Diminished carcinogen detoxification is a novel mechanism for hypoxia-inducible factor 1-mediated genetic instability.

The hypoxia-inducible factor 1 (HIF-1) pathway is induced in many tumors and associated with poorer outcome. The hypoxia-responsive transcription factor HIF-1alpha dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT), which is also an important binding partner for the aryl hydrocarbon receptor (AhR). AhR is an important mediator in the metabolic activation and detoxification of carcinogens, such as the environmental pollutant benzo[a]pyrene (BaP). We hypothesized that HIF-1alpha activation attenuates BaP-induced AhR-mediated gene expression, which may lead to increased genetic instability and malignant progression. Human lung carcinoma cells (A549) were simultaneously stimulated with CoCl(2), which leads to HIF-1alpha stabilization and varying concentrations of BaP. Both quantitative PCR and immunoblot analysis indicated that induction of the hypoxia response pathway significantly reduced the levels of AhR downstream targets CYP1A1 and CYP1B1 and AhR protein binding to ARNT. We further demonstrate that the BaP-induced hypoxanthine-guanine phosphoribosyltransferase mutation frequency and gamma-H2AX foci were markedly amplified when the HIF-1 pathway was induced. BaP-DNA adducts were only marginally increased, and transient strand breaks were diminished by HIF-1 induction, indicating changes in DNA repair. These data indicate that concurrent exposure of tumor cells to hypoxia and exogenous genotoxins can enhance genetic instability.

The role of polycyclic aromatic hydrocarbon-DNA adducts in inducing mutations in mouse skin.

Polycyclic aromatic hydrocarbons (PAH) form stable and depurinating DNA adducts in mouse skin to induce preneoplastic mutations. Some mutations transform cells, which then clonally expand to establish tumors. Strong clues about the mutagenic mechanism can be obtained if the PAH-DNA adducts can be correlated with both preneoplastic and tumor mutations. To this end, we studied mutagenesis in PAH-treated early preneoplastic skin (1 day after exposure) and in the induced papillomas in SENCAR mice. Papillomas were studied by PCR amplification of the H-ras gene and sequencing. For benzo[a]pyrene (BP), BP-7,8-dihydrodiol (BPDHD), 7,12-dimethylbenz[a]anthracene (DMBA) and dibenzo[a,l]pyrene (DB[a,l]P), the codon 13 (GGC to GTC) and codon 61 (CAA to CTA) mutations in papillomas corresponded to the relative levels of Gua and Ade-depurinating adducts, despite BP and BPDHD forming significant amounts of stable DNA adducts. Such a relationship was expected for DMBA and DB[a,l]P, as they formed primarily depurinating adducts. These results suggest that depurinating adducts play a major role in forming the tumorigenic mutations. To validate this correlation, preneoplastic skin mutations were studied by cloning H-ras PCR products and sequencing individual clones. DMBA- and DB[a,l]P-treated skin showed primarily A.T to G.C mutations, which correlated with the high ratio of the Ade/Gua-depurinating adducts. Incubation of skin DNA with T.G-DNA glycosylase eliminated most of these A.T to G.C mutations, indicating that they existed as G.T heteroduplexes, as would be expected if they were formed by errors in the repair of abasic sites generated by the depurinating adducts. BP and its metabolites induced mainly G.C to T.A mutations in preneoplastic skin. However, PCR over unrepaired anti-BPDE-N(2)dG adducts can generate similar mutations as artifacts of the study protocol, making it difficult to establish an adduct-mutation correlation for determining which BP-DNA adducts induce the early preneoplastic mutations. In conclusion, this study suggests that depurinating adducts play a major role in PAH mutagenesis.

Mitomycin-DNA adducts induce p53-dependent and p53-independent cell death pathways.

10-Decarbamoyl-mitomycin C (DMC), a mitomycin C (MC) derivative, generates an array of DNA monoadducts and interstrand cross-links stereoisomeric to those that are generated by MC. DMC was previously shown in our laboratory to exceed the cytotoxicity of MC in a human leukemia cell line that lacks a functional p53 pathway (K562). However, the molecular signal transduction pathway activated by DMCDNA adducts has not been investigated. In this study, we have compared molecular targets associated with signaling pathways activated by DMC and MC in several human cancer cell lines. In cell lines lacking wild-type p53, DMC was reproducibly more cytotoxic than MC, but it generated barely detectable signal transduction markers associated with apoptotic death. Strikingly, DMCs increased cytotoxicity was not associated with an increase in DNA double-strand breaks but was associated with early poly(ADP-ribose) polymerase (PARP) activation and Chk1 kinase depletion. Alkylating agents can induce increased PARP activity associated with programmed necrosis, and the biological activity of DMC in p53-null cell lines fits this paradigm. In cell lines with a functional p53 pathway, both MC and DMC induced apoptosis. In the presence of p53, both MC and DMC activate procaspases; however, the spectrum of procaspases involved differs for the two drugs, as does induction of p73. These studies suggest that in the absence of p53, signaling to molecular targets in cell death can shift in response to different DNA adduct structures to induce non-apoptotic cell death.

Escherichia coli RecA promotes strand invasion with cisplatin-damaged DNA.

The antitumor drug cisplatin causes intrastrand cross-linking of adjacent guanine residues that severely distorts the DNA backbone. These DNA adducts impede the progress of the replisome and may result in replication fork arrest. In Escherichia coli, the response to cisplatin involves the action of the prototypic recombinase RecA. Here we show that RecA can utilize, albeit at reduced levels, oligonucleotides that bear site-specific cisplatin-induced 1,2 d(GpG) intrastrand cross-links in strand invasion reactions. Binding of RecA to cisplatin-damaged oligonucleotides was not affected, indicating that the impediment was in the pairing step. The cognate E. coli single-strand DNA-binding protein specifically stimulated strand invasion particularly with cisplatin-damaged DNA. These results indicate that RecA is capable of processing the major cisplatin-induced lesion via a recombination mechanism.

[DNA damage induced by products of lipid peroxidation]. / Uszkodzenia DNA powodowane przez produkty peroksydacji lipidów.

The adduction of the aldehydic end-products of lipid peroxidation to DNA induces bulky adducts, leading to genome instability. The bulky-DNA adducts are miscoding and thus play a fundamental role in mutagenesis and cancerogenesis. Special attention is given to the etheno- and propanoadducts, recognized as DNA modifiers. Such DNA lesions are repaired by different DNA repair mechanisms, mainly base excision repair (BER) and nucleotide excision repair (NER), as well as nucleotide incision repair (NIR) and transcription-coupled repair (TCR).

Yeast Nhp6A/B and mammalian Hmgb1 facilitate the maintenance of genome stability.

Saccharomyces cerevisiae Nhp6A and Nhp6B are chromatin architectural factors that belong to the high-mobility group box (HMGB) superfamily and appear to be functionally related to mammalian Hmgb1. They bind to the minor groove of double-stranded DNA in a non-sequence-specific manner and thereby influence chromatin structure. Previous work has implicated these proteins in a variety of nuclear processes, including chromatin remodeling, DNA replication, transcription, and recombination . Here, we show that Nhp6A/B loss leads to increased genomic instability, hypersensitivity to DNA-damaging agents, and shortened yeast cell life span that is associated with elevated levels of extrachromosomal rDNA circles. Furthermore, we show that hypersensitivity toward UV light does not appear to reflect a decreased capacity for DNA repair but instead correlates with higher levels of UV-induced thymine dimer adducts being formed in cells lacking Nhp6A/B. Likewise, we show that mouse fibroblasts lacking Hmgb1 display higher rates of damage after UV irradiation than wild-type controls and also exhibit pronounced chromosomal instability. Taken together, these data indicate that Nhp6A/B and Hmgb1 protect DNA from damaging agents and thus guard against the generation of genomic aberrations.

The implausibility of leukemia induction by formaldehyde: a critical review of the biological evidence on distant-site toxicity.

Formaldehyde is a naturally occurring biological compound that is present in tissues, cells, and bodily fluids. It is also a potent nasal irritant, a cytotoxicant at high doses, and a nasal carcinogen in rats exposed to high airborne concentrations. The normal endogenous concentration of formaldehyde in the blood is approximately 0.1 mM in rats, monkeys, and humans, and it is 2- to 4-fold higher in the liver and nasal mucosa of the rat. Inhaled formaldehyde enters the one-carbon pool, and the carbon atom is rapidly incorporated into macromolecules throughout the body. Oxidation to formate catalyzed by glutathione-dependent and -independent dehydrogenases in nasal tissues is a major route of detoxication and generally precedes incorporation. The possibility that inhaled formaldehyde might induce various forms of distant-site toxicity has been proposed, but no convincing evidence for such toxicity has been obtained in experimental studies. This review summarizes the biological evidence that pertains to the issue of leukemia induction by formaldehyde, which includes: (1) the failure of inhaled formaldehyde to increase the formaldehyde concentration in the blood of rats, monkeys, or humans exposed to concentrations of 14.4, 6, or 1.9 ppm, respectively; (2) the lack of detectable protein adducts or DNA-protein cross-links (DPX) in the bone marrow of normal rats exposed to [3H]- and [14C]formaldehyde at concentrations as high as 15 ppm; (3) the lack of detectable protein adducts or DPX in the bone marrow of glutathione-depleted (metabolically inhibited) rats exposed to [3H]- and [14C]formaldehyde at concentrations as high as 10 ppm; (4) the lack of detectable DPX in the bone marrow of Rhesus monkeys exposed to [14C]formaldehyde at concentrations as high as 6 ppm; (5) the failure of formaldehyde to induce leukemia in any of seven long-term inhalation bioassays in rats, mice, or hamsters; and (6) the failure of formaldehyde to induce chromosomal aberrations in the bone marrow of rats exposed to airborne concentrations as high as 15 ppm or of mice injected intraperitoneally with formaldehyde at doses as high as 25 mg/kg. Biological evidence that might be regarded as supporting the possibility of leukemia induction by formaldehyde includes: (1) the detection of cytogenetic abnormalities in circulating lymphocytes in seven studies of human subjects exposed to ambient concentrations in the workplace (but not in seven other studies of human subjects or in rats exposed to 15 ppm); (2) the induction of leukemia in rats in a single questionable drinking water study with formaldehyde concentrations as high as 1.5 g/L (but not in three other drinking water studies with concentrations as high as 1.9 or 5 g/L); (3) the detection of chromosomal aberrations in the bone marrow of rats exposed to very low concentrations of formaldehyde (0.4 or 1.2 ppm) (but not in another study at concentrations as high as 15 ppm); and (4) an apparent increase in the fraction of protein-associated DNA (assumed to be due to DPX) in circulating lymphocytes of humans exposed to ambient concentrations in the workplace (1-3 ppm). This evidence is regarded as inconsequential for several reasons, including lack of reproducibility, inadequate reporting of experimental methods, inconsistency with other data, or insufficient analytical sensitivity, and therefore, it provides little justification for or against the possibility that inhaled formaldehyde may be a leukemogen. In contrast to these inconclusive findings, the abundance of negative evidence mentioned above is undisputed and strongly suggests that there is no delivery of inhaled formaldehyde to distant sites. Combined with the fact that formaldehyde naturally occurs throughout the body, and that multiple inhalation bioassays have not induced leukemia in animals, the negative findings provide convincing evidence that formaldehyde is not leukemogenic.

Mutational spectrum and genotoxicity of the major lipid peroxidation product, trans-4-hydroxy-2-nonenal, induced DNA adducts in nucleotide excision repair-proficient and -deficient human cells.

trans-4-Hydroxy-2-nonenal (4-HNE), a major product of lipid peroxidation, is able to interact with DNA to form 6-(1-hydroxyhexanyl)-8-hydroxy-1,N(2)-propano-2'-deoxyguanosine (4-HNE-dG) adducts, but its genotoxicity and mutagenicity remain elusive. It has been reported that 4-HNE treatment in human cells induces a high frequency of G.C to T.A mutations at the third base of codon 249 (AGG*) of the p53 gene, a mutational hot spot in human cancers, particularly in hepatocellular carcinoma. This G.C to T.A transversion at codon 249, however, has been thought to be caused by etheno-DNA adducts induced by the endogenous metabolite of 4-HNE, 2,3-epoxy-4-hydroxynonanal. We have recently found that 4-HNE preferentially forms 4-HNE-dG adducts at the GAGG*C/A sequence in the p53 gene including codon 249 (GAGG*C). Our finding supports the possibility that G.C to T.A mutations at codon 249 may be induced by 4-HNE-dG adducts. To investigate this possibility, we determined the mutational spectrum induced by 4-HNE-dG adducts in the supF gene of shuttle vector pSP189 replicated in human cells. We have found that 4-HNE-dG adducts are mutagenic and genotoxic in human cells, and that G.C to T.A transversions are the most prevalent mutations induced by 4-HNE-dG adducts. Furthermore, 4-HNE-dG adducts induce a significantly higher level of genotoxicity and mutagenicity in nucleotide excision repair (NER)-deficient human and Escherichia coli cells than in NER-proficient cells, indicating that NER is a major pathway for repairing 4-HNE-dG adducts in both human and E. coli cells. Together, these results suggest that 4-HNE-dG adducts may contribute greatly to the G.C to T.A mutation at codon 249 of the p53 gene, and may play an important role in carcinogenesis.

Molecular epidemiologic studies of polycyclic aromatic hydrocarbon-DNA adducts and breast cancer.

We review our studies on the role of polycyclic aromatic hydrocarbon (PAH)-DNA adducts in breast cancer. Additionally we report on analyses of the reliability of the scoring procedures used with immunohistochemical assay for PAH-DNA adducts and of potential bias arising from the use of benign breast disease (BBD) controls. We conducted a case-control study utilizing two control groups: BBD controls who donated tissue and blood samples, and healthy controls who donated blood samples. In comparisons of tumor tissue from cases and benign tissue from BBD controls, increasing adduct levels were significantly associated with case-control status [odds ratio (OR) = 2.40, 95% confidence interval (CI) 1.18-4.92], whereas in comparisons of nontumor tissue from cases and benign tissue from BBD controls the association was nonsignificant (OR = 1.97, 95% CI 0.94-4.17). We also show among cases, but not among BBD controls, that the GSTM1 null genotype is associated with increased adduct levels in breast tissue. Our reliability study found the scoring procedures used with the immunohistochemical assay to have high reliability, 0.93 in nontumor, 0.82 in tumor, and 0.74 in benign tissues. However, we found that the technician significantly contributed to the total variability of a series of data. Finally, we did not find a consistent bias to the null associated with the use of BBD controls; however, BBD controls may overestimate the prevalence of family history of breast cancer compared to that of healthy controls (18% vs.14%). We hypothesize that the higher prevalence results from a referral bias and discuss how this may influence our results.

Human urinary bladder epithelial cells lacking wild-type p53 function are deficient in the repair of 4-aminobiphenyl-DNA adducts in genomic DNA.

The effect of the tumor suppressor gene TP53 on repair of genomic DNA damage was examined in human urinary bladder transitional cell carcinoma (TCC) cell lines. Utilizing TCC10 containing wild-type p53 (wt-p53) as the parental line, an isogenic set of cell lines was derived by retroviral infection that expressed a transdominant mutant p53 (Arg --> His at codon 273, TDM273-TCC10), or the human papilloma virus 16-E6 oncoprotein (E6-TCC10). 32P-postlabeling analyses were performed on DNA from TCC cultures obtained after treatment with N-hydroxy-4-aminobiphenyl (N-OH-ABP), N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) and N-acetoxy-4-acetylaminobiphenyl (N-OAc-AABP). The major adduct was identified as N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) with all three chemicals. The amount of adducts in urothelial DNA ranged between 0.1 and 20 per 10(6) nucleotides, N-OAc-AABP yielding the highest levels, followed by N-OH-ABP and N-OH-AABP. To determine, if the functional status of p53 affects the rate of repair of dG-C8-ABP in genomic DNA, TCC10 and the TDM273-TCC10 and E6-TCC10 isotypes were exposed to N-OH-AABP for 12h and the DNA damage was allowed to repair up to 24h. The adduct levels were quantified and compared between the TCC10 isotypes. The amounts of dG-C8-ABP that remained in genomic DNA from E6-TCC10 and TDM273-TCC10 were approximately two-fold higher, as compared to the parental TCC10. At the dose used for DNA repair studies, N-OH-AABP or N-OAc-AABP did not induce apoptosis in TCC10. However, N-OAc-AABP at high doses (>5 microM) induced apoptosis, as evidenced by DNA fragmentation analyses. Furthermore, N-OAc-AABP-mediated apoptosis was independent of the functional status of wt-p53, since both E6-TCC10 and the parental TCC10 exhibited DNA fragmentation following treatment. These results suggest that p53 might modulate the repair of DNA adducts generated from the human bladder carcinogen ABP in its target human uroepithelial cells. This implies that in p53 null cells the unrepaired DNA damage could cause accumulation of mutation, which might contribute to increased genomic instability and neoplastic progression.

The chemical form of selenium influences 3,2'-dimethyl-4-aminobiphenyl-DNA adduct formation in rat colon.

There is increasing evidence that selenium can protect against tumorigenesis or preneoplastic lesion development induced by chemical carcinogens. This study examined whether selenite, selenate or selenomethionine would be protective against 3, 2'-dimethyl-4-aminobiphenyl (DMABP)-DNA adduct formation in the liver and colon of rats and sought to delineate the mechanism for the protective effects of the different chemical forms of selenium against aberrant crypt formation, a preneoplastic lesion for colon cancer. After injection of DMABP, two DNA adducts were identified in the liver and colon of rats. Supplementation with either 0.1 or 2.0 mg selenium/kg diet as either selenite or selenate but not selenomethionine resulted in significantly fewer (53-70%; P < 0.05) N-(deoxyguanosin-8-yl)-(deoxyguanosin-8-yl)-3, 2'-dimethyl-4-aminobiphenyl (C8-DMABP)-DNA adducts in the colon but not the liver than in rats fed a selenium-deficient diet. Rats supplemented with selenomethionine had greater (P < 0.05) plasma and liver selenium concentrations and glutathione peroxidase activity than those supplemented with selenite or selenate; however, they also had more DMABP-DNA adducts. The protective effect of selenite and selenate against DMABP-DNA adduct formation apparently is not a result of alterations in plasma or liver selenium concentrations or altered glutathione peroxidase or glutathione transferase activities but may be related to differences in the metabolism of the different forms of selenium.