Aflatoxin B1-induced DNA damage and its repair

Aflatoxin B(1) (AFB(1))-N(7)-guanine is the predominant adduct formed upon the reaction of AFB(1)-8,9-exo-epoxide with guanine residues in DNA. AFB(1)-N(7)-guanine can convert to the ring-opened formamidopyrimidine, or the adducted strand can undergo depurination. AFB(1)-N(7)-guanine and AFB(1)-formamidopyrimidine are thought to be predominantly repaired by nucleotide excision repair in bacteria, yeast and mammals. Although AFB(1)-formamidopyrimidine is removed less efficiently than AFB(1)-N(7)-guanine in mammals, both lesions are repaired with equal efficiencies in bacteria, reflecting differences in damage recognition between bacterial and mammalian repair systems. Furthermore, DNA repair activity and modulation of repair by AFB(1) seem to be major determinants of susceptibility to AFB(1)-induced carcinogenesis.     

Excision repair is required for genotoxin-induced mutagenesis in mammalian cells

Certain hexavalent chromium [Cr(VI)] compounds are human lung carcinogens. Although much is known about Cr-induced DNA damage, very little is known about mechanisms of Cr(VI) mutagenesis and the role that DNA repair plays in this process. Our goal was to investigate the role of excision repair (ER) pathways in Cr(VI)-mediated mutagenesis in mammalian cells. Repair-proficient Chinese hamster ovary cells (AA8), nucleotide excision repair (NER)-deficient (UV-5) and base excision repair (BER)-inhibited cells were treated with Cr(VI) and monitored for forward mutation frequency at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. BER was inhibited using methoxyamine hydrochloride (Mx), which binds to apurinic/apyrimidinic sites generated during BER. Notably, we found that both NER-deficient (UV-5 and UV-41) and BER-inhibited (AA8 + Mx) cells displayed attenuated Cr(VI) mutagenesis. To determine whether this was unique to Cr(VI), we included the alkylating agent, methylmethane sulfonate (MMS) and ultraviolet (UV) radiation (260 nm) in our studies. Similar to Cr(VI), UV-5 cells exhibited a marked attenuation of MMS mutagenesis, but were hypermutagenic following UV exposure. Moreover, UV-5 cells expressing human xeroderma pigmentosum complementation group D displayed similar sensitivity to Cr(VI) and MMS-induced mutagenesis as AA8 controls, indicating that the genetic loss of NER was responsible for attenuated mutagenesis. Interestingly, Cr(VI)-induced clastogenesis was also attenuated in NER-deficient and BER-inhibited cells. Taken together, our results suggest that NER and BER are required for Cr(VI) and MMS-induced genomic instability. We postulate that, in the absence of ER, DNA damage is channeled into an error-free system of DNA repair or damage tolerance.     

Comparative formation and removal of aflatoxin B1-DNA adducts in cultured mammalian tracheal epithelium

Aflatoxin B1 (AFB1) DNA binding, adduct formation, and AFB1-DNA adduct repair were studied in tracheal explants from rabbit, hamster, and rat. These species vary in populations of cytochrome P-450-containing nonciliated tracheal epithelial cells. Explants were cultured in media containing 0.5 microM AFB1 for 12 h. After the 12-h treatment, the explants were cultured for time intervals up to 84 h and then analyzed for AFB1-DNA adducts. Binding of AFB1 to DNA was highest in rabbit tracheal explants (78 pmol/mg DNA), followed by the hamster (28 pmol/mg DNA), with the rat (3 pmol/mg DNA) showing minimal AFB1-DNA binding. Repair rates in the hamster and rat were constant over time with removal of the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 accounting for the majority of adduct disappearance. The rabbit demonstrated biphasic repair of adducts; all adduct types [8,9-dihydro-8-(2-amino-6-formamido-4-oxo-3,4-dihydropyrimid-5- ylamino)-9- hydroxyaflatoxin B1] were rapidly removed during the first 12 h posttreatment with AFB1, followed by a slower removal phase of primarily 8,9-dihydro-8-N7-guanyl)-9-hydroxyaflatoxin B1. After 84 h, 90, 72, and 55% of the initial adducts were removed in the rabbit, hamster, and rat, respectively. Labeled thymidine studies showed that cells of the tracheal epithelium did not turn over sufficiently to bias the apparent repair rates. These results demonstrated that carcinogen activation and repair capabilities of tracheal epithelium vary among species and that these processes likely relate to the presence of smooth endoplasmic reticulum containing nonciliated tracheal epithelial cells in those species.     

Repair of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced DNA pyridyloxobutylation by nucleotide excision repair

The tobacco-specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) forms DNA methylating and pyridyloxobutylating species. In this study, the involvement of nucleotide excision repair (NER) in the repair of pyridyloxobutyl adducts was assessed using an in vitro NER assay with pyridyloxobutylated plasmid DNA. Nuclear extracts from NER-deficient xeroderma pigmentosum (XP) cells, XPA and XPC, were less active at repairing pyridyloxobutyl adducts than were extracts from normal cells, while combining NER-deficient extracts reconstituted activity. Also, NER-deficient cells were more susceptible to NNKOAc-induced cytotoxicity than were normal cells. Results demonstrate a role for NER in the repair of NNK-induced pyridyloxobutylation.     

Enzymology of the repair of free radicals-induced DNA damage

A number of intrinsic and extrinsic mutagens induce structural damage in cellular DNA. These DNA damages are cytotoxic, miscoding or both and are believed to be at the origin of cell lethality, tissue degeneration, ageing and cancer. In order to counteract immediately the deleterious effects of such lesions, leading to genomic instability, cells have evolved a number of DNA repair mechanisms including the direct reversal of the lesion, sanitation of the dNTPs pools, mismatch repair and several DNA excision pathways including the base excision repair (BER) nucleotide excision repair (NER) and the nucleotide incision repair (NIR). These repair pathways are universally present in living cells and extremely well conserved. This review is focused on the repair of lesions induced by free radicals and ionising radiation. The BER pathway removes most of these DNA lesions, although recently it was shown that other pathways would also be efficient in the removal of oxidised bases. In the BER pathway the process is initiated by a DNA glycosylase excising the modified and mismatched base by hydrolysis of the glycosidic bond between the base and the deoxyribose of the DNA, generating a free base and an abasic site (AP-site) which in turn is repaired since it is cytotoxic and mutagenic.     

Induction of DNA adducts and mutations in spleen, liver and lung of XPA- deficient/lacZ transgenic mice after oral treatment with benzo[a]pyrene: correlation with tumour development

We were interested to study the relationship between DNA lesions, DNA repair, mutation fixation, and tumour development. Therefore, mice harbouring lacZ reporter genes and being either wild-type or defective in the DNA excision repair gene XPA, were treated with the genotoxic carcinogen benzo[a]pyrene at an oral dose of 13 mg/kg b.w. (3 times/week). At different time points, i.e. 1, 5, 9 or 13 weeks after start of the oral administration, levels of BPDE-N2-dG adducts (the major formed DNA adduct by benzo[a]pyrene in mice), and lacZ mutation frequencies were measured both in target (spleen) and non-target (lung and liver) tissues. Both in wild-type and XPA-deficient mice, benzo[a]pyrene treatment resulted in increased BPDE-N2-dG adduct levels in all three tissues analysed. In XPA-deficient mice, BPDE-N2-dG adduct levels still increased up to 13 weeks of oral benzo[a]pyrene treatment, whereas in DNA repair proficient mice steady-state levels were reached after 5 weeks of treatment. After 13 weeks, the BPDE-N2-dG adduct levels observed in XPA-/- mice, were 2- to 3-fold higher than the steady state levels observed in XPA+/+ mice in the same tissues. Mutation frequencies in the lacZ reporter gene were the same in wild- type and XPA-deficient mice that were treated with the solvent only. Oral benzo[a]pyrene treatment resulted in an increase in mutation frequency in the lacZ marker gene in all three tissues, but this increase was most profound in the spleen. After 13 weeks of treatment, a 7-fold increase in lacZ mutation frequency was detected in the spleen of wild-type mice as compared to mutation frequencies in control mice. At the same time point, a 15-fold increase in lacZ mutation frequency was observed in the spleen of XPA-deficient mice. The data presented here show, that a defect in NER mainly results in enhanced mutation frequencies in lymphocytic cells after oral treatment with the genotoxic compound benzo[a]pyrene. Interestingly, as we established in a previously performed carcinogenicity assay, the same oral treatment with benzo[a]pyrene induced lymphomas residing in the spleen of XPA- deficient mice.      

Repair of DNA lesions induced by polycyclic aromatic hydrocarbons in human cell-free extracts: involvement of two excision repair mechanisms in vitro

Polycyclic aromatic hydrocarbons (PAHs) are significant environmental pollutants representing an important risk factor in human cancers. DNA adducts formed by the ultimate carcinogens of PAHs are potentially toxic, mutagenic and carcinogenic. DNA repair represents an important defense system against these genotoxic insults. Using a human cell-free system we have examined repair of DNA lesions induced by several PAH dihydrodiol epoxides, including anti-(+/-)-benzo[a]pyrene-trans-7,8- dihydrodiol-9,10-epoxide, anti-(+/-)-benz[a]anthracene-trans-3,4- dihydrodiol-1,2-epoxide, anti-(+/-)-benz[a]anthracene-trans-8,9- dihydrodiol-10,11-epoxide, anti-(+/-)-benzo[b]fluoranthene-trans-9,10- dihydrodiol-11,12-epoxide and anti-(+/-)-chrysene-trans-1,2-dihydrodiol- 3,4-epoxide. Effective repair of DNA damage induced by these five PAH metabolites was detected. Two distinct mechanisms of excision repair were observed. The major repair mechanism is nucleotide excision repair (NER). The other mechanism is independent of NER and correlated with the presence of apurinic/apyrimidinic sites in the damaged DNA, thus presumably reflecting base excision repair (BER). However, the contribution of BER to different PAH lesions varied in vitro. These results suggest the possibility that BER may also play an important role in repair of certain PAH-induced DNA lesions.      

Structural characterization of the major adducts obtained after reaction of an ultimate carcinogen aflatoxin B1-dichloride with calf thymus DNA in vitro

The major adduct formed on acid hydrolysis of calf thymus DNA which has been reacted with 8,9-dichloro-8,9-dihydroaflatoxin B1, a chemical model of the ultimate carcinogen 8,9-dihydro-8,9-epoxyaflatoxin B1 (AFB1-epoxide), has been characterized by proton nuclear magnetic resonance and fast atom bombardment mass spectroscopy. This adduct has been identified as an N7-substituted guanine adduct analogous to that formed on reaction of AFB1-8,9-epoxide with DNA in vivo and in vitro, namely trans-8,9-dihydro-8-(7-guanyl)-9-hydroxy AFB1. This 8,9-dichloro-8,9-dihydroaflatoxin B1 adduct in DNA, like its equivalent B1 adduct in DNA, like its equivalent AFB1-epoxide adduct, is prone to quantitative imidazole ring opening of the substituted guanine in mildly alkaline conditions and to substantial depurination under mildly acidic conditions.     

Role of p53 in sensing oxidative DNA damage in response to reactive oxygen species-generating agents

The tumor suppressor p53 plays an important role in the regulation of cellular response to DNA damage. Recent studies suggest that p53 is able to bind DNA with certain structural alterations in a sequence-independent manner and to interact with several molecules involved in DNA repair. This study was undertaken to test the hypothesis that p53 may participate in sensing oxidative DNA damage, the most frequently occurring spontaneous DNA lesion, and modulate its repair by the base excision repair (BER) machinery. Using synthetic DNA containing 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG), we showed that p53 was pulled down together with two BER proteins, human 8-oxoguanine glycosylase (hOGG1) and AP endonuclease (APE). Functional analysis showed that p53 significantly enhanced the sequential activities of hOGG1 and APE in excising the 8-oxoG nucleotide from DNA in vitro. The ability of p53 to enhance the removal of oxidized DNA bases was further demonstrated in vivo using a pair of p53 isogenic lines. HCT116 p53+/+ cells exhibit a more rapid removal of 8-oxoG from DNA than p53-/- cells exposed to the same levels of reactive oxygen species (ROS) stress. Together, these results suggest that p53 participates in sensing oxidative DNA damage and modulates BER function in response to persistent ROS stress.     

Effect of S. cerevisiae APN1 protein on mammalian DNA base excision repair

Mammalian cells transfected with the S. cerevisiae APN1 protein acquire resistance to oxidizing agents, the damage of which are mainly repaired via DNA base excision repair (BER). We have recently hypothesized that this effect might be linked to the possible capacity of APN1 to accelerate mammalian BER by its 3' diesterase activity. We have investigated here the effect of pure APN1 protein on BER performed by mouse embryonic fibroblast extracts. No significant acceleration was observed in the repair of either a single AP site cleaved by the bifunctional glycosylase NTH of E. coli or the repair of a single 8-oxoguanine, initiated by the bifunctional glycosylase OGG1. Similarly, no significant effect was observed on the repair of a single U (initiated by the monofunctional glycosylase U DNA glycosylase) or the repair of a single natural abasic site. The inability of APN1 to increase the efficiency of BER initiated by bifunctional glycosylases indicates that removal of 3' blocking fragments is not the rate limiting step of this repair pathway.     

Genetic variants in DNA repair pathways are not associated with disease progression among multiple myeloma patients

DNA damage induced by high dose melphalan and autologous transplantation is repaired by the nucleotide excision repair (NER) and base excision repair (BER) pathways. We evaluated the association between single nucleotide polymorphisms (SNPs) (n = 311) in the NER and BER pathways and disease progression in 695 multiple myeloma patients who underwent autologous transplantation. None of the SNPs were associated with disease progression. Pathway based analyses showed that the NER pathway had a borderline association with disease progression (p = 0.09). These findings suggest that common variation in the NER and BER pathways do not substantially influence disease progression in multiple myeloma patients.     

Analysis of nucleotide excision repair by detection of single-stranded DNA transients.

Nucleotide excision repair (NER) removes bulky DNA lesions and is thus crucial for the protection against environmental carcinogens and UV light exposure. Deficiencies in NER cause increased mutation rates and chromosomal aberrations. Current methods for studying NER are mostly based on either quantitation of lesion removal or detection of repair DNA synthesis. Both have their limitations: lesion removal is inaccurate at very short times post-lesion, where the fraction of removal is low. Repair synthesis is difficult to apply to normally cycling cells due to the need to discriminate repair from replicative DNA synthesis. To overcome these problems we developed a method for analysis of NER based on detection of transient single-stranded (ss) DNA stretches generated at the nucleotide excision step. Cells are metabolically labelled with BrdU, exposed to UV-irradiation and the ssDNA transients generated during excision repair are detected using an anti-BrdU antibody. The method allows single-cell microscopic analysis of the distribution of DNA repair sites as well as kinetic analysis of the DNA repair response. Studies using various DNA repair-deficient cell lines indicate that the detection method integrates a number of pre-synthesis nucleotide excision repair stages. Thus, assembled repair sites can be detected even when they may not lead to complete resolution of the DNA lesion. Using this approach, we show that repair helicase-deficient cells differ from endonuclease-deficient cells.     

Opposite base-dependent excision of 7,8-dihydro-8-oxoadenine by the Ogg1 protein of Saccharomyces cerevisiae

The yOgg1 protein of Saccharomyces cerevisiae is a DNA glycosylase/AP lyase that excises guanine lesions such as 7,8-dihydro-8-oxoguanine (8- OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (me-Fapy- G) and incises apurinic/apyrimidinic sites (AP sites) in damaged DNA. The yOgg1 protein displays a marked preference for DNA duplexes containing 8-OxoG or AP sites placed opposite cytosine. In this paper, we show that yOgg1 can also excise an adenine lesion, 7,8-dihydro-8- oxoadenine (8-OxoA), when paired with cytosine or 5-methylcytosine. In contrast, yOgg1 does not release 8-OxoA when placed opposite thymine, adenine, guanine or uracil. The specificity constants (Kcat/Km) for repair of 8-OxoG/C and 8-OxoA/C duplexes are (50 +/- 18) x 10(-3) and (13 +/- 3) x 10(-3)/min/nM, respectively. The catalytic mechanism for strand cleavage at 8-OxoA/C involves excision of 8-OxoA by the DNA glycosylase activity of yOgg1, followed by incision at the newly formed AP site via a beta-elimination reaction. Furthermore, cleavage of 8- OxoA/C involves formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydride (NaBH4). The yOgg1 protein binds strongly to the 8-OxoA/C duplex, as demonstrated by an apparent dissociation constant (Kdapp) value of 45 nM, as determined by gel mobility shift assay. In contrast, the yOgg1 protein has a very low binding affinity for the 8-OxoA/T duplex, a Kdapp value of 680 nM, which in turn can explain the lack of repair of 8-OxoA in this duplex. The capacity of other DNA glycosylases/AP lyases to repair 8-OxoA has also been investigated. The results show that human hOgg1 protein efficiently repairs 8-OxoA placed opposite cytosine or 5-methylcytosine. On the other hand, the Fpg protein of Escherichia coli cleaves 8-OxoA/C at a very slow rate as compared with yOgg1.      

A high pressure liquid chromatography study on the removal of DNA-bound aflatoxin B1 in rat liver and in vitro

Following intraperitoneal administration of [3H] aflatoxin B1 (AFB1) to young adult male rats, there is rapid uptake of the carcinogen by the liver, the target organ for carcinogenesis, leading to DNA covalent binding. Acid hydrolysis of this DNA shows that after 2h, the major DNA adduct is trans 8,9-dihydro-8-(7-guanyl)-9-hydroxy AFB1 (AFB1-gua). By 24h after AFB1 administration the major DNA adduct is no longer AFB1-gua but a product with the identical retention time on h.p.l.c. to 8,9-dihydro-8-(N5-formyl-2',5',6' triamino-4' oxo-N5-pyrimidyl)-9-hydroxy AFB1 (AFB1-triamino-Py). 48h after carcinogen administration, only a small amount of AFB1-gua remains and the major product is AFB1-triamino-Py. The half-life of removal of AFB1-gua is 22h, while AFB1-triamino-Py is much more persistent. In vitro incubation studies on DNA isolated from rats treated 2h previously with [3H] AFB1 show that at pH 7.4 AFB1-gua is the major product released from the DNA with some release of 8,9-dihydro-8,9-dihydroxy AFB1, (AFB1-diol). If more extensively reacted AFB1-DNA is used than that obtained from in vivo administration, then the rate of AFB1-diol release is enhanced while that of AFB1-gua is reduced. It would appear, therefore, that much of the release of AFB1 from DNA in vivo within the first 24h is probably not through a DNA repair process but through chemical release arising from the positively charged N7-guanine. There is considerable conversion of AFB1-gua to AFB1-triamino-Py on in vitro incubation of DNA as well as AFB1-gua and AFB1-diol release. By 24h approximately 66% of the bound AFB1 is in the form of AFB1-triamino-Py and after 48h the conversion is complete. The complex pattern of AFB1-release from DNA may have important consequences in both the induction of mutations and in tumour initiation.     

Susceptibility to aflatoxin B1-induced carcinogenesis correlates with tissue-specific differences in DNA repair activity in mouse and in rat

To investigate the mechanisms responsible for species- and tissue-specific differences in susceptibility to aflatoxin B(1) (AFB(1))-induced carcinogenesis, DNA repair activities of nuclear extracts from whole mouse lung and liver and rat liver were compared, and the ability of in vivo treatment of mice with AFB(1) to alter repair of AFB(1)-DNA damage was determined. Plasmid DNA containing AFB(1)-N(7)-guanine or AFB(1)-formamidopyrimidine adducts were used as substrates for the in vitro determination of DNA repair synthesis activity, detected as incorporation of radiolabeled nucleotides. Liver extracts from CD-1 mice repaired AFB(1)-N(7)-guanine and AFB(1)-formamidopyrimidine adducts 5- and 30-fold more effectively than did mouse lung, and approximately 6- and 4-fold more effectively than did liver extracts from Sprague-Dawley rats. The susceptibility of mouse lung and rat liver to AFB(1)-induced carcinogenesis correlated with lower DNA repair activity of these tissues relative to mouse liver. Lung extracts prepared from mice treated with a single tumorigenic dose of 50 mg/kg AFB(1) i.p. and euthanized 2 hours post-dosing showed minimal incision and repair synthesis activities relative to extracts from vehicle-treated mice. Conversely, repair activity towards AFB(1)-N(7)-guanine damage was approximately 3.5-fold higher in liver of AFB(1)-treated mice relative to control. This is the first study to show that in vivo treatment with AFB(1) can lead to a tissue-specific induction in DNA repair. The results suggest that lower DNA repair activity, sensitivity of mouse lung to inhibition by AFB(1), and selective induction of repair in liver contribute to the susceptibility of mice to AFB(1)-induced lung tumorigenesis relative to hepatocarcinogenesis.     

Lack of involvement of nucleotide excision repair gene polymorphisms in colorectal cancer

DNA repair has an essential role in protecting the genome from damage by endogenous and environmental agents. Polymorphisms in DNA repair genes and differences in repair capacity between individuals have been widely documented. For colorectal cancer, the loss of mismatch repair gene activity is a key genetic determinant. Nucleotide excision repair (NER), recombination repair (RR) and base excision repair (BER) pathways have critical roles in protection against other cancers, and we wished to investigate their role in colorectal cancer. We have compared the frequency of polymorphisms in the NER genes, XPD, XPF, XPG, ERCC1; in the BER gene, XRCC1; and in the RR gene, XRCC3; in colorectal cancer patients and in a control group. No significant associations were found for any of the NER gene polymorphisms or for the XRCC1 polymorphism. The C allele (position 18067) of the XRCC3 gene was weakly but significantly associated with colorectal cancer (odds ratio 1.52, 95% confidence interval 1.04-2.22, P=0.03). For all patients who were heterozygous for any of the repair genes studied, tumour tissue was investigated for loss of heterozygosity (LOH). Only one example of LOH was found for all the genes examined. From the association and LOH data, we conclude that these genes do not have an important role in protection against colorectal carcinogenesis.     

Polymorphisms of DNA repair genes and risk of non-small cell lung cancer

Lung cancer is a leading cause of cancer mortality with an inter-individual difference in susceptibility to the disease. The inheritance of low-efficiency genotypes involved in DNA repair and replication may contribute to the difference in susceptibility. We investigated 44 single nucleotide polymorphisms (SNPs) in 20 DNA repair genes including nucleotide excision repair (NER) genes XPA, ERCC1, ERCC2/XPD, ERCC4/XPF and ERCC5/XPG; base excision repair (BER) genes APE1/APEX, OGG1, MPG, XRCC1, PCNA, POLB, POLiota, LIG3 and EXO1; double-strand break repair (DSB-R) genes XRCC2, XRCC3, XRCC9, NBS1 and ATR; and direct damage reversal (DR) gene MGMT/AGT. The study included 343 non-small cell lung cancer (NSCLC) cases and 413 controls from Norwegian general population. Our results indicate that SNPs in the NER genes ERCC1 (Asn118Asn, 15310G>C, 8902G>T), XPA (-4G>A), ERCC2/XPD (Lys751Gln) and ERCC5/XPD (His46His); the BER genes APE1/APEX (Ile64Val), OGG1 (Ser326Cys), PCNA (1876A>G) and XRCC1 (Arg194Trp, Arg280His, Arg399Gln); and the DSB-R genes ATR (Thr211Met), NBS1 (Glu185Gln), XRCC2 (Arg188His) and XRCC9 (Thr297Ile) modulate NSCLC risk. The level of polycyclic aromatic hydrocarbon-DNA (PAH-DNA) adducts in normal lung tissue from 211 patients was analysed. The variant alleles of XRCC1(Arg280His), XRCC1 (Arg399Gln), ERCC1(G8092T), ERCC5(His46His) and MGMT/AGT(Lys178Arg) were more frequent in patients with PAH-DNA adduct levels lower than the mean whereas the XRCC1(Arg194Trp) variant was more frequent in cases with higher adduct levels than the mean.