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.      

ERCC2/XPD基因缺失对苯并[a]芘所致DNA损伤修复的影响

目的： 探讨核苷酸切除修复交叉互补基因2 (excision repair cross complementation group 2/Xeroderma pigmentosum D,ERCC2/XPD)在苯并[a]芘所诱导的细胞DNA损伤与修复过程中的作用。方法：应用中国仓鼠卵巢细胞系CHO野生型AA8和ERCC2表达缺失型UV5作为细胞对照模型,MTT法比较两种细胞经苯并[a]芘处理后细胞抑制率的差别;彗星试验和Rad51免疫荧光试验检测不同浓度苯并[a]芘处理及修复24 h后细胞DNA损伤修复的情况。结果：与野生型AA8细胞相比,UV5细胞对苯并[a]芘所致损伤更加敏感,细胞存活率降低 (P<0.05)。彗星试验和Rad51免疫荧光试验结果显示,UV5细胞由于缺失ERCC2/XPD基因,修复苯并[a]芘所致DNA损伤能力降低 (P<0.05)。 结论：ERCC2/XPD蛋白在核苷酸切除修复中发挥解旋作用,对苯并[a]芘所致DNA损伤修复至关重要。     

Cigarette smoke condensate-induced level of adenomatous polyposis coli blocks long-patch base excision repair in breast epithelial cells

Our previous studies have shown that treatment with cigarette smoke condensate (CSC) transforms normal breast epithelial cell line, MCF-10A. In the present study, the mechanism of CSC-induced transformation of breast epithelial cells was examined. We first determined whether benzo[a]pyrene (B[a]P)- and CSC-induced levels of APC are capable of inhibiting long-patch base excision repair (LP-BER) since our earlier studies had shown that an interaction of APC with DNA polymerase beta (pol-beta) blocks strand-displacement synthesis. With the use of a novel in vivo LP-BER assay, it was demonstrated that increased and decreased APC levels in different breast cancer cell lines were associated with a decrease or increase in LP-BER activity, respectively. The effect of APC on LP-BER in malignant and pre-malignant breast epithelial cell lines was produced by either overexpression or knockdown of APC. Furthermore, it was shown that the decreased LP-BER in B[a]P- or CSC-treated pre-malignant breast epithelial cells is associated with an increased level of APC and decreased cell growth. Our results suggest that the decreased growth allows cells to repair the damaged DNA before mitosis, and failure to repair damaged DNA has the potential to transform pre-malignant breast epithelial cells.     

Kinetics of DNA adduct formation and removal in mouse hepatocytes following in vivo exposure to 5,9-dimethyldibenzo[c,g]carbazole

5,9-Dimethyldibenzo[c,g]carbazole (DMDBC), a potent mouse hepatocarcinogen, has been shown to induce a non-linear increase in mutant frequency in the liver of the transgenic MutaMouse. To gain insight into the mechanisms underlying the mutagenicity of DMDBC in vivo, DNA damage formation and removal were monitored in mouse hepatocytes over 4-144 h after a single skin application of 10 or 90 mg/kg DMDBC. DNA adducts were measured by (32)P-post-labeling. DNA repair was assessed by: (i) the unscheduled DNA synthesis (UDS) assay, which measures [(3)H]thymidine incorporation into hepatocyte DNA undergoing excision repair; (ii) the Comet assay, which detects DNA strand breaks transiently produced between the incision and rejoining steps of the excision repair process. A plateau of approximately 400 DNA adducts/10(8) nucleotides was reached 24 h after treatment with 10 mg/kg and remained unchanged until 144 h. UDS activity was significantly induced at 15 and 24 h, while no DNA strand breaks were observed at any sampling time. These results suggest that DNA repair mechanisms were efficiently induced and the formation of a high degree of DNA damage was avoided at this dose level. Following exposure to 90 mg/kg DMDBC, the number of DNA adducts increased sharply to a maximum at 24 h ( approximately 8000/10(8) nucleotides) and then declined to approximately 500/10(8) nucleotides at 144 h. UDS activity was markedly induced from 15 to 72 h. Low levels of DNA strand breaks were observed at 24 and 48 h. The formation of large numbers of DNA adducts and the emergence of DNA strand breaks despite a strong initial induction of UDS activity suggested that DNA repair mechanisms were saturated at this dose level. This phenomenon could partly account for the non-linear induction of gene mutations previously reported in the liver of the transgenic MutaMouse.     

Genotypes and haplotypes of ERCC1 and ERCC2/XPD genes predict levels of benzo[a]pyrene diol epoxide-induced DNA adducts in cultured primary lymphocytes from healthy individuals: a genotype-phenotype correlation analysis

Benzo[a]pyrene diol epoxide (BPDE)-induced DNA adducts are a risk factor for tobacco-related cancers. Excision repair cross-complementing complementation group 1 (ERCC1) and excision repair cross-complementing complementation group 2/xeroderma pigmentosum D (ERCC2/XPD) participate in the nucleotide excision repair (NER) pathway that removes BPDE-DNA adducts; however, few studies have provided population-based evidence for this association. Therefore, we assayed for levels of in vitro BPDE-induced DNA adducts and genotypes of single-nucleotide polymorphisms (SNPs) of the NER genes ERCC1 (rs3212986 and rs11615) and ERCC2/XPD (rs13181, rs1799793 and rs238406) in 707 healthy non-Hispanic whites. The linear trend test of increased adduct values in never to former to current smokers was statistically significant (P(trend) = 0.0107). The median DNA adduct levels for the ERCC2 rs1799793 GG, GA and AA genotypes were 23, 29 and 30, respectively (P(trend) = 0.057), but this trend was not observed for other SNPs. After adjustment for covariates, adduct values larger than the median value were significantly associated with the genotypes ERCC1 rs3212986TT [odds ratio (OR) = 1.89, 95% confidence interval (CI) = 1.03-3.48] and ERCC2/XPD rs238406AA (OR = 0.64, 95% CI = 0.41-0.99) and rs238406CA (OR = 0.63, 95% CI = 0.45-0.89) compared with their corresponding wild-type homozygous genotypes. The results of haplotype analysis further suggested that haplotypes CAC and CGA of ERCC2/XPD, TC of ERCC1 and CACTC of ERCC2/XPD and ERCC1 were significantly associated with high levels of DNA adducts compared with their most common haplotypes. Our findings suggest that the genotypes and haplotypes of ERCC1 and ERCC2/XPD may have an effect on in vitro BPDE-induced DNA adduct levels.     

A modified host-cell reactivation assay to measure repair of alkylating DNA damage for assessing risk of lung adenocarcinoma

The nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces lung adenocarcinoma through formation of DNA adducts. Our previous research on susceptibility to tobacco-induced carcinogenesis focused on benzo[a]pyrene diol epoxide (BPDE) as the in vitro mutagen for phenotype measurements of DNA repair capacity (DRC) in mammalian cells. Here, we present a modified host-cell reactivation (HCR) assay to measure lymphocytic DRC for alkylating DNA damage as is induced by the tobacco-specific nitrosamine, NNK. We substituted dimethyl sulfate (DMS) to create alkylating damage in pCMVluc plasmid DNA and established the damage-repair dose-response curves in both normal and nucleotide excision repair-deficient lymphoblastoid cell lines and in phytohemagglutinin (PHA)-stimulated primary lymphocytes. We then successfully measured the DRC in PHA-stimulated lymphocytes from 48 patients with lung adenocarcinoma and 45 cancer-free controls and tested our hypothesis that lower DRC for alkylating damage is associated with an increased risk of lung adenocarcinoma. The cases exhibited a lower mean DRC than did the controls. A >3-fold increased risk (odds ratio = 3.21; 95% confidence interval = 1.25-8.21) was found for those with DRC levels below the control median. There was no correlation between the DRC measured with this DMS-HCR assay and that from the parallel BPDE-HCR assay. Interestingly, risk increased to >10-fold for those with sub-optimal DRC measured by both DMS- and BPDE-HCR assays. We conclude that variability in DRC is a risk factor for lung cancer and our results provide proof of principle for a new assay that can assess DRC for NNK-induced DNA damage.     

3-Amino-1, 4-dimethyl-5H-pyrido[4, 3-b]indole (Trp-P-1) inhibits the binding activity of T4 endonuclease V to UV-damaged DNA

3-Amino-1, 4-dimethyl-5H-pyrido[4, 3-b]indole (Trp-P-1) is amutagen/carcinogen derived from cooked foods which enhancesthe induction of mutations and chromosome aberrations by UVwithout microsomal activation. These co-mutagenic effects areconsidered to arise from inhibition of DNA excision repair atthe incision step. However, the inhibition mechanism has notbeen clarified. in this study we show, using agarose gel electrophoresis,thatTrp-P-1 inhibits incision by T4 endonuclease V, which cleavesDNA at the site of cyclobutane dimers. Trp-P-1 also inhibitsthebinding of this enzyme to UV-damaged DNA in a gel shift assay.In addition, the results of DNA unwinding assay with topoisomeraseI suggest that Trp-P-1 intercalates into DNA molecules. Theknown intercalators ethidium bromide and acriflavine demonstratesimilar effects in these experiments. However, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which showed no co-mutagenic effects inour previous study, does not demonstrate such effects. Theseresultssuggest that Trp-P-1 changes DNA conformation by intercalation,causing inhibition of binding of repair enzymes to UV-damagedDNA, and this in turn leads to inhibition of DNA excision repairand to co-mutagenic effects.     

Quantitation of carcinogen-DNA adducts by a standardized high-sensitive enzyme immunoassay

A highly sensitive competitive enzyme immunoassay has been developed, allowing accurate determination of DNA containing the adducts N-(deoxyguanosin-8-yl)-N-acetyl-2-amino-fluorene, N-(deoxyguanosin-8-yl)-2-aminofluorene, 1-[6-(2,5-diamino-4-oxopyrimidinyl-N6-deoxyriboside)]-3-(2-fluo renyl)urea or trans-(7R)-N2-[10-(7 beta,8 alpha,9 alpha-trihydroxy-7,8, 9,10-tetrahydrobenzo[a]pyren)yl]deoxyguanosine. Standard amounts of the carcinogen-modified DNA preparations (25 fmol/500 ng) were coated on the wells of microtitre plates. Various amounts of the modified DNA preparations were used as inhibitors and added before binding of the antibodies (dilution 1:10(6). As second antibody, goat anti-rabbit IgG coupled to alkaline phosphatase was used. The amount of enzyme was determined with 4-methylumbelliferyl phosphate as substrate (Van der Laken et al., 1982). The 50% inhibition values of the standard curves are in the range of 2-16 fmol for the enzyme immunoassays. It is possible to add up to 50 micrograms of unknown DNA to the wells, allowing for comparison with the same quantity of modified DNA. This extends the limit of detection to 1-6 adducts per 10(8) nucleotides. The sensitivity of the assay seems sufficient to demonstrate exposure of humans to known chemical carcinogens.     

Excessive base excision repair of 5-hydroxymethyluracil from DNA induces apoptosis in Chinese hamster V79 cells containing mutant p53

We have demonstrated previously that the toxicity of 5-hydroxymethyl-2'-deoxyuridine (hmdUrd) to Chinese hamster fibroblasts (V79 cells) results from enzymatic removal of large numbers of hydroxymethyluracil residues from the DNA backbone [Boorstein,R. et al. (1992) Mol. Cell. Biol., 12, 5536-5540]. Here we report that a significant portion of the hmdUrd-induced cell death that is dependent on DNA base excision repair in V79 cells is apoptosis. Incubation of V79 cells with pharmacologically relevant concentrations of hmdUrd resulted in the characteristic changes of apoptosis as measured by gel electrophoresis, flow cytometry and phase contrast microscopy. However, hmdUrd did not induce apoptosis in V79mut1 cells, which are deficient in DNA base excision repair of 5-hydroxymethyluracil (hmUra). Apoptosis was not prevented by addition of 3-aminobenzamide, which inhibits synthesis of poly(ADP-ribose) from NAD, indicating that apoptosis was not the direct consequence of NAD depletion. Pulsed field gel electrophoresis indicated that hmdUrd treatment resulted in high molecular weight (2.2-4.5 Mb) DNA double-strand breaks prior to formation of internucleosomal ladders in V79 cells. Simultaneous measurement of DNA strand breaks with bromodeoxyuridine/terminal deoxynucleotidyl transferase-fluorescein isothiocyanate labeling and of cell cycle distribution indicated that cells with DNA strand breaks accumulated in late S/G(2) and that hmdUrd-treated cells underwent apoptosis after arrest in late S/G(2) phase. Our results indicate that excessive DNA base excision repair results in the generation of high molecular weight DNA double-strand breaks and eventually leads to apoptosis in V79 cells. Thus, delayed apoptosis following DNA damage can be a consequence of excessive DNA repair activity. Immunochemical analysis showed that both V79 and V79mut1 cells contained mutant p53, indicating that apoptosis induced by DNA base excision repair can be independent of p53.     

Modulation of repair of ultraviolet damage in the host-cell reactivation assay by polymorphic XPC and XPD/ERCC2 genotypes

DNA repair capacity (DRC) plays an important role in genetic susceptibility to cancer. Polymorphisms of a number of DNA repair genes involved in several distinct pathways have been identified. However, their effects on repair function have not been well characterized. We demonstrated previously that DRC for removal of benzo[a]pyrene diol epoxide-induced DNA damage measured by a host-cell reactivation assay was modulated by two XPD/ERCC2 polymorphisms in lung cancer. In this report, we investigated the association between the repair phenotype of ultraviolet (UV)-induced damage and genotypes of three DNA repair genes, XPC and XPD [involved in nucleotide excision repair (NER)] and XRCC1 [involved in base excision repair (BER)]. We measured DRC for removal of UV photoproducts by the host-cell reactivation assay in cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. We also typed these subjects for five polymorphisms in these three DNA repair genes (at intron 9 of XPC; exons 6, 10 and 23 of XPD and exon 10 of XRCC1). Compared with wild-type homozygotes, subjects homozygous for polymorphisms of the two NER genes consistently had suboptimal DRC. The DRC was consistently lower in subjects homozygous for XPC, XPD or both than in subjects with other genotypes, although the difference was not statistically significant for XPD variants. In contrast, the polymorphic allele of the BER gene, XRCC1, had no consistent effect on DRC. We concluded that these NER polymorphisms may modulate DRC and may be useful biomarkers for identifying individuals at risk of developing cancer.     

Development and field-test validation of an assay for DNA repair in circulating human lymphocytes

A method for measuring nucleotide excision repair in response to UV irradiation and chemical-induced DNA damage has been developed, validated, and field tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiological studies seeking to investigate associations between DNA repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the suggestion that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum is manifested as a result of the reduced capacity of patients' cells to repair DNA damaged by UV-mimetic agents. For the assay, damaged, nonreplicating, recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (cat) reporter gene is introduced into lymphocytes by using a DEAE-dextran/DNA complex short-term transfection conditions. Excision repair of the damaged bacterial cat gene is monitored proportionately as a function of reactivated CAT enzyme activity following a 40-h repair/expression incubation period. The validity of the approach was indicated by the ability of the assay to discriminate xeroderma pigmentosum virus-transformed lymphocyte cell lines of both severe (complementation groups A and D) and moderate (complementation group C) excision repair deficiencies from repair-proficient cell lines. Similar results were observed when a mitogen-stimulated peripheral blood lymphocyte culture from an xeroderma pigmentosum A patient was assayed concurrently with mitogen-stimulated peripheral blood lymphocytes obtained from healthy individuals. Adaptation of this DNA repair assay as a field test in a pilot-tested select group of basal cell carcinoma patients and cancer-free controls led to the preliminary identification of a specific subset at risk for this disease as a consequence of significant reduction to the repair of photochemically (UV)-damaged plasmid DNA.     

Decreased DNA repair gene expression among individuals exposed to arsenic in United States drinking water

Arsenic is well established as a human carcinogen, but its precise mechanism of action remains unknown. Arsenic does not directly damage DNA, but may act as a carcinogen through inhibition of DNA repair mechanisms, leading indirectly to increased mutations from other DNA damaging agents. The molecular mechanism underlying arsenic inhibition of nucleotide excision repair after UV irradiation (Hartwig et al., Carcinogenesis 1997;18:399-405) is unknown, but could be due to decreased expression of critical genes involved in nucleotide excision repair of damaged DNA. This hypothesis was tested by analyzing expression of repair genes and arsenic exposure in a subset of 16 individuals enrolled in a population based case-control study investigating arsenic exposure and cancer risk in New Hampshire. Toenail arsenic levels were inversely correlated with expression of critical members of the nucleotide excision repair complex, ERCC1 (r(2) = 0.82, p < 0.0001), XPF (r(2) = 0.56, p < 0.002), and XPB (r(2) = 0.75, p < 0.0001). The internal dose marker, toenail arsenic level, was more strongly associated with changes in expression of these genes than drinking water arsenic concentration. Our findings, based on human exposure to arsenic in a US population, show an association between biomarkers of arsenic exposure and expression of DNA repair genes. Although our findings need verification in a larger study group, they are consistent with the hypothesis that inhibition of DNA repair capacity is a potential mechanism for the co-carcinogenic activity of arsenic.     

Parp-1 deficiency does not enhance liver carcinogenesis induced by 2-amino-3-methylimidazo[4,5-f]quinoline in mice

The susceptibility of poly(ADP-ribose) polymerase-1 (Parp-1) knockout mice to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced liver carcinogenesis was analyzed. Twelve-week-old male Parp-1(+/+), Parp-1(+/-) and Parp-1(-/-) mice of the C57BL/6 congenic strain were fed a diet containing IQ at a concentration of 300 ppm or a control diet for 60 weeks. Hepatocellular carcinomas were observed only in 1/19, 2/18 and 1/17 of the Parp-1(-/-), Parp-1(+/-) and Parp-1(+/+) mice, respectively. Parp-1 deficiency did not affect the susceptibility of mice to carcinogenicity of IQ, which produces bulky DNA adducts that are repaired mainly through the nucleotide excision repair pathway. This result is in sharp contrast to the increased susceptibility of Parp-1(-/-) mice to carcinogenesis induced by alkylating agents that produce DNA damage repaired mainly through base excision repair and DNA strand break repair pathways.     

The influence of inhibitors on the repair of benzo[a]pyrene-damaged DNA in hamster tracheal epithelial cells

We investigated, in a cloned hamster tracheal epithelial cell line HTE-B, the effects of inhibitors of DNA topoisomerase, novobiocin and nalidixic acid; of DNA polymerase, 1-beta-arabinofuranosylcytosine (ara-C) and 2',3'-dideoxythymidine; of ribonucleotide reductase, hydroxyurea; and of poly(ADP-ribose)synthetase, 3-aminobenzamide, upon the removal of benzo[a]pyrene adducted to DNA [B[a]P--DNA]. A substantial reduction in the rate of removal of the polycyclic hydrocarbon-adducts occurred when nalidixic acid was added to the HTE-B cells that had been previously incubated with B[a]P for 8 h. Novobiocin produced a similar, but less marked, effect. The rate of disappearance of the individual B[a]P--DNA adducts was measured by analysis of the h.p.l.c. profiles. Of the 5 major adducts observed under the h.p.l.c. conditions, 4 were reduced in control cells to 30% of the original levels by 24 h after removal of the B[a]P from the medium; adduct 5 was almost completely removed. In the presence of nalidixic acid, during the 24 h repair period, only the removal of adduct 5 was unimpaired; the removal of the other 4 adducts was significantly retarded. On the other hand, 3-aminobenzamide addition did not affect the rate of removal of B[a]P--DNA adducts from the HTE-B cells. We employed the combinations of ara-C and dideoxythymidine or ara-C and hydroxyurea to allow the accumulation of single strand breaks after incubation of the HTE-B cells with B[a]P. These breaks were assayed by alkaline elution analysis. Inclusion of these inhibitors during the 2 h after removal of the B[a]P from the medium resulted in the accumulation of 4-5 single strand breaks/10(10) daltons of HTE-B DNA. This compares with a minimum estimate of the number of adducts removed during this period of 3 adducts/10(7) daltons. This discrepancy may indicate that the majority of lesions are not repaired by a pathway sensitive to polymerase inhibitors. In the presence of 3-aminobenzamide, we routinely observed a 10% increase in the alkaline elution of the DNA obtained from B[a]P-treated cells (1-2 breaks/10(10) daltons). Our results indicate that an excision repair process may be involved in the removal of at least some of the B[a]P-induced damage to DNA. However, the repair of the multiple adducts is complex and may involve pathways other than classical excision repair.     

Binding of human single-stranded DNA binding protein to DNA damaged by the anticancer drug cis-diamminedichloroplatinum (II).

The chemotherapeutic drug cis-diamminedichloroplatinum (II) covalently binds to DNA resulting in a variety of adducts and cross-links which are thought to be responsible for the toxicity of the drug. We have used the gel mobility shift assay to detect proteins which bind to DNA treated in vitro with cis-diamminedichloroplatinum (II) and have identified two complexes which bind with increased affinity to cis-diamminedichloroplatinum (II)-damaged DNA. Using monoclonal antibodies we have shown that one complex, B1, contains human single-stranded DNA binding protein, a protein known to be involved in the in vitro repair synthesis assay of mammalian excision repair.     

Fanconi anemia gene mutations in young-onset pancreatic cancer

Genes of the Fanconi complementation groups [Fanconi anemia (FA) genes] are suggested to be involved in homologous DNA recombination and produce FA when two allelic mutations are inherited. BRCA2 is an FA gene and additionally conveys an inherited risk for breast, ovarian, and pancreatic cancer for individuals carrying a single mutated allele [N. G. Howlett et al., Science (Wash. DC), 297: 606-609, 2002]. Here we report inherited and somatic mutations of FANCC and FANCG present in young-onset pancreatic cancer. This may imply a general involvement of Fanconi genes with an inherited risk of cancer. The known hypersensitivity of Fanconi cells to mitomycin and other therapeutic agents [M. S. Sasaki, Nature (Lond.), 257: 501-503, 1975] suggests a therapeutic utility for a more complete characterization of the DNA repair defects and their causative genetic mutations in pancreatic cancer.     

Unrepaired fjord region polycyclic aromatic hydrocarbon-DNA adducts in ras codon 61 mutational hot spots

The fjord region diol-epoxide metabolites of polycyclic aromatic hydrocarbons display stronger tumorigenic activities in rodent studies than comparable bay region diol-epoxides, but the molecular basis for this difference between fjord and bay region derivatives is not understood. Here we tested whether the variable effects of these genotoxic metabolites of polycyclic aromatic hydrocarbons may result from different DNA repair reactions. In particular, we compared the repairability of DNA adducts formed by bay region benzo[a]pyrene (B[a]P) diol-epoxides and the structurally similar but significantly more tumorigenic fjord region diol-epoxide metabolites of benzo[c]phenanthrene (B[c]Ph). For that purpose, we incorporated both types of polycyclic aromatic hydrocarbon adducts into known hot spot sites for carcinogen-induced proto-oncogene activation. Synthetic DNA substrates were assembled using a portion of human N-ras or H-ras that includes codon 61, and stereospecific B[a]P or B[c]Ph adducts were synthesized on adenine N6 at the second position of these two ras codon 61 sequences. DNA repair was determined by incubating the site-directed substrates in human cell extracts, followed by electrophoretic visualization of radiolabeled oligonucleotide excision products. These cell-free assays showed that all tested bay region B[a]P-N6-dA adducts are removed by the human nucleotide excision repair system, although excision efficiency varied with the particular stereochemical configuration of each B[a]P residue. In contrast, all fjord region B[c]Ph-N6-dA adducts located in the identical sequence context and with exactly the same stereochemical properties as the corresponding B[a]P lesions were refractory to the nucleotide excision repair process. These findings indicate that the exceptional tumorigenic potency of B[c]Ph or related fjord region diol-epoxides may be attributed, at least in part, to slow repair of the stable base adducts deriving from the reaction of these compounds with DNA.     

Effect of soluble and particulate nickel compounds on the formation and repair of stable benzo[a]pyrene DNA adducts in human lung cells.

Nickel compounds are well-known human carcinogens, but the underlying mechanisms are still not fully understood. Even though only weakly mutagenic, nickel chloride has been shown previously to impair the repair of UV-induced DNA damage as well as oxidative DNA damage. However, the carcinogenic potential depends largely on solubility, with poorly water-soluble nickel subsulfide and nickel oxide being strong carcinogens. Within the present study we investigated the effects of particulate black NiO and soluble NiCl(2) on the induction and removal of stable DNA adducts formed by benzo[a]pyrene (B[a]P) measured by a highly sensitive high performance liquid chromatography (HPLC)/fluorescence assay. With respect to adduct formation, NiO but not NiCl(2) reduced the generation of DNA lesions by approximately 30%. Regarding their repair, in the absence of nickel compounds, most lesions were removed within 24 h; nevertheless, between 20 and 35% of induced adducts remained even 48 h after treatment. NiCl(2) and NiO reduced the removal of adducts in a dose-dependent manner. Thus, 100 microM NiCl(2) led to approximately 80% residual repair capacity; after 500 microM the repair was reduced to approximately 36%. Also, even at the completely non-cytotoxic concentration of 0.5 microg/cm(2) black NiO, lesion removal was reduced to approximately 35% of control and to 15% at 2.0 microg/cm(2). Furthermore, both nickel compounds increased the benzo[a]pyrene-7,8-diol 9,10-epoxide (BPDE)-induced cytotoxicity. Taken together, our results indicate that the nucleotide excision repair pathway is affected in general by water-soluble and particulate nickel compounds and provide further evidence that DNA repair inhibition may be one predominant mechanism in nickel-induced carcinogenicity.     

Trichothiodystrophy, a human DNA repair disorder with heterogeneity in the cellular response to ultraviolet light

Trichothiodystrophy (TTD) is an autosomal recessive disorder characterized by brittle hair with reduced sulfur content, ichthyosis, peculiar face, and mental and physical retardation. Some patients are photosensitive. A previous study by Stefanini et al. (Hum. Genet., 74: 107-112, 1986) showed that cells from four photosensitive patients with TTD had a molecular defect in DNA repair, which was not complemented by cells from xeroderma pigmentosum, complementation group D. In a detailed molecular and cellular study of the effects of UV light on cells cultured from three further TTD patients who did not exhibit photosensitivity we have found an array of different responses. In cells from the first patient, survival, excision repair, and DNA and RNA synthesis following UV irradiation were all normal, whereas in cells from the second patient all these responses were similar to those of excision-defective xeroderma pigmentosum (group D) cells. With the third patient, cell survival measured by colony-forming ability was normal following UV irradiation, even though repair synthesis was only 50% of normal and RNA synthesis was severely reduced. The excision-repair defect in these cells was not complemented by other TTD cell strains. These cellular characteristics of patient 3 have not been described previously for any other cell line. The normal survival may be attributed to the finding that the deficiency in excision-repair is confined to early times after irradiation. Our results pose a number of questions about the relationship between the molecular defect in DNA repair and the clinical symptoms of xeroderma pigmentosum and TTD.