Epigenetics in radiation-induced fibrosis.

Radiotherapy is a major cancer treatment option but dose-limiting side effects such as late-onset fibrosis in the irradiated tissue severely impair quality of life in cancer survivors. Efforts to explain radiation-induced fibrosis, for example, by genetic variation remained largely inconclusive. Recently published molecular analyses on radiation response and fibrogenesis showed a prominent role of epigenetic gene regulation. This review summarizes the current knowledge on epigenetic modifications in fibrotic disease and radiation response, and it points out the important role for epigenetic mechanisms such as DNA methylation, microRNAs and histone modifications in the development of this disease. The synopsis illustrates the complexity of radiation-induced fibrosis and reveals the need for investigations to further unravel its molecular mechanisms. Importantly, epigenetic changes are long-term determinants of gene expression and can therefore support those mechanisms that induce and perpetuate fibrogenesis even in the absence of the initial damaging stimulus. Future work must comprise the interconnection of acute radiation response and long-lasting epigenetic effects in order to assess their role in late-onset radiation fibrosis. An improved understanding of the underlying biology is fundamental to better comprehend the origin of this disease and to improve both preventive and therapeutic strategies.

The traditional Chinese medical compound Rocaglamide protects nonmalignant primary cells from DNA damage-induced toxicity by inhibition of p53 expression.

One of the main obstacles of conventional anticancer therapy is the toxicity of chemotherapeutics to normal tissues. So far, clinical approaches that aim to specifically reduce chemotherapy-mediated toxicities are rare. Recently, a number of studies have demonstrated that herbal extracts derived from traditional Chinese medicine (TCM) may reduce chemotherapy-induced side effects. Thus, we screened a panel of published cancer-inhibiting TCM compounds for their chemoprotective potential and identified the phytochemical Rocaglamide (Roc-A) as a candidate. We show that Roc-A significantly reduces apoptotic cell death induced by DNA-damaging anticancer drugs in primary human and murine cells. Investigation of the molecular mechanism of Roc-A-mediated protection revealed that Roc-A specifically blocks DNA damage-induced upregulation of the transcription factor p53 by inhibiting its protein synthesis. The essential role of p53 in Roc-A-mediated protection was confirmed by siRNA knockdown of p53 and by comparison of the effects of Roc-A on chemoprotection of splenocytes isolated from wild-type and p53-deficient mice. Importantly, Roc-A did not protect p53-deficient or -mutated cancer cells. Our data suggest that Roc-A may be used as an adjuvant to reduce the side effects of chemotherapy in patients with p53-deficient or -mutated tumors.

Early epigenetic downregulation of WNK2 kinase during pancreatic ductal adenocarcinoma development.

Pancreatic ductal adenocarcinoma (PDAC) is usually incurable. Contrary to genetic mechanisms involved in PDAC pathogenesis, epigenetic alterations are ill defined. Here, we determine the contribution of epigenetically silenced genes to the development of PDAC. We analyzed enriched, highly methylated DNAs from PDACs, chronic pancreatitis (CP) and normal tissues using CpG island microarrays and identified WNK2 as a prominent candidate tumor suppressor gene being downregulated early in PDAC development. WNK2 was further investigated in tissue microarrays, methylation analysis of early pancreatic intraepithelial neoplasia (PanIN), mouse models for PDAC and pancreatitis, re-expression studies after demethylation, and cell growth assays using WNK2 overexpression. Demethylation assays confirmed the link between methylation and expression. WNK2 hypermethylation was higher in tumor than in surrounding inflamed tissues and was observed in PanIN lesions as well as in a PDAC mouse model. WNK2 mRNA and protein expressions were lower in PDAC and CP compared with normal tissues both in patients and mouse models. Overexpression of WNK2 led to reduced cell growth, and WNK2 expression in tissues correlated negatively with pERK1/2 expression, a downstream target of WNK2 responsible for cell proliferation. Downregulation of WNK2 by promoter hypermethylation occurs early in PDAC pathogenesis and may support tumor cell growth via the ERK-MAPK pathway.

Epigenetic screen of human DNA repair genes identifies aberrant promoter methylation of NEIL1 in head and neck squamous cell carcinoma.

Aberrant promoter methylation of different DNA repair genes has a critical role in the development and progression of various cancer types, including head and neck squamous cell carcinomas (HNSCCs). A systematic analysis of known human repair genes for promoter methylation is however missing. We generated quantitative promoter methylation profiles in single CpG units of 160 human DNA repair genes in a set of DNAs isolated from fresh frozen HNSCC and normal tissues using MassARRAY technology. Ninety-eight percent of these genes contained CpG islands (CGIs) in their promoter region; thus, DNA methylation is a potential regulatory mechanism. Methylation data were obtained for 145 genes, from which 15 genes exhibited more than a 20% difference in methylation levels between tumor and normal tissues, manifested either as hypermethylation or as hypomethylation. Analyses of promoter methylation with mRNA expression identified the DNA glycosylase NEIL1 (nei endonuclease VIII-like 1) as the most prominent candidate gene. NEIL1 promoter hypermethylation was confirmed in additional fresh frozen HNSCC samples, normal mucosa, HNSCC cell lines and primary human skin keratinocytes. The investigation of laser-microdissected tissues further substantiated increased methylation levels in tumor versus matched non-tumor cells. Immunohistological analysis revealed significantly less NEIL1 protein expression in tumor tissues. 5-Aza-2'-deoxycytidine treatment and DNMT1 knockdown resulted in the re-expression of NEIL1 in HNSCC cell lines, which initially carried hypermethylated promoter regions. In conclusion, our results suggest that DNA methylation contributes to the downregulation of NEIL1 expression and might thus have a role in modulating the response to therapies of HNSCC.

Suberoylanilide hydroxamic acid affects γH2AX expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation.

PURPOSE: Osteosarcoma and atypical teratoid rhabdoid tumors are tumor entities with varying response to common standard therapy protocols. Histone acetylation affects chromatin structure and gene expression which are considered to influence radiation sensitivity. The aim of this study was to investigate the effect of the combination therapy with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and irradiation on atypical teratoid rhabdoid tumors and osteosarcoma compared to normal tissue cell lines. METHODS: Clonogenic assay was used to determine cell survival. DNA double-strand breaks (DSB) were examined by pulsed-field electrophoresis (PFGE) as well as by γH2AX immunostaining involving flow cytometry, fluorescence microscopy, and immunoblot analysis. RESULTS: SAHA lead to an increased radiosensitivity in tumor but not in normal tissue cell lines. γH2AX expression as an indicator for DSB was significantly increased when SAHA was applied 24 h before irradiation to the sarcoma cell cultures. In contrast, γH2AX expression in the normal tissue cell lines was significantly reduced when irradiation was combined with SAHA. Analysis of initial DNA fragmentation and fragment rejoining by PFGE, however, did not reveal differences in response to the SAHA pretreatment for either cell type. CONCLUSION: SAHA increases radiosensitivity in tumor but not normal tissue cell lines. The increased H2AX phosphorylation status of the SAHA-treated tumor cells post irradiation likely reflects its delayed dephosphorylation within the DNA damage signal decay rather than chromatin acetylation-dependent differences in the overall efficacy of DSB induction and rejoining. The results support the hypothesis that combining SAHA with irradiation may provide a promising strategy in the treatment of solid tumors.

Genetic polymorphisms in DNA repair and damage response genes and late normal tissue complications of radiotherapy for breast cancer.

Breast-conserving surgery followed by radiotherapy is effective in reducing recurrence; however, telangiectasia and fibrosis can occur as late skin side effects. As radiotherapy acts through producing DNA damage, we investigated whether genetic variation in DNA repair and damage response confers increased susceptibility to develop late normal skin complications. Breast cancer patients who received radiotherapy after breast-conserving surgery were examined for late complications of radiotherapy after a median follow-up time of 51 months. Polymorphisms in genes involved in DNA repair (APEX1, XRCC1, XRCC2, XRCC3, XPD) and damage response (TP53, P21) were determined. Associations between telangiectasia and genotypes were assessed among 409 patients, using multivariate logistic regression. A total of 131 patients presented with telangiectasia and 28 patients with fibrosis. Patients with variant TP53 genotypes either for the Arg72Pro or the PIN3 polymorphism were at increased risk of telangiectasia. The odds ratios (OR) were 1.66 (95% confidence interval (CI): 1.02-2.72) for 72Pro carriers and 1.95 (95% CI: 1.13-3.35) for PIN3 A2 allele carriers compared with non-carriers. The TP53 haplotype containing both variant alleles was associated with almost a two-fold increase in risk (OR 1.97, 95% CI: 1.11-3.52) for telangiectasia. Variants in the TP53 gene may therefore modify the risk of late skin toxicity after radiotherapy.

[Epigenetic aspects in carcinomas of the head and neck]. / Epigenetische Aspekte bei Karzinomen der Kopf-Hals-Region.

For years, head and neck squamous cell carcinomas (HNSCC) have been among the leading cancers worldwide. Despite considerable efforts, the 5-year survival rate for HNSCC has not changed significantly. To improve this situation, it is necessary to understand the fundamental biological processes leading to the disease and its progression. In addition to known genetic changes in HNSCC, molecular cytogenetic investigations have identified chromosomal regions of gains and losses, but many of the responsible candidate genes have yet to be identified. Furthermore, recent results indicate the importance of epigenetic modifications in HNSCC, such as DNA methylation. Several genes, including the tumor suppressor CDKN2A and other candidates such as DAPK1, MGMT, TIMP3, TCF21, and C/EBPalpha, have been found to harbor hypermethylated regulatory sequences that lead to reduced expression or gene silencing. Hypermethylation in such genes could be used not only as biomarkers for the early detection of HNSCC but also to improve prevention strategies and therapy outcomes.

UVA radiation causes DNA strand breaks, chromosomal aberrations and tumorigenic transformation in HaCaT skin keratinocytes.

The role of UVA-radiation-the major fraction in sunlight-in human skin carcinogenesis is still elusive. We here report that different UVA exposure regime (4 x 5 J/cm(2) per week or 1 x 20 J/cm(2) per week) caused tumorigenic conversion (tumors in nude mice) of the HaCaT skin keratinocytes. While tumorigenicity was not associated with general telomere shortening, we found new chromosomal changes characteristic for each recultivated tumor. Since this suggested a nontelomere-dependent relationship between UVA irradiation and chromosomal aberrations, we investigated for alternate mechanisms of UVA-dependent genomic instability. Using the alkaline and neutral comet assay as well as gamma-H2AX foci formation on irradiated HaCaT cells (20-60 J/cm(2)), we show a dose-dependent and long lasting induction of DNA single and double (ds) strand breaks. Extending this to normal human skin keratinocytes, we demonstrate a comparable damage response and, additionally, a significant induction and maintenance of micronuclei (MN) with more acentric fragments (indicative of ds breaks) than entire chromosomes particularly 5 days post irradiation. Thus, physiologically relevant UVA doses cause long-lasting DNA strand breaks, a prerequisite for chromosomal aberration that most likely contribute to tumorigenic conversion of the HaCaT cells. Since normal keratinocytes responded similarly, UVA may likewise contribute to the complex karyotype characteristic for human skin carcinomas.

DNA damaging capability of hematoporphyrin towards DNAs of various accessibilities.

In this work we wanted to verify that photoactivation of DNA-non-binding porphyrin derivative hematoporphyrin IX (Hp) is able to induce damages in DNAs of various accessibilities such as B-conformation and superhelical isolated DNA, nucleoprotein complex and intracellular DNAs. It was found that photodynamic reaction of Hp results significant changes in thermal stability of isolated T7 DNA and induces single strand breaks in supercoiled Bluescript plasmid isolated from Escherichia coli cells. As optical melting measurements revealed, the irradiation of photosensitized T7 nucleoprotein complex leads to a destabilization of the protein capsid. The photodynamic reaction affected both the protein structure and DNA-protein interaction, however, the parameters corresponding to the DNA denaturation are not influenced. The accumulation of Hp in HeLa cells was followed by laser scanning confocal microscopy. The picture received is typical for lipophilic dyes. When Hp loaded cells were irradiated, a reduction of viability could be observed in a concentration and a light dose dependent manner; 12microM porphyrin induced almost complete cell killing after 30min irradiation. After similar treatment, alkaline agarose gel electrophoresis of isolated nuclear DNA did not show the presence of single strand breaks. The alkaline comet assay also failed to demonstrate any DNA damage in HeLa cells. We also considered the possibility of the generation of damages in intracellular SV40 DNA. According to the electropherograms there was no difference between the patterns of DNAs from treated and control samples.

Genotoxicity studies on permethrin, DEET and diazinon in primary human nasal mucosal cells.

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.

Glutathione-S-transferase M1, M3, T1 and P1 polymorphisms and susceptibility to non-small-cell lung cancer subtypes and hamartomas.

Polymorphic glutathione-S-transferase (GST) genes causing variations in enzyme activity may influence individual susceptibility to lung cancer. In this case-control study (consisting of 389 Caucasian lung cancer patients, including 151 adenocarcinomas (ACs) and 172 squamous cell carcinomas (SCCs), and 353 hospital control subjects without malignant disease, genotype frequencies for GSTM1, GSTM3, GSTP1 and GSTT1 were determined by polymerase chain reaction (PCR)/ restriction fragment length polymorphism (RFLP)-based methods. While adjusted odds ratios (ORs) indicated no significantly increased risk for lung cancer overall due to any single GST genotype, the risk alleles for GSTM1, GSTM3 and GSTP1 conferring reduced enzyme activity were present at higher frequency in SCC than in AC patients. This is consistent with a reduced detoxification of carcinogenic polycyclic aromatic hydrocarbons (PAHs) from cigarette smoke that are more important for the development of SCC than for AC. An explorative data analysis also identified statistically significantly increased ORs for the combinations GSTT1 non-null and GSTP1 GG or AG for lung cancer overall (OR 2.23, CI 1.11-4.45), and for SCC (OR 2.69, CI 1.03-6.99). For lung cancer overall, and especially among SCC patients, the GSTT1 null genotype was underrepresented (SCC 11.2% v. control subjects 19%, P = 0.026, OR 0.57, CI 0.30-1.06). Additionally, in 28 patients with hamartomas, the GSTT1 null genotype was also protective (P = 0.013), while GSTP1 variant allele carriers were overrepresented (OR 2.48, CI 1.06-6.51). In conclusion, GST genotypes may act differently, either by detoxifying harmful tobacco carcinogens and/or by eliminating lung cancer chemopreventive agents. The latter role for GSTT1 would explain the observed lower risk of SCC and hamartoma associated with GSTT1 null. Further confirmatory studies are required.

N-nitrososdimethylamine is activated in microsomes from hepatocytes to reactive metabolites which damage DNA of non-parenchymal cells in rat liver.

The liver carcinogen N-nitrosodimethylamine (NDMA) has to be metabolically activated by specific cytochromes before it can react with cellular macromolecules (e.g. proteins or DNA). Although hepatocytes are believed to be responsible for this activation, the liver tumours originate mainly from non-parenchymal cells (NPC). To investigate their activation capacity we determined NDMA-demethylase activity in isolated microsomes from both liver cell types. The results demonstrate that only hepatocytes have activation capacity. Additional experiments were performed with hepatocytes and NPC using the single cell microgel electrophoresis assay (MGE). DNA damage appears in both cell types following in vivo exposure. Tested in vitro, however, the carcinogens induce DNA damages only in hepatocytes (the cells which activate these compounds). N-nitroso-hydroxymethyl-methylamine could be the responsible metabolite as it is stable enough to be transported from hepatocytes to NPC in an intact liver.

[Genotoxic effect of the insecticides pentachlorophenol and lindane on human nasal mucosal epithelium]. / Genotoxische Wirkung der Insektizide Pentachlorphenol und Lindan auf menschliche Nasenschleimhautepithelien.

BACKGROUND AND OBJECTIVE: In numerous experimental and epidemiologic studies Pentachlorphenol (PCP) and Hexachlorcyclohexan (Lindane) have been shown to be of potential carcinogenic risk for human epithelial cells. In the past, these two substances have been used for both, military and non-military purposes, e.g. for impregnation of textiles and uniforms. In this study we investigated the genotoxic effect of PCP and Lindane on human mucosal tissue from the middle and lower nasal turbinate. METHODS: In biopsy samples obtained from nasal epithelia during surgery cell vitality was evaluated by trypan-blue-staining. The specimens were incubated for 60 minutes with PCP (0.3; 0.75 und 1.2 mumol/l) and Lindane (0.5; 0.75; and 1.0 mumol/ml). The induction of DNA-damage (single-strand-breaks and double-strand-breaks) caused by PCP and Lindane was measured using single-cell microgel electrophoresis. Evaluation was performed by fluorescence microscopy. RESULTS: Especially in mucosa cells from the middle turbinate severe DNA-damages were recognized after exposition to PCP and Lindane proposing a strong genotoxic effect. In cells from the lower turbinate DNA-changes caused by PCP and Lindane were significantly lower. However a considerable genotoxic effect was also present. CONCLUSION: This study shows for the first time that there are clear facts indicating mutagenic effects of PCP and Lindane on nasal epithelia. Furthermore, this is the first study showing different susceptibility of two anatomic subsites in the nose for different pesticides. Concerning the biological plausibility, this study offers important arguments for evaluating the role of PCP and Lindane in the induction of upper aerodigestive tract cancer.

The male rat carcinogens limonene and sodium saccharin are not mutagenic to male Big Blue rats.

Limonene and sodium saccharin are male rat specific carcinogens giving rise to renal and bladder tumours, respectively. Both compounds give negative results in genetic toxicity assays suggesting a non-genotoxic mode of action for their carcinogenicity. The alpha 2U-globulin accumulation theory has been invoked to explain the renal carcinogenicity of limonene: the accumulation of micro masses of calcium phosphate in the bladder, coupled with a high pH environment in the male rat bladder, has been suggested to be responsible for the bladder carcinogenicity of sodium saccharin. The implication of these proposed mechanisms is that limonene and sodium saccharin will not be mutagenic to the rat kidney and bladder, respectively. This proposal has been evaluated by assessing the mutagenic potential of the two chemicals to male lacI transgenic (Big Blue) rats. Male Big Blue rats were exposed for 10 consecutive days to either limonene in diet, at a dose level in excess of that used in the original National Toxicology Program gavage carcinogenicity bioassay, or to sodium saccharin in diet at the dose known to induce bladder tumours. The multi-site rat carcinogen 4-aminobiphenyl was used as a positive control for the experiment. Limonene failed to increase the mutant frequency in the liver or kidney of the rats, and sodium saccharin failed to increase the mutant frequency in the liver or bladder of the rats. 4-Aminobiphenyl was mutagenic to all three of these tissues. These results add further support to a non-genotoxic mechanism of carcinogenic action for both limonene and sodium saccharin.

Preliminary assessment of the skin sensitizing activity of selected rodent carcinogens using the local lymph node assay.

It has been demonstrated previously that there exists an incomplete correlation between the skin sensitizing potential of chemicals and their mutagenic properties as judged by activity in the Salmonella mutation assay. More recently, it has been proposed that there may exist a broader association between carcinogenicity in rodents (including non-genotoxic carcinogenesis) and skin sensitizing activity. To explore further these putative relationships we have here examined the skin sensitizing potential of two non-genotoxic rodent carcinogens which are generally considered not to represent a carcinogenic hazard in humans (limonene and saccharin) and of three genotoxic rodent carcinogens (vinylidene dichloride, ethyl acrylate and bisphenol A diglycidyl ether). For this purpose we have used the local lymph node assay (LLNA), a method for the identification and characterization of skin sensitizing chemicals that has recently been recognized as a stand-alone method for hazard identification purposes. Activity in the LLNA was compared with the results of Salmonella tests conducted previously. This small series of investigations reveals that there exists no general relationship between skin sensitizing potential and rodent carcinogenicity. Furthermore, although a general correlation does exist between mutagenic activity and skin sensitization, this association is not universal and activity in the Salmonella mutation assay does not necessarily imply skin sensitizing potential. Collectively these data suggest that it is inappropriate currently to recommend the use of skin sensitization tests as an adjunct to conventional approaches to the evaluation of potential carcinogenicity.

Carcinogenicity assays of wood dust and wood additives in rats exposed by long-term inhalation.

OBJECTIVE: This study evaluates whether wood dust and/or wood preservatives develop a carcinogenic potential against the tissues of the airways of rats. METHODS: The formation of tumors of the respiratory tract after exposure to wood dust was studied in six groups of approximately 60 female Fischer 344 rats exposed by long-term inhalation to mean concentrations of (1) 18 mg/m3 of untreated oak wood dust, (2) wood preservatives containing ca. 1 microgram/m3 lindane and 0.2 microgram/m3 of pentachlorophenol (PCP) in the exposure air, or lindane and 18 micrograms/m3 of PCP (group lindane/PCP vapors, and group oak wood treated with lindane/PCP), (3) 21 or 39 micrograms/m3 of sodium dichromate (calculated as CrO3, group chromate aerosol and group oak wood with chromate), and 72 micrograms/m3 of N-nitrosodimethylamine vapors as positive control. The negative control group consisted of 115 animals (sham-exposed). RESULTS: Tumors of the nasal cavity developed in two rats exposed to chromate aerosol or in combination with wood dust (2/102, 2%). Malignant tumors of the lower respiratory tract were induced only in exposed groups of rats (three adenocarcinomas of the lung and four bronchiolar lung carcinomas, 7/254, 2.8%). More respiratory tract tumors were observed in rats exposed to chromate or wood with chromate (5/102, 5%), also in groups exposed to oak wood dust (oak untreated, oak + chromate, oak + lindane/PCP; together 5/155, 3.2%). Analysis of 'unpreserved' oak wood dust revealed up to 5 micrograms/m3 of chromate. When this exposure was taken into account, eight of nine animals with respiratory tract tumors (including nasal cavity) had exposure to chromate, while only one tumor occurred in the group lindane/PCP. Otherwise the incidence of systemic tumors was increased in animals exposed to lindane/PCP, due in particular to a significantly increased incidence of liver tumors (OR = 3.7; 1.24-11.3; P = 0.019). Fatal (mucoepidermoid) tumors were induced by N-nitrosodimethylamine (NDMA) in the positive control (14/46, 30%). No such tumors of the respiratory tract were observed in the negative control. CONCLUSIONS: Tumors in the respiratory tract were found only in exposed animals, predominantly in the groups which inhaled oak wood dust and chromate stain. Chromate may play a decisive role for the etiology of tumors of the nasal cavity in wood workers. This assumption should be supported by further dose-response studies.

Relevance of N-acetyltransferase 1 and 2 (NAT1, NAT2) genetic polymorphisms in non-small cell lung cancer susceptibility.

The highly polymorphic N-acetyltransferases (NAT1 and NAT2) are involved in both activation and inactivation reactions of numerous carcinogens, such as tobacco derived aromatic amines. The potential effect of the NAT genotypes in individual susceptibility to lung cancer was examined in a hospital based case-control study consisting of 392 Caucasian lung cancer patients [152 adenocarcinomas, 173 squamous cell carcinomas (SCC) and 67 other primary lung tumours] and 351 controls. In addition to the wild-type allele NAT1*4, seven variant NAT1 alleles (NAT1*3, *10, *11, *14, *15, *17 and *22) were analysed. A new method based on the LightCycler (Roche Diagnostics Inc.) technology was applied for the detection of the polymorphic NAT1 sites at nt 1088 and nt 1095. The NAT2 polymorphic sites at nt 481, 590, 803 and 857 were detected by polymerase chain reaction-restriction fragment length polymorphism or LightCycler. Multivariate logistic regression analyses were performed taking into account levels of smoking, age, gender and occupational exposure. An increased risk for adenocarcinoma among the NAT1 putative fast acetylators [odds ratio (OR) 1.92 (1.16-3.16)] was found but could not be detected for SCC or the total case group. NAT2 genotypes alone appeared not to modify individual lung cancer risk, however, individuals with combined NAT1 fast and NAT2 slow genotype had significantly elevated adenocarcinoma risk [OR 2.22 (1.03-4.81)] compared to persons with other genotype combinations. These data clearly show the importance of separating different histological lung tumour subtypes in studies on genetic susceptibility factors and implicate the NAT1*10 allele as a risk factor for adenocarcinoma.

Altered DNA repair capacity and bleomycin sensitivity as risk markers for non-small cell lung cancer.

DNA repair capacity in human peripheral blood lymphocytes was monitored by the repair rate of bleomycin-induced DNA damage using an alkaline single-cell gel electrophoresis assay (comet assay). DNA repair capacity, after 15 min repair time, in lymphocytes of non-small cell lung cancer patients (n = 160) and controls (n = 180) was 67% and 79.3%, respectively (p < 0.0004). Bleomycin sensitivity defined as the tail moment of bleomycin-treated peripheral blood lymphocytes, without allowing time for DNA repair, was significantly higher in lung cancer patients than in tumor-free hospital controls (p < 0.0001). There was no correlation, in either patient or control group, between the bleomycin sensitivity and DNA repair capacity with age or gender. The median values of DNA repair capacity and sensitivity in controls were used as the cut-off points for calculating odds ratios (OR). After adjustment for age, gender and smoking status, the cases vs. controls had reduced DNA repair capacity (OR = 2.1; 95% confidence limit [CL] 1.1-4.0) and increased bleomycin sensitivity (OR = 4; 95% CL 2.2-7.4). For current smokers, the adjusted risk associated with bleomycin sensitivity was 2.3 (95% CL 1.1-4.9). We conclude that our standard comet assay as a phenotypical repair test has sufficient sensitivity and rapidity allowing application to both native and cryopreserved lymphocytes. Bleomycin sensitivity and DNA repair capacity were found to be 2 independent susceptibility markers for non-small cell lung cancer, confirming similar investigations with different marker end points. The latter were much more time consuming than the method used in our study. Thus, the comet assay is more suitable for screening large numbers of individuals in epidemiological studies. Validation of this assay in large prospective studies for the identification of subjects at high risk for non-small cell lung cancer is now warranted.

Rapid screening assay for mutagen sensitivity and DNA repair capacity in human peripheral blood lymphocytes.

Individual susceptibility to carcinogens, an important determinant of disease risk, is influenced by host factors such as the ability to repair DNA lesions. In order to identify subjects who are at high risk, we have developed a microgel electrophoresis assay for use in molecular epidemiological studies. The assay was validated in a pilot case-control study: Peripheral blood lymphocytes were collected from 100 patients with lung cancer and 110 control patients without cancer and from the same hospital, and stored at -80 degrees C. After thawing, phytohaemagglutinin-stimulated cells were treated with bleomycin at 20 microg/ml for 30 min and the extent of DNA damage and DNA repair capacity were determined by microgel electrophoresis. Peripheral blood lymphocytes from patients with lung cancer were significantly more sensitive to mutagens than those from controls and showed reduced DNA repair capacity (both P < 0.001). Both endpoints were independent risk factors for smoking-related lung cancer. Repeated analysis of peripheral blood lymphocytes from the same individual demonstrated good reproducibility of the assay. Cryopreservation of the lymphocytes for less than or = 12 months did not significantly affect their sensitivity. Our standardized microgel electrophoresis assay is suitable for determining individual sensitivity to mutagens and DNA repair capacity: it is sensitive and faster than cytogenetic assays, and can be applied to native and cryopreserved peripheral blood lymphocytes.

hOGG1 polymorphism and loss of heterozygosity (LOH): significance for lung cancer susceptibility in a caucasian population.

Oxidative damage is implicated in several chronic diseases including cancer. 8-Hydroxyguanine (8-oxoG) is one of the major promutagenic DNA lesions, which is produced by reactive oxygen species, causes G:C to T:A transversions and is excised by OGG1, an 8-oxoG specific DNA glycosylase/AP-Lyase. In a nested case-control study, gDNA from 105 Caucasian primary non-small cell lung cancer cases and 105 matched controls was screened for 6 possible new polymorphic sites in the human OGG1 gene, detected previously mainly in tumour tissue. The previously described Ser(326)Cys polymorphism was found to be common (allele frequency 0.22) in Caucasians. However, no major difference in Ser(326)Cys genotype distribution could be detected between cases and controls. Two 5;-end polymorphisms previously found in Japanese as well as Arg(131)Gln could not be detected in this population. An Ala(85)Ser polymorphism was found in 2 controls, whereas Arg(46)Gln was detected in only 1 case. As the hOGG1 gene is mapped (3p26.2) to a region frequently lost in primary lung tumours, the frequency of loss of heterozygosity (LOH) was investigated. Forty-three percent of the studied lung tumours exhibited loss of one of the hOGG1 alleles. The wt Ser(326) allele was not predominantly lost in our sample set, which suggests a minor role of this polymorphism in tumourgenesis. Our results show that LOH at the hOGG1 gene locus is a very common occurrence in lung tumourgenesis, possibly leading to increased mutational damage due to ROS in smokers. However, the hOGG1 polymorphisms studied are probably not major contributors to individual lung cancer susceptibility in Caucasians.