Significance of amino acid substitution variants of DNA repair genes in radiosusceptibility of cervical cancer patients; a pilot study.

The present pilot study was designed to elucidate the functional significance of amino acid substitution variants of DNA repair genes. Using the peripheral blood lymphocytes (PBLs) from healthy donors and cervical cancer patients, the contribution of four non-synonymous single nucleotide polymorphisms (SNPs) in three base excision repair genes (BER), XRCC1 (Arg194Trp and Arg399Gln), hOGG1 (Ser326Cys), and APE1 (Asp148Glu), to the susceptibility to ionizing radiation were evaluated. The level of initial, oxidative and residual DNA damage produced by 2 Gy was measured by the alkaline single cell gel electrophoresis (the comet assay), and the SNPs were determined by PCR-restriction fragment length polymorphism (RFLP) assay. No significant differences in the allele frequencies between cancer patients and controls for any of these four SNPs were detected. Although the initial DNA damage levels were approximately similar, significantly higher level of Fpg-sensitive sites were found in patients compared with controls (p<0.001) irrespective of genotype distribution. A trend towards increased values of EndoIII-sensitive sites was determined in PBLs from cancer patients compared with healthy women, mainly carriers of the XRCC1 and OGG1 variant alleles; however, the mean value of EndoIII-sensitive sites does not reach any significance. A substantial delay in DNA strand-break rejoining was ascertained in patients who carried APE1 Glu variant allele in comparison with healthy donors 15 and 60 minutes after irradiation (p< 0.05 and p< 0.01, respectively). In contrast, slightly higher but statistically significant level of residual DNA damage was estimated in controls (APE1Asp/Asp) compared with patients. An association between single nucleotide polymorphism (SNP) of two DNA repair genes functioning in the same biochemical pathway and susceptibility to radiation was found. In the combined genotype APE1/XRCC1 and APE1/hOGG1, a decreased level of residual DNA damage was detected in carriers of wild type APE1 genotype. In addition, a possible modulating effect of hOGG1 gene on the kinetics of strand-break rejoining was estimated. The lowest residual DNA damage level was determined in subjects with the combined APE1(Asp/Asp)/hOGG1(Ser/Cys+Cys/Cys) genotypes. Based on these preliminary data we suppose that a combination of several amino acid substitution variants of DNA repair genes involved in the same repair pathway rather than one low-penetrance SNP in a single gene may contribute to DNA repair outcomes. Larger study with more subjects is needed to verify these findings.

Radiosensitivity of peripheral blood lymphocytes from healthy donors and cervical cancer patients; the correspondence of in vitro data with the clinical outcome.

The reliability of a particular in vitro parameter as potential prognostic biomarker of individual radiosensitivity is still discussed. Therefore, several in vitro radiation-induced cellular endpoints including initial, oxidative and residual DNA damage and the rate of DNA repair were assessed in peripheral blood lymphocytes (PBL) from healthy donors and patients with carcinoma of the cervix using the alkaline single cell gel electrophoresis (the comet assay). PBL from cancer patients were analyzed three times during the course of therapy, prior, in the middle (25-27 Gy) and after the radiotherapy. Interindividual differences in radiation-induced DNA damage and in the kinetics of strand break rejoining were determined within both groups. Significantly higher level of mean background and oxidative DNA damage was estimated in the cancer patient cohort than in the healthy subject group; however similar mean values of the initial DNA damage and the rate of DNA repair kinetics were found in both groups. No adaptive response was determined in PBL from cancer patients due to radiotherapy. The acute radiation toxicity and the clinical outcome were scored according to the criteria as proposed by the National Cancer Institute. A substantial delay in DNA strand break rejoining was determined in cancer patients suffering from adverse side effects (G2+) in comparison to persons with no or very mild radiation toxicity (G0-G1) (p<0.05). The recurrence of disease has been associated with a lower initial DNA damage and slope value of dose-response effect, and increased rate of DNA repair. Results from this pilot study suggest that the residual DNA damage level might be a promising prognostic biomarker of acute radiation morbidity; however, further study is necessary to validate this finding.

Radiosensitivity of cervical cancer cell lines: the impact of polymorphisms in DNA repair genes.

The aim of this study was to evaluate radiosensitivity of cervical cancer cells in vitro and to assess the relationship between genetic polymorphisms in DNA repair genes and the response of cells to ionizing radiation. The alkaline comet assay as a predictive assay of radiosensitivity was used to examine the susceptibility of four human cervical cancer cell lines (CaSki, C-33A, HeLa and SiHa) to radiation damages. The initial DNA damage and the residual DNA damage at 15, 30, 45 and 60 min after irradiation were assessed. Genotypes of DNA repair genes (XRCC1, hOGG1, PARP, XPD, XRCC3 and XRCC4) were analyzed by PCR-RFLP assays. The comet data clearly indicate a variable but dose-dependent increase in the initial DNA damage in all cell lines. The highest slope of dose response curve was observed in C-33A cells and this cell line was assumed to be radiosensitive. All cell lines repaired DNA damage in a similar manner, the level of DNA strand breakage has returned near the background level within 45 min after irradiation. According to the genotype we found that C-33A cells are polymorphic in the majority of analyzed DNA repair genes. This pilot study indicated associations between polymorphisms in DNA repair genes and cell radiosensitivity.

Induction of micronuclei by 7H-dibenzo[c,g]carbazole and its tissue specific derivatives in Chinese hamster V79MZh1A1 cells.

The clastogenicity/aneugenicity of N-heterocyclic polycyclic aromatic pollutant 7H-dibenzo[c,g]carbazole (DBC) and its two synthetic derivatives N-methyl DBC (MeDBC) and 5,9-dimethyl DBC (diMeDBC) was evaluated in the genetically engineered Chinese hamster V79 cell line V79MZh1A1 with stable expression of human cytochrome P4501A1 and in the parental V79MZ cell line without any cytochrome P450 activity. While none of the three carbazoles changed significantly the level of micronuclei in the parental V79MZ cells, a variable, but statistically significant rise of micronucleus frequencies was assessed in V79MZh1A1 cells. DBC induced dose-dependent increase in the number of micronuclei at harvest times of 24 and 48h and MeDBC at sampling time of 48h in V79MZh1A1 cells in comparison to untreated cells, however, no significant time-dependent increase in micronucleus frequencies was found. The use of the antikinetochore immunostaining revealed that DBC and MeDBC induced approximately equal levels of both kinetochore positive (C+) and kinetochore negative (C-) micronuclei. DiMeDBC, a strict hepatocarcinogen, did not manifest any effect on micronucleus induction in V79MZh1A1 cells. These studies suggest that genetically engineered Chinese hamster V79 cell lines expressing individual CYP cDNAs are a useful in vitro model for evaluation the role of particular cytochromes P450 in biotransformation of DBC and its tissue and organ specific derivatives.

Role of cytochrome P4501A1 in biotransformation of a tissue specific sarcomagen N-methyldibenzo[c,g]carbazole.

7H-dibenzo[c,g]carbazole (DBC) is a potent liver and skin carcinogen, while its synthetic methyl derivative N-methyldibenzo[c,g]carbazole (MeDBC) is tissue specific sarcomagen. It is supposed that sarcomagenic activity of DBC depends on biotransformation at ring-carbon atoms, as with PAH, whereas the heterocyclic nitrogen plays an important role in liver carcinogenicity. The objective of this study was to elucidate the role of cytochrome P4501A1 in metabolic activation of sarcomagenic derivatives of DBC and to characterize the DNA damage profiles induced by DBC and MeDBC in relation to the mode of metabolic activation. The genetically engineered V79MZh1A1 cell line with stable expression of cDNA of human cytochrome P4501A1, the parental V79MZ cell line lacking any cytochrome P450 activity and human hepatocarcinoma Hep G2 cells were used as a model cells. Dose-dependent decrease in colony forming ability (CFA) was found in the V79MZh1A1 cell line after treatment of cells with DBC and MeDBC; however, no change in CFA was induced in parental V79MZ cells. These results were in a good correlation with DNA damaging effects of these two derivatives measured by the alkaline DNA unwinding (ADU) and the modified single cell gel electrophoresis (SCGE) techniques. Differences in DNA damage profiles induced by DBC and MeDBC were found in V79MZh1A1 and Hep G2 cells. These differences were probably the result of different reactive metabolite formation depending on chemical structure of the molecule and ways of biotransformation. This study showed that the cytochrome P4501A1 took part in activation of sarcomagenic DBC derivatives. Moreover, V79 cell lines with stable expression of different cytochromes P450 in combination with DNA repair endonucleases should be a useful tool for characterization of the role of individual cytochromes in metabolic activation pathways of DBC and MeDBC.

Cytotoxic and genotoxic effect of inhibitor of vulcanisation N-cyclohexylthiophthalimide in a battery of in vitro assays.

Mutagenicity of N-cyclohexylthiophthalimide (Duslin P) was tested first by the Ames test in the bacteria, Salmonella typhimurium. The negative results of the Ames test suggested that this compound does not induce mutations in the genome of S. typhimurium under the conditions used. To estimate the cytotoxicity of Duslin P to human cells, we measured cellular DNA and protein as well as cell proliferation, i.e., the mitotic index of treated and control cells. The genotoxic effects were assayed by two biochemical methods developed for detection of single-strand breaks of DNA in mammalian cells, i.e., by the alkaline single cell gel electrophoresis (comet assay) and by the DNA unwinding method, respectively. The DNA unwinding method showed that this compound did not induce DNA damage at concentrations < 7 micrograms/ml. Alkaline single cell gel electrophoresis revealed approximately double the level of DNA damage (in comparison to untreated control DNA) at a concentration of 2 micrograms/ml, which reduced proliferation to approximately 30%, and triple the level of DNA damage at higher concentrations (6 and 7 micrograms/ml), which inhibited completely both DNA synthesis and proteosynthesis. Cells with moderately damaged DNA were more common than cells with heavily damaged DNA. Parallel experiments with the strong mutagen and carcinogen MNNG showed that MNNG induced in cells a high level of DNA damage at concentrations which did not reduce the mitotic index or proteosynthesis, while DNA synthesis inhibited only partially. After treatment with MNNG, cells with heavily damaged DNA were more common than cells with moderately damaged DNA. Duslin P-treated VH10 cells were also tested cytogenetically, confirming that Duslin P induced neither chromosomal aberrations nor aneuploidy. We conclude that Duslin P has no mutagenic effect on bacteria, does not induce chromosomal aberrations and CREST positive or CREST negative micronuclei in human cells and induces only a small increase of DNA damage in human cells which is consistent with DNA fragmentation due to cell death.

Preculturing of Chinese hamster V79 cells with sublethal concentration of theophylline sensitizes cells to cytotoxic effects of MNNG.

In our previous work concerning the biologic effects of theophylline, we found that cells incubated during 48 h at low concentrations of theophylline (0.3 mg/ml of medium) manifested short-term deviations in the rate of DNA replication; however, this short-term inhibition of DNA replication did not reduce either the growth rate or the colony-forming ability of cells. In the present study, we concentrated on cytotoxic and DNA-damaging effects of MNNG on V79 cells precultured with sublethal concentration of methylxanthine theophylline. Cytotoxicity was evaluated on the basis of growth rate of treated cells as well as by colony-forming ability (plating efficiency) test and by trypan blue exclusion test. The level of DNA lesions (strand breaks) induced by MNNG was measured by alkaline DNA unwinding and by the comet assay. In an effort to explain higher cytotoxic effects of MNNG on precultured cells, we studied rejoining of damaged parental DNA after 4 h incubation post-MNNG-treatment as well. We found differences as against the controls in theophylline-precultured cells after treatment with the mutagen and carcinogen MNNG. The higher cytotoxic effect of MNNG in precultured cells was accompanied by a higher level of ss breaks of DNA and by more unrepaired lesions which remained after 4 h in parental DNA. Our results demonstrate that theophylline belongs to the group of agents inhibiting repair of potentially lethal DNA lesions.

Detection of MNNG-induced DNA lesions in mammalian cells; validation of comet assay against DNA unwinding technique, alkaline elution of DNA and chromosomal aberrations.

Human cells (VH10 or Hep G2) and hamster cells V79 were exposed to different concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the level of DNA lesions was evaluated by the DNA unwinding technique, alkaline elution of DNA and the comet assay. All three methods were able to detect the effects of MNNG but with a clear difference in sensitivity. At low concentrations of MNNG the most sensitive method appeared to be the comet assay. After the short-term treatment the comet assay was able to detect the lesions induced by MNNG at approx. 0.1 microgram/ml, alkaline elution of DNA at 1 microgram/ml and DNA unwinding at 1-2 micrograms/ml. MNNG treated VH10 cells, human lymphocytes and V79 cells were also tested cytogenetically, confirming that MNNG induced chromosomal aberrations at concentrations > 1 microgram/ml in VH10 cells (short-term treatment): > 0.2 microgram/ml in V79 cells (long-term treatment) and > 8 micrograms/ml in human lymphocytes (long-term treatment). In some experiments we tried to increase the level of MNNG-induced DNA breaks with help of DNA repair inhibitors cytosine arabinoside (Ara C) and hydroxyurea (HU) which were applied either after or during MNNG treatment. Our results showed that the level of MNNG-induced lesions was increased by simultaneous treatment of cells with MNNG and Ara C and HU. 2 x 10(-5) M Ara C and 2 x 10(-3) MHU were as effective as 10-times higher concentrations of inhibitors. Ara C and HU increased the level of MNNG-induced DNA breaks mainly in combination with lower concentrations of MNNG (< 2 micrograms/ml). Rejoining of DNA breaks was observed in human cells VH10 and Hep G2 as well as in Chinese hamster cells V79 damaged by both lower and higher MNNG-concentrations. All methods showed that MNNG-induced DNA breaks had been gradually rejoined.

Testing of toxic and DNA-damaging effects of N-cyclohexylthiophthalimide (Duslin P) on hamster V79 cells.

N-cyclohexylthiophthalimide, commercial name Duslin P, at concentrations 0.5-2 micrograms/ml inhibited proliferation of V79 cells and at concentrations > 2 micrograms/ml acted cytotoxically. Inhibition of cumulative DNA synthesis correlated well with the deleterious effects of Duslin P on growth activity and plating efficiency. DNA synthesis was not renewed even 6 h after the treatment of cells. Alkaline elution of DNA of V79 cells treated with Duslin P did not confirm our expectation that this chemical compound has a DNA-damaging effect. Duslin P strongly inhibited protein synthesis at concentrations > 2 micrograms/ml. We suggest that the cytotoxic effects of Duslin P are not accompanied by any genotoxic effects.

Measurement of DNA strand breakage and DNA repair induced with hydrogen peroxide using single cell gel electrophoresis, alkaline DNA unwinding and alkaline elution of DNA.

Three techniques: single cell gel electrophoresis (SCGE), alkaline elution of DNA (AE), and alkaline DNA unwinding (ADU) were chosen to compare the sensitivity among these methods in detection of DNA damage and repair in human diploid VH10 cell line after short-term exposure to hydrogen peroxide. Using SCGE technique a dose-dependent increase in DNA migration was found in cells exposed to hydrogen peroxide in concentration range from 10 micromol/l to 100 micromol/l. Alkaline DNA unwinding method detected increased level of single strand breaks (ssb) in concentration range from 25 micromol/l to 100 micromol/l of H2O2, and alkaline elution of DNA estimated increased DNA elution rate from concentration 50 micromol/l of H2O2. In a time course study to evaluate the kinetics of DNA repair, both SCGE and ADU techniques showed that the repair of DNA strand breaks is very rapid; the level of ssb in treated cells has returned to near the background level within two hours. After this time damage remaining in the DNA was in the form of oxidised bases as revealed the incubation of treated cells with specific DNA repair endonuclease, formamidopyrimidine-DNA glycosylase.