host cell reactivation assay, we found that the repair of BPDE-DNA adduct in a luciferase reporter gene is greatly inhibited after Cr(VI) exposure in NER-proficient cells while not in NER-deficient cells. Together these results clearly demonstrate that Cr(VI) exposure can greatly enhance the mutagenicity and cytotoxicity of PAHs by inhibiting the cellular NER pathway, and this may constitute an important mechanism for Cr(VI)-induced human carcinogenesis.

Biologically effective dose markers--DNA and protein adducts--are classified among exposure biomarkers, and are currently used to assess the biologically active fraction of xenobiotics, which is capable of interacting with cellular macromolecules at the target site. Macromolecular adducts should not only be considered as exposure indicators; indeed, their biological significance can also be extended to biomarkers of effect and of susceptibility. The achievement of such a goal needs research programs aimed both at studying molecular mechanisms related to each step along the continuum of events between exposure and disease, and at establishing quantitative relationships between exposure levels and adduct formation, between adducts and early biological effects, effects and cellular structural/functional modifications, leading to the development and eventual increase in incidence of specific diseases. Moreover, different factors must be considered during data evaluation, such as interindividual variability, the background levels of biomarkers in non occupationally exposed population, the gradually decreasing doses of genotoxic agents involved in occupational exposure, and confounding factors such as diet and smoking habits. Despite the large body of literature documenting DNA and protein adduct molecular dosimetry for many carcinogen exposures, many authors highlight the need for systematic interlaboratory comparison and collaboration by measuring the same biomarkers using different techniques and/or different biomarkers related to the same exposure levels. There is also general agreement about reducing costs, so that dosimetric analyses can become economically more advantageous and accessible in all cases where they prove to be useful in preventing health risks.

It has been proposed that the DNA repair protein O6-alkylguanine-DNA alkyltransferase increases the mutagenicity of 1,2-dibromoethane by reacting with it at its cysteine acceptor site to form a highly reactive half-mustard, which can then react with DNA (Liu, L., Pegg, A. E., Williams, K. M., and Guengerich, F. P. (2002) J. Biol. Chem. 277, 37920-37928). Incubation of Escherichia coli-expressed human alkyltransferase with 1,2-dibromoethane and single-stranded oligodeoxyribonucleotides led to the formation of covalent transferaseoligo complexes. The order of reaction determined was Gua>Thy>Cyt>Ade. Mass spectrometry analysis of the tryptic digest of the reaction product indicated that some of the adducts led to depurination with the release of the Gly136-Arg147 peptide cross-linked to a Gua at the N7 position, with the site of reaction being the active site Cys145 as established by chromatographic retention time and the fragmentation pattern determined by tandem mass spectrometry of a synthetic peptide adduct. The alkyltransferase-mediated mutations produced by 1,2-dibromoethane were predominantly Gua to Ade transitions but, in the spectrum of such rifampicin-resistant mutations in the RpoB gene, 20% were Gua to Thy transversions. The latter are likely to have arisen from the apurinic site generated from the Gua-N7 adduct. Support exists for an additional adduct/mutagenic pathway because evidence was obtained for DNA adducts other than at the Gua N7 atom and for mutations other than those attributable to depurination. Thus, chemical and biological evidence supports the existence of at least two alkyltransferase-dependent pathways for 1,2-dibromoethane-induced mutagenicity, one involving Gua N7-alkylation by alkyltransferase-S-CH2CH2Br and depurination, plus another as yet uncharacterized system(s).

The dihalomethane CH(2)Cl(2) is an industrial solvent of potential concern to humans because of its potential genotoxicity and carcinogenicity. To characterize DNA damage by dihalomethanes, a rapid DNA digestion under acidic conditions was developed to identify alkali labile DNA-dihalomethane nucleoside adducts using HPLC-electrospray mass spectrometry. DNA digestion worked best using pH 5.0 sodium acetate buffer, a 30 min incubation with DNase II and phosphodiesterase II, and a 2 h acid phosphatase digest. DNA was modified with S-(1-acetoxymethyl)glutathione (GSCH(2)OAc), a reagent modeling activated dihalomethanes. Adducts to G, A, and T were detected at high ratios of GSCH(2)OAc/DNA following digestion of the DNA with the procedure used here. The relative efficacy of adduct formation was G > T > A >> C. The four DNA nucleosides were also reacted with the dihalomethanes CH(2)Cl(2) and CH(2)Br(2) in the presence of glutathione (GSH) and GSH S-transferases from bacteria (DM11), rat (GST 5-5), and human (GST T1-1) under conditions that produce mutations in bacteria. All enzymes formed adducts to all four nucleosides, with dGuo being the most readily modified nucleoside. Thus, the pattern paralleled the results obtained with the model compounds GSCH(2)OAc and DNA. CH(2)Cl(2) and CH(2)Br(2) yielded similar amounts of adducts under these conditions. The relative efficiency of adduct formation by GSH transferases was rat 5-5 > human T1-1 > bacterial DM11, showing that human GSH transferase T1-1 can form dihalomethane adducts under the conditions used. Although the lability of DNA adducts has precluded more sophisticated experiments and in vivo studies have not yet been possible, the work collectively demonstrates the ability of several GSH transferases to generate DNA adducts from dihalomethanes, with G being the preferred site of adduction in both this and the GSCH(2)OAc model system.

Instability of repetitive sequences is a hallmark of human cancer, and its enhancement has been linked to oxidative stress. Malondialdehyde is an endogenous product of oxidative stress that reacts with guanine to form the exocyclic adduct, pyrimido[1,2- alpha]purin-10(3H)-one (M1G). We used site-specifically modified single- and double-stranded vectors to investigate the mutagenic potential of M1G in bacteria and mammalian cells. M1G induced frameshift mutations (-1 and -2) when positioned in a reiterated (CpG)4 sequence but not when positioned in a nonreiterated sequence in Escherichia coli and in COS-7 cells. The frequency of frameshift mutations was highest when M1G was placed at the third G in the sequence. M1G induced base pair substitutions at comparable frequencies in both sequence contexts in COS-7 cells. These studies indicate that M1G, an endogenously generated product of oxidative stress, induces sequence-dependent frameshift mutations and base pair substitutions in bacteria and in mammalian cells. This finding suggests a potential role for the M1G lesion in the induction of mutations commonly associated with human diseases.

Acrolein is a bifunctional electrophile, present as an ubiquitous environmental pollutant and an endogenous cellular product of lipid peroxidation. Reaction of acrolein with deoxyguanosine produces two regioisomeric DNA adducts, specifically gamma-hydroxypropanodeoxyguanosine (gamma-HOPdG) and alpha-hydroxypropanodeoxyguanosine (alpha-HOPdG). While previous investigations have focused on the major gamma-HOPdG adduct, little is known about the properties of the minor alpha-HOPdG adduct. Therefore, this comparative investigation has assessed the following: the ability of each adduct to undergo secondary chemical reactions with biomolecules to form various cross-linked species, in vitro translesion DNA synthesis, and mutagenic properties, following replication in mammalian cells. In contrast to gamma-HOPdG, which is capable of forming DNA-DNA, DNA-peptide, and DNA-protein cross-links, alpha-HOPdG did not form any of these cross-linked species. These results can be attributed to the inability of the alpha-HOPdG adduct to undergo ring opening, whereas the gamma-HOPdG adduct forms the ring open, acyclic N(2) oxopropyl in duplex DNA, which readily reacts with nucleophilic functions. Consistent with this interpretation, when polymerase eta replication bypass of DNA containing alpha-HOPdG was assayed, this lesion posed a stronger block to replication than the gamma-HOPdG adduct, closely resembling the results for polymerase eta bypass of propanodeoxyguanosine in which the exocyclic adduct remains permanently ring-closed. Cellular replication and mutagenesis assays in COS-7 cells using single-stranded DNA containing a site specific alpha-HOPdG revealed that this adduct was significantly mutagenic, yielding a nearly identical frequency and spectrum of mutations as compared with the gamma-HOPdG adduct.

Exposure of cells or animals to carcinogenic chromium(VI) (Cr(VI)) produces Cr(III)-DNA adducts. The relevance of these lesions to Cr(VI)-induced tumors is unclear. Various Cr(III) complexes have been used to model the products resulting from Cr(VI) metabolism in order to gain mechanistic insights. The purpose of this study was to characterize interactions of Cr(III) complexes with DNA in order to evaluate their use as models for these purposes. The reactivity of DNA with chromic chloride hexahydrate (CrCl(3)) and sodium bis(l-cysteinato)chromium(III) dihydrate (Cr(cys)(2)(-)) was compared to that with cis-diamminedichloroplatinum(II) (cis-platin). Both Cr(III) and Pt(II) cause unwinding of supercoiled DNA that can be visualized as a mobility shift by gel electrophoresis. Chromic chloride was much less distorting than cis-platin, unwinding DNA by only 1-2 degrees, and Cr(cys)(2)(-) interacted with DNA only weakly. Consistent with in vitro studies, CrCl(3) produced Cr-DNA adducts in CHO AA8 cells at levels equivalent to those obtained with Cr(VI), whereas Cr(cys)(2)(-) did not produce significant adducts. Lesions produced by CrCl(3) were not mutagenic in the hypoxanthine-Gua-phosphoribosyl-transferase assay. These data are consistent with CrCl(3) producing a nondistorting lesion, perhaps by association with the phosphate backbone. There are two possible interpretations of these results: Either the Cr(III) products formed by Cr(VI) metabolism are not modeled by CrCl(3) and Cr(cys)(2)(-) complexes, or Cr(III) is not an active species for Cr(VI)-induced DNA damage. This study provides the first structural evidence for Cr(III)-DNA adducts. A molecular understanding of Cr(III)-DNA interactions will be necessary before we can determine their relevance in Cr(VI)-induced cancers.

Reduction of carcinogenic Cr(VI) by vitamin C generates ascorbate-Cr(III)-DNA cross-links, binary Cr(III)-DNA adducts, and can potentially cause oxidative DNA damage by intermediate reaction products. Here, we examined the mutational spectrum and the importance of different forms of DNA damage in genotoxicity and mutagenicity of Cr(VI) activated by physiological concentrations of ascorbate. Reduction of Cr(VI) led to a dose-dependent formation of both mutagenic and replication-blocking DNA lesions as detected by propagation of the pSP189 plasmids in human fibroblasts. Disruption of Cr-DNA binding abolished mutagenic responses and normalized the yield of replicated plasmids, indicating that Cr-DNA adducts were responsible for both mutagenicity and genotoxicity of Cr(VI). The absence of DNA breaks and abasic sites confirmed the lack of a significant production of hydroxyl radicals and Cr(V)-peroxo complexes in Cr(VI)-ascorbate reactions. Ascorbate-Cr(III)-DNA cross-links were much more mutagenic than smaller Cr(III)-DNA adducts and accounted for more than 90% of Cr(VI) mutagenicity. Ternary adducts were also several times more potent in the inhibition of replication than binary complexes. The Cr(VI)-induced mutational spectrum consisted of an approximately equal number of deletions and G/C-targeted point mutations (51% G/C --> T/A and 30% G/C --> A/T). In Escherichia coli cells, Cr(VI)-induced DNA adducts were only highly genotoxic but not mutagenic under either normal or SOS-induced conditions. Lower toxicity and high mutagenicity of ascorbate-Cr(III)-DNA adducts in human cells may result from the recruitment of an error-prone bypass DNA polymerase(s) to the stalled replication forks. Our results suggest that phosphotriester-type DNA adducts could play a more important role in human than bacterial mutagenesis.

Progressive decline in fertility and sperm quality has been reported over the last few decades, especially in industrialized nations. It has been proposed that exposure to factors that induce damage in DNA of spermatogenic cells may significantly contribute to impaired fertility. Here, the (32)P-postlabelling method was used to analyze the levels of bulky DNA adducts in sperm cells in a group of 179 volunteers, either healthy subjects or patients with an impaired fertility. The levels of DNA adducts were 1.35-fold higher in the infertile group as compared to healthy individuals (P = 0.012). Similarly, a significant negative correlation between the levels of DNA adducts and measures of semen quality (sperm concentration and motility) has been observed (P

Mitomycin C (MC), a cytotoxic anticancer drug and bifunctional DNA DNA alkylating agent, induces cross-linking of the complementary strands of DNA. The DNA interstrand cross-links (ICLs) are thought to be the critical cytotoxic lesions produced by MC. Decarbamoyl mitomycin C (DMC) has been regarded as a monofunctional mitomycin, incapable of causing ICLs. Paradoxically, DMC is slightly more toxic than MC to hypoxic EMT6 mouse mammary tumor cells as well as to CHO cells. To resolve this paradox, EMT6 cells were treated with MC or DMC under hypoxia at equimolar concentrations and the resulting DNA adducts were analyzed using HPLC and UV detection. MC treatment generated both intrastrand and interstrand cross-link adducts and four monoadducts, as shown previously. DMC generated two stereoisomeric monoadducts and two stereoisomeric ICL adducts, all of which were structurally characterized; one was identical with that formed with MC, the other was new and unique to DMC. Overall, adduct frequencies were strikingly higher (20-30-fold) with DMC than with MC. Although DMC monoadducts greatly exceeded DMC cross-link adducts ( approximately 10:1 ratio), the latter were equal or higher in number than the cross-link adducts from MC. DMC displayed a much higher monoadduct:cross-link ratio than MC. The similar cytotoxicities of the two drug show a correlation with their similar DNA cross-link adduct frequencies, but not with their total adduct or monoadduct frequencies. This provides specific experimental evidence that the ICLs rather than the monoadducts are critical factors in the cell death induced by MC. In vitro, overall alkylation of calf thymus DNA by DMC was much less efficient than by MC. Nevertheless, ICLs formed with DMC were clearly detectable. The chemical pathway of the cross-linking was shown to be analogous to that occurring with MC. These results also suggest that the differential sensitivity of Fanconi's Anemia cells to MC and DMC is related to factors other than a selective defect in cross-link repair.

Possible genotoxic effects exerted by three widely used pesticides, permethrin, N,N-diethyl-m-toluamide (DEET) and diazinon, in primary human nasal mucosal cells were investigated. Primary nasal mucosa cells were prepared from tissue biopsies taken from 21 patients who underwent nasal surgery. Cells were exposed to 0.5-1.0 mM concentrations of permethrin, DEET and diazinon for 60 min. Genotoxic effects were detected by the alkaline microgel electrophoresis assay ("comet assay"). Within the concentration range, no significant cytotoxic effects were observed, but all three tested pesticides showed a significant genotoxic response that was concentration dependent. More pronounced genotoxic effects were observed in mucosal cells from the middle turbinate than in the inferior turbinate. The results provide some evidence for the potential carcinogenicity of these agents to human nasal mucosal cells. This should be further investigated.

8-(Hydroxymethyl)-3,N(4)-etheno-C (8-HM-epsilonC) is an exocyclic adduct resulting from the reaction of dC with glycidaldehyde, a mutagen and animal carcinogen. This compound has now been synthesized and its phosphoramidite incorporated site-specifically into a defined 25-mer oligonucleotide. In this study, the mutagenic potential of this adduct in the 25-mer oligonucleotide was investigated in an in vitro primer-template extension assay using four mammalian DNA polymerases. The miscoding potentials were also compared to those of an analogous derivative, 3,N(4)-etheno C (epsilonC), in the same sequence. Both adducts primarily blocked replication by calf thymus DNA polymerase alpha at the modified base, while human polymerase beta catalyzed measurable replication synthesis through both adducts. Nucleotide insertion experiments showed that dA and dC were incorporated by pol beta opposite either adduct, which would result in a C --> T transition or C --> G transversion. Human polymerase eta, a product of the xeroderma pigmentosum variant (XP-V) gene, catalyzed the most efficient bypass of the two lesions with 25% and 32% for 8-HM-epsilonC and epsilonC bypassed after 15 min. Varying amounts of all four bases opposite the modified bases resulted with pol eta. Human polymerase kappa primarily blocked synthesis at the base prior to the adduct. However, some specific misincorporation of dT resulted, forming an epsilonC.T or 8-HM-epsilonC.T pair. From these data, we conclude that the newly synthesized glycidaldehyde-derived adduct, 8-HM-epsilonC, is a miscoding lesion. The bypass efficiency and insertion specificity of 8-HM-epsilonC and epsilonC were similar for all four polymerases tested, which could be attributed to the similar planarity and sugar conformations for these two derivatives as demonstrated by molecular modeling studies.

Site-specific mutagenesis by O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine (O(6)-pobGua), a product of DNA pyridyloxobutylation by metabolites of the tobacco-specific nitrosamines N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), was studied in Escherichia coli strain DH10B and human kidney cells (293) when the modified base was incorporated in either a double-stranded or a gapped shuttle vector. In the repair-competent E. coli strain, less than 3% of the colonies produced by double-stranded vectors harboring the modified base were mutant whereas 96% were mutant when DH10B cells were transformed with modified gapped vectors. By contrast, transformation of DH10B cells with plasmids derived from O(6)-pobGua-containing double-stranded and gapped vectors previously replicated in 293 cells produced 7 and 16% mutant colonies, respectively. These percentages increased to 42 and 82%, respectively, when the 293 cells were pretreated with O(6)-benzylguanine to inactivate the O(6)-alkylguanine-DNA alkyltransferase protein. These findings confirm that the adduct is readily repaired by the human O(6)-alkylguanine-DNA alkyltransferase in both double-stranded and gapped vectors and suggest that it is also highly mutagenic in both human cells and E. coli. In the E. coli strain, the adduct produced exclusively G --> A transition mutations although in human 293 cells it also produced G --> T transversions and more complex mutations in addition to G --> A transitions. These data suggest that O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine can contribute significantly to the mutagenic risk posed by exposure to both NNN and NNK in tobacco smoke.

The effects of turmeric feeding before and after benzo(a)pyrene [B(a)P] exposure on the levels of B(a)P-derived DNA adducts were studied in tissues of Swiss mice employing (32)P-postlabelling analysis. A reduction in the levels of B(a)P-derived DNA adducts in liver, lung, and forestomach was observed in animals pre-treated with 0.2 or 1% turmeric diet and exposed to B(a)P by oral intubation when compared to animals receiving standard laboratory diet and B(a)P. The observed decrease was not due to dilution caused by nascent DNA synthesis. Comparative evaluation of levels of B(a)P-derived DNA adducts in tissues of animals shifted to 0.2 or 1% turmeric diet after 24 h of oral intubation of B(a)P with those continued on standard laboratory diet did not suggest enhanced disappearance/repair of B(a)P-derived DNA adducts due to exposure to turmeric. Further, pre-treatment of mice with 1% turmeric diet significantly reduced the B(a)P-induced increase in activity of cytochrome P450 (CYP450) isozymes CYP 1A1 and 1A2 in liver, lung, and forestomach of mice. In addition, hepatic glutathione S-transferase (GST) was found to be elevated in turmeric pre-treated mice. Thus turmeric-mediated decrease in induction of phase-I enzymes in liver, lung, and forestomach of mice and enhancement of hepatic GST appear to play an important role in reducing the B(a)P-induced DNA damage in target and non-target tissues.

1-Nitropyrene, a common environmental pollutant, forms a major DNA adduct, N-(deoxyguanosin-8-yl)-1-amino- pyrene (dG(AP)). Mutational spectra of randomly introduced dG(AP) in Escherichia coli included different types of mutations, which depended on the base sequence surrounding the adduct. In earlier works we investigated the DNA sequence context effects of the adduct in repetitive CpG and non-repetitive CpGpC sequences. In the current work this adduct was incorporated into a non-repetitive GpGpC sequence in single-stranded M13mp7L2 DNA with the adduct located at either the 5' or 3' G. Potent genotoxicity of dG(AP) was evident from a significant reduction in the population of progeny phage following replication of these constructs in repair-competent E.coli cells. However, progeny derived from the 3'-G(AP) construct were much larger than those from the 5'-G(AP) construct. We suspect that a more facile translesion synthesis past the adduct at the 3' G relative to that at the 5' G, presumably due to a difference in conformation of dG(AP) in these two sites, might be responsible for this effect. With both adducted constructs, >95% of the progeny did not show any mutations at or near the adduct site, indicating highly efficient error-free translesion synthesis. However, a small population of mutants with one base deletions and base substitutions were detected. While the adduct induced -1 frameshifts (<1%) in each G site, base substitutions (1-2%), exhibiting predominantly G-->C transversions, were detected only when the adduct was located at the 5' G. A comparison of the data from this study with a prior study in the CpGpC sequence suggests that dG(AP) mutagenesis is highly sensitive to the local DNA sequence and that a 5'-pyrimidine base might be important for targeted base substitutions by this adduct in E.coli.

Site-specifically modified oligodeoxynucleotides were used to explore the influence of neighboring base sequence context on the mutagenic potential of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF) in mammalian cells. Oligodeoxynucleotides ((5)(')TCCTCCTNXNCTCTC, where X is dG-AAF, dG-AF, or dG and N is C, A, G, or T) with different bases flanking the lesion were incorporated into a single-strand shuttle plasmid vector and used to establish the mutational frequency and specificity of dG-AAF and dG-AF adducts in simian kidney (COS-7) cells. Vectors containing dG-AAF promote preferential incorporation of dCMP at the site of the lesion; misincorporation of dAMP and dTMP also was observed. Mutational frequencies range from 11 to 23%. High mutational frequencies (18-23%) were observed when G or T was positioned 5' to dG-AAF and a lower frequency (11%) when C was 5' to the lesion. dCMP was predominantly incorporated opposite the dG-AF adduct when C, A, or T was 5' to the lesion; dAMP and dTMP were misincorporated at a frequency of 2-4%. With G 5' to the lesion, the overall mutational frequency for dG-AF ranged between 11 and 70%; the highest value occurred when C was the 3' flanking base, and the predominant mutation event was G --> T transversion (59%). We conclude from these experiments that dG-AAF and dG-AF promote G --> T transversions and G --> A transitions in mammalian cells. The mutational frequency and specificity of dG-AF vary significantly, depending on the nature of the bases flanking the lesion.

Acrolein is produced extensively in the environment by incomplete combustion of organic materials, and it arises endogenously in humans as a metabolic by-product. Acrolein reacts with DNA at guanine residues to form the exocyclic adduct, 8-hydroxypropanodeoxyguanosine (HOPdG). Acrolein is mutagenic, and a correlation exists between HOPdG levels in Salmonella typhimurium treated with acrolein and a resultant increase in mutation frequency. Site-specifically modified oligonucleotides were used to explore the mutagenic potential of HOPdG in Escherichia coli strains that were either wild-type for repair or deficient in nucleotide excision repair or base excision repair. Oligonucleotides modified with HOPdG were inserted into double-stranded bacteriophage vectors using the gapped-duplex method or into single-stranded bacteriophage vectors and transformed into SOS-induced E. coli strains. Progeny phage were analyzed by oligonucleotide hybridization to establish the mutation frequency and the spectrum of mutations produced by HOPdG. The correct base, dCMP, was incorporated opposite HOPdG in all circumstances tested. In contrast, in vitro lesion bypass studies showed that HOPdG causes misincorporation opposite the modified base and is a block to replication. The combination of these studies showed that HOPdG is not miscoding in vivo at the level of sensitivity of these site-specific mutagenesis assays.

In this review, a mechanism of estrogen-induced cancer has been examined which features a dual role of estrogen as hormone and carcinogen. Evidence exists that estrogens are metabolically activated to 4-hydroxylated metabolites by a specific cytochrome P450 in tissues prone to estrogen-induced cancer. These metabolites and their semiquinone/quinone oxidation products may cause various types of DNA damage. Preliminary data also exist that estrogens induce various genetic mutations. Tumors may develop from cells carrying such mutations and responding to receptor-mediated proliferation signals., (Copyright 2000 Academic Press.)

Site-specific bay region benzo[a]pyrene (7R,8S,9R,10S)-N(6)-[10-(7,8, 9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyrenyl)]-2'-deoxyadeno syl, (7S,8R,9S,10R)-N(6)-[10-(7,8,9,10-tetrahydro-7,8, 9-trihydroxybenzo[a]pyrenyl)]-2'-deoxyadenosyl, (7S,8R,9R, 10S)-N(6)-[10-(7,8,9,10-tetrahydro-7,8, 9-trihydroxybenzo[a]pyrenyl)]-2'-deoxyadenosyl, and (7R,8S,9S, 10R)-N(6)-[10-(7,8,9,10-tetrahydro-7,8, 9-trihydroxybenzo[a]pyrenyl)]-2'-deoxyadenosyl adducts, bay region benz[a]anthracene (1R,2S,3R,4S)-N(6)-[1-(1,2,3,4-tetrahydro-2,3, 4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl and (1S,2R,3S, 4R)-N(6)-[1-(1,2,3,4-tetrahydro-2,3, 4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adducts, non-bay region benz[a]anthracenyl (8S,9R,10S,11R)-N(6)-[11-(8,9,10, 11-tetrahydro-8,9,10-trihydroxybenz[a]anthracenyl)]-2'-de oxyadenosyl and (8R,9S,10R,11S)-N(6)-[11-(8,9,10,11-tetrahydro-8,9, 10-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adducts, and the R- and S-adducts of styrene oxide were located in the ras61 oligodeoxynucleotide and examined with respect to electrophoretic mobility. The results were compared to NMR structural data, and to site-specific mutagenesis data and in vitro DNA replication assays for the same adducts. There was a correlation between adducts having lower electrophoretic mobility and greater disorder at the adduct site as monitored by NMR. The disorder combined with the lower electrophoretic mobilities suggested that these adducts induced flexible hinge joints in the DNA rather than static bending. Usually, these were adenine N(6) adducts having S-stereochemistry at the benzylic carbon. The results also revealed a possible role for the bay region ring in stabilizing adenyl N(6) benz[a]anthracene adducts with respect to hinging at the adduct site. On the other hand, there was not a simple relationship between altered electrophoretic mobility and mutagenesis or DNA replication.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a mutagenic and carcinogenic heterocyclic amine found in cooked meat. Hepatic DNA adduct formation, in vivo mutagenicity, and hepatocarcinogenicity of MeIQx were examined in mice harboring the lacZ mutation reporter gene (Muta mice) and bitransgenic mice overexpressing the c-myc oncogene. C57Bl/lambda lacZ and bitransgenic c-myc (albumin promoter)/lambda lacZ mice were bred and weaned onto an American Institute of Nutrition-76-based diet containing 0.06% (w/w) MeIQx or onto control diet. After 30 weeks on diet, only male bitransgenic mice on MeIQx developed hepatocellular carcinoma (100% incidence). By 40 weeks, hepatic tumor incidence was 100%/75% (17%/0%) and 44%/17% (0%/0%) in male c-myc/lambda lacZ and C57Bl/lambda lacZ mice who were given MeIQx (or control) diet, respectively, supporting a synergism between MeIQx and c-myc overexpression in hepatocarcinogenesis. At either time point, mutant frequency in the lacZ gene was at least 40-fold higher in MeIQx-treated mice than in control mice of either strain. These findings suggest that MeIQx-induced hepatocarcinogenesis is associated with MeIQx-induced mutations. Elevated mutant frequency in MeIQx-treated mice also occurred concomitant with the formation of MeIQx-guanine adducts, as detected by the 32P-postlabeling assay. Irrespective of strain or diet, sequence analysis of the lacZ mutants from male mouse liver showed that the principal sequence alterations were base substitutions at guanine bases. Adenine mutations, however, were detected only in animals on control diet. MeIQx-fed mice harboring the c-myc oncogene showed a 1.4-2.6-fold higher mutant frequency in the lacZ gene than mice not carrying the transgene. Although there was a trend toward higher adduct levels in c-myc mice, MeIQx-DNA adduct levels were not significantly different between c-myc/lambda lacZ and C57Bl/lambda lacZ mice after 30 weeks on diet. Thus, it seemed that factors in addition to MeIQx-DNA adduct levels, such as the enhanced rate of proliferation associated with c-myc overexpression, may have accounted for a higher mutant frequency in c-myc mice. In the control diet groups, the lacZ mutant frequency was significantly higher in c-myc/lambda lacZ mice than in C57Bl/lambda lacZ mice. The findings are consistent with the notion that c-myc overexpression is associated with an increase in mutagenesis. The mechanism for the synergistic effects of c-myc overexpression on MeIQx hepatocarcinogenicity seems to involve an enhanced expression of MeIQx-induced mutations.

Breast cancer patients treated with the antiestrogen tamoxifen (TAM) show an increased risk of developing endometrial cancer. We have recently detected TAM-DNA adducts in endometrium obtained from patients treated with TAM and identified them as trans- and cis-forms of alpha-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM). To explore the mutagenic properties of these TAM-DNA adducts, we prepared site-specifically modified oligodeoxynucleotides containing a single isomer of dG-N2-TAM by reacting a 15-mer oligodeoxynucleotide containing a single dG (5'-TCCTCCTCGCCTCTC) with tamoxifen alpha-sulfate. These modified oligodeoxynucleotides were inserted into a single-stranded shuttle vector to investigate mutagenic specificities of the adducts in simian kidney (COS-7) cells. An epimer of dG-N2-trans-TAM showed targeted mutations ranging from 0.7 to 1.5%. The other dG-N2-trans-TAM adduct showed 9.6% G-->T transversions, accompanied by 2.8% G-->A transitions. Both dG-N2-cis-TAM adducts showed similar mutation spectra, where G-->T transversions (11-12%) predominated, along with a small number of G-->A transitions and G-->C transversions. Thus, dG-N2-TAMs are mutagenic lesions in mammalian cells. The tamoxifen-DNA adducts detected in patient endometrium may cause mutations and initiate endometrial cancer.

Nitrogen mustard (bis(2-chloroethyl) methylamine, HN2) inhibited the binding of upstream factors Sp1 and AP2 to their consensus sequences. At concentrations where 50% of the consensus sequence DNA contained at least one lesion, HN2 inhibited formation of the Sp1 complex by 37% (40 microM HN2) and the AP2 complex by 40% (50 microM HN2). The binding of the TATA binding protein (TBP) to the TATA element was also inhibited by HN2, whereas sulphur mustard and the monofunctional sulphur mustard 2-chloroethyl ethyl sulphide (CEES) resulted in a disproportional extent of inhibition with respect to the level of alkylation. The level of alkylation of the TBP oligonucleotide varied significantly at 100 microM drug, with 80, 42 and 15% of HN2, sulphur mustard and CEES, respectively. However, this level of alkylation inhibited formation of the TBP-DNA complex by 70, 70 and 45%, respectively. This differential sensitivity of transcription factors to mustard-induced DNA damage therefore appears to reside dominantly in the stereochemical differences between the specific mustard lesions.

Carcinogenic Cr(VI) compounds were previously found to induce amino acid/glutathione-Cr(III)-DNA crosslinks with the site of adduction on the phosphate backbone. Utilizing the pSP189 shuttle vector plasmid we found that these ternary DNA adducts were mutagenic in human fibroblasts. The Cr(III)-glutathione adduct was the most potent in this assay, followed by Cr(III)-His and Cr(III)-Cys adducts. Binary Cr(III)-DNA complexes were only weakly mutagenic, inducing a significant response only at a 10 times higher number of adducts compared with Cr(III)-glutathione. Single base substitutions at the G:C base pairs were the predominant type of mutations for all Cr(III) adducts. Cr(III), Cr(III)-Cys and Cr(III)-His adducts induced G:C-->A:T transitions and G:C-->T:A transversions with almost equal frequency, whereas the Cr(III)-glutathione mutational spectrum was dominated by G:C-->T:A transversions. Adduct-induced mutations were targeted toward G:C base pairs with either A or G in the 3' position to the mutated G, while spontaneous mutations occurred mostly at G:C base pairs with a 3' A. No correlation was found between the sites of DNA adduction and positions of base substitution, as adducts were formed randomly on DNA with no base specificity. The observed mutagenicity of Cr(III)-induced phosphotriesters demonstrates the importance of a Cr(III)-dependent pathway in Cr(VI) carcinogenicity.

To determine the mutagenic and genotoxic properties of the major guanine N2-adduct formed by the antitumor drug mitomycin C, we have synthesized a decanucleotide, d(TTACG[MC]TATCT), containing the adduct, which was inserted into a gapped bacteriophage M13 genome. Analysis of the constructed genome indicated that 41% ligation of the adducted 10-mer occurred on both sides of the gap, whereas the control 10-mer ligated with 34% efficiency. After transfection of the adducted single-stranded M13 DNA into Escherichia coli, the adduct was found to be highly genotoxic. Viability of the adducted genome in a repair-competent strain was only 7%, which increased to 12% and 15% upon induction of SOS by irradiating the cells with 254-nm light at 20 and 50 J/m2, respectively. Even lower viability of 2%, 4.6%, and 0.2% was observed in uvrA, uvrB, and uvrC strains, respectively, which increased up to 10-fold with SOS. An examination of the surviving phage populations revealed that the adduct was not detectably mutagenic. No mutants from the repair-proficient strain were detected after analysis of more than 2500 progeny phage. Only 0.2% of the survivors were mutants in the uvrA strain. It is uncertain, however, if they were induced by the adduct, since all the mutants showed untargeted mutations. We conclude that the major guanine N2-adduct formed by mitomycin C is cytotoxic but not appreciably mutagenic in E. coli.

In order to provide a quantitative basis for pretreatment and therapy of intoxications with sulfur mustard (SM) the toxicokinetics of this agent as well as its major DNA-adduct were studied in male hairless guinea pigs for the intravenous, respiratory and percutaneous routes. The study comprised measurement of the concentration-time course of SM in blood and measurement of the concentrations of intact SM and its adduct to guanine in various tissues at several time points after administration of, or exposure to SM. SM was analyzed in blood and tissues by gas chromatography with automated thermodesorption injection and mass-spectrometric detection. DNA-adducts were measured via an immuno-slot-blot method. In contrast with nerve agents of the phosphofluoridate type, SM partitions strongly to various organs, especially the lung, spleen, liver and bone marrow. The respiratory toxicity of SM appears to be local, rather than systemic. Surprisingly, the maximum concentration of SM in blood upon percutaneous exposure to 1 LCt50 (10,000 mg.min.m-3, estimated) is approximately 6-fold higher than that for nose--only exposure to 3 LCt50 (2,400 mg.min.m-3). Pretreatment of hairless guinea pigs with the potential scavengers N-acetyl cysteine or cysteine isopropyl ester did not significantly increase the LCt50-value for nose--only exposure to SM vapor.

The 1-halogen substituted 2,4-dinitrobenzenes have been found to be mutagenic in Salmonella TA98 with an activity order of 1-fluoro > 1-chloro > 1-bromo > 1-iodo. This specific activity was not lowered in the nitroreductase deficient strain TA98NR and O-acetyltransferase deficient strain TA98/1,8-DNP6, indicating that the mutagenicity of these compounds is not dependent upon -NO2 reduction to -NHOH and its subsequent esterification. The activity was, rather, higher in the former and remained almost equal in the latter strain compared to TA98, suggesting these compounds to react directly with bacterial DNA. Further, the reaction of these halodinitrobenzenes with methionine, at physiological pH, resulting in the formation of 1-S-methyldinitrobenzene showed that these compounds have the ability to attack DNA directly through nucleophilic substitution of the halogen atom and the role of the bacterial nitroreductases for production of mutagenic lesions was not considered. It was, further, proposed that these compounds interact with DNA through SNAr mechanism instead of SN2 cited in literature.