Mutagenesis - current issue

Type II topoisomerases--inhibitors, repair mechanisms and mutations

Heisig, P. — Mon, 02 Nov 2009 03:04:52 PST

Type II topoisomerases are ubiquitous enzymes that play an essential role in the control of replicative DNA synthesis and share structural and functional homology among different prokaryotic and eukaryotic organisms. Antibacterial fluoroquinolones target prokaryotic topoisomerases at concentrations 100- to 1000-fold lower than mammalian enzymes, the preferred targets of anticancer drugs such as etoposide. The mechanisms of action of both of these types of inhibitors involve the fixation of an intermediate reaction step, where the enzyme is covalently bound to an enzyme-mediated DNA double-strand break (DSB). The resulting ternary drug–enzyme–DNA complexes can then be converted to cleavage complexes that block further movement of the DNA replication fork, subsequently inducing stress responses. In haploid prokaryotic cells, stress responses include error-free and error-prone DNA damage repair pathways, such as homologous recombination and translesion synthesis, respectively. The latter can result in the acquisition of point mutations. Diploid mammalian cells are assumed to preferentially use recombination mechanisms for the repair of DSBs, an example of which, non-homologous end joining, is a major error-prone repair mechanism associated with an increased frequency of detectable small deletions, insertions and translocations. However, results obtained from safety testing of novel fluoroquinolones at high concentrations indicate that point mutations may also occur in mammalian cells. Recent data provide evidence for translesion synthesis catalysed by error-prone repair polymerases as a damage-tolerance repair mechanism of DSBs in eukaryotic cells. This paper discusses possible roles of different mechanisms for the repair of DSBs operating in both eukaryotic and prokaryotic cells that result in recombinational rearrangements, deletions/insertions as well as point mutations.

Chromosome instability in Mediterranean Italian buffaloes affected by limb malformation (transversal hemimelia)

Albarella, S., Ciotola, F., Dario, C., Iannuzzi, L., Barbieri, V., Peretti, V. — Mon, 02 Nov 2009 03:04:52 PST

For several years, a genetic disease called transversal hemimelia (TH), also known as congenital amputation, has been spreading in Mediterranean Italian buffalo. TH is characterized by the lack of limb distal structures, normally developing proximally to the malformed limb and being amputated at different points distally. A sample of 13 animals affected by TH was examined using the chromosome aberration (CA) test to better characterize chromosome instability already emerging in a preliminary study where we found a significantly higher difference (P < 0.001) in the mean rate of sister chromatid exchange/cell (8.80 ± 3.19) performed in 10 malformed animals, when compared with the control (6.61 ± 2.73). The percentage of aneuploid cells was higher in animals with TH (12.76) than in control animals (7.85). Mean gaps are greater in cells of animals with TH (6.62 ± 2.38) than those found in the control (2.86 ± 1.01) and similar results were obtained in chromatid breaks (0.13 ± 0.31 and 0.07 ± 0.06, respectively), chromosome breaks (0.11 ± 0.27 and 0.06 ± 0.13, respectively) and CAs excluding gaps (0.24 ± 0.47 and 0.13 ± 0.18, respectively). All these differences are statistically highly significant (P < 0.001).

Relationship between TP53 tumour suppressor gene mutations and smoking-related bulky DNA adducts in a lung cancer study population from Hungary

Mon, 02 Nov 2009 03:04:52 PST

Lung cancer rate in Hungary is one of the highest in the world among men and also very high among women, for reasons not clearly understood yet. The aim of the study was to explore characteristics of DNA damage and TP53 gene mutations in lung cancer from Hungary. Tissue samples from 104 lung resections for lung cancer patients, both men and women, operated on for non-small cell lung cancer, specifically, primary squamous cell carcinoma or adenocarcinoma were studied. Of the cases, 37% smoked up to the surgery, 24% stopped smoking within 1 year before the surgery, 26% stopped smoking more than a year before the surgery and 13% never smoked. TP53 mutations were detected by denaturant gradient gel electrophoresis, automated capillary electrophoresis single-strand conformation polymorphism and sequencing. Bulky DNA adduct levels were determined by ³²P-post-labelling in non-tumorous lung tissue. In total, 45% (47/104) of the cases carried TP53 mutation. The prevalence of TP53 mutations was statistically significantly associated with duration of smoking, tumour histology and gender. Smokers had approximately twice as high bulky adduct level as the combined group of former- and never-smokers (10.9 ± 6.5 versus 5.5 ± 3.4 adducts/10⁸ nucleotides). The common base change G -> T transversion (8/43; 19%) was detected exclusively in smokers. For the first time, we demonstrate that most carriers of G -> T transversions had also a high level of bulky DNA adducts in their non-tumourous lung tissue. Our study provides evidence for a high burden of molecular alterations occurring concurrently in the lung of lung cancer patients.

Signalling pathways involved in 1-nitropyrene (1-NP)-induced and 3-nitrofluoranthene (3-NF)-induced cell death in Hepa1c1c7 cells

Mon, 02 Nov 2009 03:04:52 PST

We previously reported that 1-nitropyrene (1-NP) and 3-nitrofluoranthene (3-NF) elicited apoptotic cell death as well as non-apoptotic programmed cell deaths (PCDs) with paraptotic and necroptotic characteristics, respectively. In the present study, we have further confirmed and extended these findings. Flow cytometric analyses of 1-NP-exposed/3NF-exposed Hepa1c1c7 cells revealed that caspase-3 was only activated in the subpopulation of cells corresponding to that with classic apoptotic morphology. Immunocytochemical analysis indicated that leucocyte elastase inhibitor-derived DNaseII (LEI/L-DNaseII), apoptosis-inducing factor (AIF) and endonuclease G (EndoG) were more clearly translocated to the nucleus following 3-NF exposure than after 1-NP. These 3-NF-induced changes in AIF and EndoG translocation were reduced by necrostatin-1, an inhibitor of necroptotic cell death. Both compounds lead to accumulation of lipid droplets and induced DNA damage. Activation of checkpoint kinase (CHK) 1 and H2AX, but not ataxia telangiectasia mutated and CHK2, were observed. Furthermore, inhibition of p53 using pifithrin- reduced the cell death induced by both compounds, suggesting a role of DNA damage/CHK1/p53 pathway in the death process. 1-NP-induced cell death was in addition characterized by increased oxidative damage and intracellular accumulation of Ca²⁺. These findings further support the notion that 1-NP elicited apoptotic cell death and PCD with paraptotic characteristics, while 3-NF induced apoptosis and a PCD with necroptotic features.

Cytoprotection against Cr6+-induced DNA damage by alpha-lipoic acid: implications in reducing occupational cancer risk

Kumar, S., Budhwar, R., Nigam, A., Priya, S. — Mon, 02 Nov 2009 03:04:52 PST

Alpha-lipoic acid (LA), the metabolic antioxidant, was evaluated for its potential to protect against Cr⁶⁺-induced DNA damage. Potassium dichromate was administered to Swiss albino mice orally ad libitum at the doses of 5, 10 or 25 mg/kg body weight in drinking water to set DNA damage in cells, which was characterized in mouse peripheral blood mononuclear cells and bone marrow cells using single-cell gel electrophoresis and analyses of generated comets for Tail moment, Tail DNA and Tail length. DNA damage was dose dependent. Cytoprotection by LA was remarkable. LA (5, 10 and 25 mg/kg body weight intraperitoneally) in pre-, co- and post-toxicant administration schedule abrogated DNA damage substantially in both cell types. Protection by LA was also dose dependent. LA annulled DNA damage by Cr⁶⁺ in plasmid relaxation assay. A negligible DNA damage resulted during interaction of Cr⁶⁺ and LA. Compared to ascorbate, LA emerged as a better antioxidant and least DNA damaging. In conclusion, our study advocated an experimental therapeutic research potential in LA against Cr⁶⁺-induced DNA damage for reduction of occupational cancer risk in humans.

Ethanolic extract of Casearia sylvestris and its clerodane diterpen (caseargrewiin F) protect against DNA damage at low concentrations and cause DNA damage at high concentrations in mice's blood cells

Mon, 02 Nov 2009 03:04:52 PST

Casearia sylvestris is used in Brazil as a popular medicine to treat ulcer, inflammation and tumour. Caseargrewiin F is a clerodane diterpene isolated from the ethanolic leaf extract of C.sylvestris. The aim of the study was to assess the capacity of the ethanolic extract of C.sylvestris leaves and caseargrewiin F to protect DNA and verify if both the compounds cause some DNA damage, using the micronucleus (MN) test and comet assay in mice. Balb-C mice were treated with the extract [3.13, 6.25, 12.5, 25, 50 and 75 mg/kg body weight (b.w.)] and caseargrewiin F (0.16, 0.32, 0.63, 1.3, 2.5 and 3.8 mg/kg b.w.) for 14 days. On day 15, DNA damage was induced by intra-peritoneal (i.p.) injection of cyclophosphamide (CP) (i.p.) at 50 mg/kg b.w. after the MN test and comet assay were performed. A protective effect of ethanolic extract was observed in MN test (6.25 and 12.5 mg/kg b.w.) and the comet assay (3.13 and 6.25, 12.5 and 25 mg/kg b.w.). Caseargrewiin F showed protective effect at 0.63, 1.3 and 2.5 mg/kg b.w. only in comet assay. We also tested the ability of compounds of C.sylvestris to induce MN and to increase the comet assay tail moment. The experimental design was similar to the DNA protection assay except that in test groups we omitted the CP challenge. We observed increased damage at 50 and 75 mg/kg b.w. of ethanolic extract of C.sylvestris and caseargrewiin F at 3.18 mg/kg b.w. in both the MN test and comet assay. We conclude that ethanolic extract of C. sylvestris and caseargrewiin F can protect cells against DNA damage induced by CP at low concentrations, but at high concentrations these compounds also induce DNA damage.

The DNA repair gene APE1 T1349G polymorphism and cancer risk: a meta-analysis of 27 case-control studies

Gu, D., Wang, M., Wang, M., Zhang, Z., Chen, J. — Mon, 02 Nov 2009 03:04:53 PST

Published data regarding the association between the apurinic/apyrimidinic endonuclease 1 (APE1) T1349G (Asp148Glu) polymorphism and cancer risk show inconclusive results. To derive a more precise estimation of the relationship, we performed a meta-analysis of 27 published studies that included 12 432 cancer cases and 17 349 controls. We used odds ratios (ORs) and 95% confidence intervals (CIs) to evaluate the strength of the associations. The overall results suggested that the variant genotypes were associated with a moderately increased risk of all cancer types (OR = 1.09, 95% CI = 1.01–1.18 for TG versus TT; OR = 1.08, 95% CI = 1.00–1.18 for GG/TG versus TT). In the stratified analyses, the risk remained for studies of colorectal cancer, European populations and population-based studies. Although some modest bias could not be eliminated, this meta-analysis supported that the APE1 T1349G polymorphism is a low-penetrance risk factor for cancer development.

In pancreatic ductal adenocarcinoma blood concentrations of some organochlorine compounds and coffee intake are independently associated with KRAS mutations

Mon, 02 Nov 2009 03:04:53 PST

While KRAS activation is a fundamental initiating event in the aetiopathogenesis of pancreatic ductal adenocarcinoma (PDA), environmental factors influencing the occurrence and persistence of KRAS mutations remain largely unknown. The objective was to test the hypothesis that in PDA there are aetiopathogenic relationships among concentrations of some organochlorine compounds (OCs) and the mutational status of the KRAS oncogene, as well as among the latter and coffee intake. Incident cases of PDA were interviewed and had blood drawn at hospital admission (N = 103). OCs were measured by high-resolution gas chromatography with electron capture detection. Cases whose tumours harboured a KRAS mutation had higher concentrations of p,p'-dichlorodiphenyltrichloroethane (DDT), p,p'-dichlorodiphenyldichloroethene (DDE) and polychlorinated biphenyls (PCBs) 138, 153 and 180 than cases with wild-type KRAS, but differences were statistically significant only for p,p'-DDT and PCBs 138 and 153. The association between coffee intake and KRAS mutations remained significant (P-trend < 0.015) when most OCs where accounted for. When p,p'-DDT, PCB 153, coffee and alcohol intake were included in the same model, all were associated with KRAS (P = 0.042, 0.007, 0.016 and 0.025, respectively). p,p'-DDT, p,p'-DDE and PCB 138 were significantly associated with the two most prevalent KRAS mutations (Val and Asp). OCs and coffee may have independent roles in the aetiopathogenesis of PDA through modulation of KRAS activation, acquisition or persistence, plausibly through non-genotoxic or epigenetic mechanisms. Given that KRAS mutations are the most frequent abnormality of oncogenes in human cancers, and the lifelong accumulation of OCs in humans, refutation or replication of the findings is required before any implications are assessed.

Abstracts of the 32nd Annual Meeting of the United Kingdom Environmental Mutagen society, 12-15 July 2009 at the University of Leeds, UK

Mon, 02 Nov 2009 03:04:53 PST