Marianne Dybdahl

DNA repair capacity: inconsistency between effect of over-expression of five NER genes and the correlation to mRNA levels in primary lymphocytes.

We have previously shown that high DNA repair capacity protects psoriasis patients against chemically induced basal cell carcinoma [Dybdahl et al. Mutat. Res. 433 (1999) 15-22]. We have used the same study persons to investigate the correlation between expression of eight genes involved in nucleotide excision repair and DNA repair capacity. mRNA levels of XPA, XPB, XPC, XPD, XPF, XPG, CSB and ERCC1 in primary lymphocytes from 33 individuals were quantified by dot-blots and normalized to beta-actin. ERCC1 and XPD mRNA quantities were highly correlated (r=0.89; P<10(-11)) while XPA, XPB, XPC, XPG, XPFand CSB mRNAs were moderately correlated (r=0.2-0.7). Thus, the mRNA expressions seem to fall in at least two groups. There was a three to sevenfold variation in the expression levels of the mRNAs. This is in contrast to the more than a hundredfold variation in mRNA levels reported in cancer patients.DNA repair capacity was measured in a host cell reactivation assay, where primary lymphocytes were transfected with an UV-irradiated plasmid encoding firefly-luciferase. Only ERCC1 and XPD mRNA levels correlated with the DNA repair capacity (P<0.03). In order to see if ERCC1 or XPD activity was limiting for DNA repair, we cotransfected with plasmids encoding NER genes, thus over-expressing either XPB, XPC, XPD, CSB or ERCC1 in the host cell reactivation assay. Only XPB over-expression increased DNA repair capacity. Thus, there is no indication that neither XPD nor ERCC1 limits the DNA repair capacity. However, our results indicate that ERCC1 and XPD mRNA levels may be used as a proxy for DNA repair capacity in lymphocytes.

Seasonal variation of DNA damage and repair in patients with non-melanoma skin cancer and referents with and without psoriasis

Quadruples of skin cancer patients with and without psoriasis and referents with and without psoriasis (4 x 20 study persons) were identified and examined for DNA damage by single cell gel electrophoresis (comet-assay) and DNA-repair by UV-induced unscheduled DNA synthesis (UDS) in mononuclear blood cells (lymphocytes and monocytes). DNA damage (strand breaks and alkaline labile sites) as assessed by the comet assay and DNA repair as assessed by UDS were significantly associated with the season in which blood sampling took place. This variation might be explained by an increased exposure to solar radiation. When the comet tail moment data were stratified by sampling period, an interaction between psoriasis and skin cancer was detected, with patients with psoriasis and skin cancer exhibiting more DNA damage. Patients with psoriasis and skin cancer also had lower UDS compared to healthy study persons, suggesting that the more DNA damage may be caused by a lower rate of DNA repair. In all study persons, the extent of UDS correlated positively with the amount of DNA damage determined by the comet assay.

Inhalation of ozone induces DNA strand breaks and inflammation in mice

Ozone (O3) is a well-known oxidant pollutant present in photochemical smog. Although ozone is suspected to be a respiratory carcinogen it is not regulated as a carcinogen in most countries. The genotoxic and inflammatory effects of ozone were investigated in female mice exposed to ozone for 90 min. The tail moment in bronchoalveolar lavage (BAL) cells from BALB/c mice was determined by the comet assay as a measure of DNA strand breaks. Within the first 200 min after exposure, the BAL cells from the mice exposed to 1 or 2 ppm ozone had 1.6- and 2.6-fold greater tail moments than unexposed mice. After 200 min there was no effect. It could be ruled out that the effect during the first 200 min was due to major infiltration of lymphocytes or neutrophils. Unexpectedly, ozone had no effect on the content of 8-oxo-deoxyguanosine (8-oxo-dG) in nuclear DNA or on oxidised amino acids in the lung tissue. The mRNA level of the repair enzyme ERCC1 was not increased in the lung tissue. Inflammation was measured by the cytokine mRNA level in lung homogenates. An up to 150-fold induction of interleukin-6 (IL-6) mRNA was detected in the animals exposed to 2 ppm ozone compared to the air-exposed control mice. Also at 1 ppm ozone, the IL-6 mRNA was induced. The large induction of IL-6 mRNA in the lung took place after DNA strand breaks were induced in BAL. This does not support the notion that inflammatory reactions are the cause of DNA damage. To determine whether these exposures were mutagenic, Muta Mice were exposed to 2 ppm ozone, 90 min per day for 5 days. No treatment-related mutations could be detected in the cII transgene. These results indicate that a short episode of ozone exposure at five times the threshold limit value (TLV) in US induces lung inflammatory mediators and DNA damage in the cells in the lumen of the lung. This was not reflected by an induction of mutations in the lung of Muta Mice.

Cytokine expression in mice exposed to diesel exhaust particles by inhalation. Role of tumor necrosis factor

BackgroundParticulate air pollution has been associated with lung and cardiovascular disease, for which lung inflammation may be a driving mechanism. The pro-inflammatory cytokine, tumor necrosis factor (TNF) has been suggested to have a key-role in particle-induced inflammation.We studied the time course of gene expression of inflammatory markers in the lungs of wild type mice and Tnf-/- mice after exposure to diesel exhaust particles (DEPs). Mice were exposed to either a single or multiple doses of DEP by inhalation. We measured the mRNA level of the cytokines Tnf and interleukin-6 (Il-6) and the chemokines, monocyte chemoattractant protein (Mcp-1), macrophage inflammatory protein-2 (Mip-2) and keratinocyte derived chemokine (Kc) in the lung tissue at different time points after exposure.ResultsTnf mRNA expression levels increased late after DEP-inhalation, whereas the expression levels of Il-6, Mcp-1 and Kc increased early. The expression of Mip-2 was independent of TNF if the dose was above a certain level. The expression levels of the cytokines Kc, Mcp-1 and Il-6, were increased in the absence of TNF.ConclusionOur data demonstrate that Tnf is not important in early DEP induced inflammation and rather exerts negative influence on Mcp-1 and Kc mRNA levels. This suggests that other signalling pathways are important, a candidate being one involving Mcp-1.

Low DNA repair is a risk factor in skin carcinogenesis: a study of basal cell carcinoma in psoriasis patients

We have studied DNA repair in patients with psoriasis aiming at investigating the importance of repair in chemically induced cancer. An increased risk of non-melanoma skin cancer has been observed in psoriasis patients extensively treated with tar, methotrexate and photochemotherapy (psoralen + UVA). We measured the DNA repair capacity (DRC) by a host cell reactivation (HCR) assay in lymphocytes from psoriasis patients with and without basal cell cancer and non-psoriatic persons with and without basal cell cancer (4 x 20 study persons). Among psoriasis patients we observed a significant lower DRC in patients with skin cancer compared to patients without skin cancer (P = 0.015; Mann-Whitney, one-sided). Using the median of the healthy control group (group 4) as a cutoff value to divide the psoriasis patients into groups of high and low repair, we found that individuals who had a low repair capacity had a 6.4-fold increased skin cancer risk compared to individuals with high repair (95% confidence interval (CI), 1.44-28.5). The level of DNA repair was correlated with the age at which the psoriasis patients got their first skin cancer. The lower the level of DNA repair, the earlier the psoriasis patients had their first skin tumor (P = 0.070 Spearman; one-sided). Psoriasis patients without BCC had marginally higher repair than healthy controls (P = 0.11, Mann-Whitney, two-sided). We found no difference between BCC patients without psoriasis and healthy controls. In conclusion, these findings suggest a protective role of DNA repair in a predominantly chemically induced cancer.

Repeated inhalations of diesel exhaust particles and oxidatively damaged DNA in young oxoguanine DNA glycosylase (OGG1) deficient mice.

DNA repair may prevent increased levels of oxidatively damaged DNA from prolonged oxidative stress induced by, e.g. exposure to diesel exhaust particles (DEP). We studied oxidative damage to DNA in broncho-alveolar lavage cells, lungs, and liver after 4 × 1.5 h inhalations of DEP (20 mg/m3) in Ogg1− / − and wild type (WT) mice with similar extent of inflammation. DEP exposure increased lung levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in Ogg1− / − mice, whereas no effect on 8-oxodG or oxidized purines in terms of formamidopyrimidine DNA glycosylase (FPG) sites was observed in WT mice. In both unexposed and exposed Ogg1− / − mice the level of FPG sites in the lungs was 3-fold higher than in WT mice. The high basal level of FPG sites in Ogg1− / − mice probably saturated the assay and prevented detection of DEP-generated damage. In conclusion, Ogg1− / − mice have elevated pulmonary levels of FPG sites and accumulate genomic 8-oxodG after repeated inhalations of DEP.

Psoriasis patients with basal cell carcinoma have more repair-mediated DNA strand-breaks after UVC damage in lymphocytes than psoriasis patients without basal cell carcinoma ☆ ☆☆

We have investigated the formation of strand-breaks following UVC irradiation in lymphocytes from psoriasis patients with or without basal cell carcinoma (BCC). Isolated lymphocytes were irradiated with UVC light at a dose of 3.6 J/m(2), and the level of DNA strand-breaks were measured 25 min after the irradiation by the alkaline comet assay. The generation of strand-breaks following UVC irradiation indicates DNA-repair-mediated incisions, as UVC light does not generate strand-breaks per se. We found that psoriasis patients with BCC had more DNA-repair incisions than non-cancer patients. The incision level correlated to two polymorphisms of the XPD gene. At present, it is not clear if the association is a primary effect that is related to differences of the XPD protein. Genes encoding for other repair proteins, namely XRCC1, ERCC1, and LIG1 are located close to the XPD gene, and it is possible that the association is due to a cosegregation with a polymorphism in one of these genes.

Twelve single nucleotide polymorphisms on chromosome 19q13.2-13.3: Linkage disequilibria and associations with basal cell carcinoma in Danish psoriatic patients

The genetic susceptibility to basal cell carcinoma (BCC) among Danish psoriatic patients was investigated in association studies with 12 single nucleotide polymorphisms on chromosome 19q13.2-3. The results show a significant association between BCC and the A-allele of a polymorphism in ERCCI exon4 (Odds ratio 12;95% Confidence Interval 1.17–124; p(χ2, two-side) = 0.019) and to a lesser extent with XPD exon6 (p = 0.06). This is in accordance with recent studies of a different group of BCC cases (Rockenbauer et al. (in press) Carcinogenesis; Yin et al. (manuscript submitted for publication). Cancer Epidemiol. Biomarkers Prev), which places two highly influential markers between these two genes. The analysis also confirmed that considerable linkage disequilibrium exists between SNPs both within genes and between genes in this region. The combined studies suggest that genetic variation in nucleotide excision repair is of importance for the development of BCC.

Proficient deoxyribonucleic acid repair of methylation damage in hamster ERCC-gene mutants.

Three major pathways, nucleotide excision repair (NER), base excision repair (BER) and O6-methylguanine-DNA methyltransferase (MGMT), are responsible for the removal of most adducts to DNA and thus for the survival of cells influenced by deoxyribonucleic acid (DNA) adduct-forming chemicals. We have evaluated host cell reactivation and cell survival of wild type Chinese hamster ovary cells and of mutants in the NER-genes ERCC1, ERCC2, and ERCC4 after treatment with the methylating compounds dimethylsulfate and methylnitrosourea. No effect of the three genes could be demonstrated, i.e., survival and host cell reactivation after methylation damage in the mutants and the wild type cells were similar. Gene-specific repair experiments confirmed the proficient removal of methyl lesions. We conclude that the three nucleotide excision repair genes are immaterial to the repair of methylation damage. This suggests that NER does not play a role in the removal of methylation in mammalian cells and that BER and MGMT are responsible for the survival of such cells, when they are challenged with methylation of DNA.