Mutagenesis - recent issues

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

Increasing the resolution of the comet assay using fluorescent in situ hybridization--a review

Shaposhnikov, S., Frengen, E., Collins, A. R. — Fri, 28 Aug 2009 10:40:08 PDT

The comet assay (single-cell gel electrophoresis) is now the most popular method for measuring low levels of damage in cellular DNA. Cells are embedded in agarose on a microscope slide and lysed to produce nucleoids of supercoiled DNA attached to the nuclear matrix. Breaks in the DNA relax the supercoiling and allow DNA loops to expand, and on electrophoresis to move towards the anode, giving the appearance of a comet tail. The % of DNA in the tail reflects the break frequency. Digestion of nucleoid DNA with lesion-specific endonucleases extends the usefulness of the method to investigate different kinds of damage. DNA repair can be studied by treating cells with a genotoxic agent, incubating them and using the comet assay to follow the removal of the damage. An important feature of the assay is that damage is detected at the level of individual cells. The comet assay can be combined with fluorescent in situ hybridization, using labelled probes to particular DNA sequences, and DNA damage and repair can be examined at an even finer level of resolution. Here, we provide a general review of the technique, answer some technical and theoretical questions and give examples of applications of the method.

Is tamoxifen a genotoxic carcinogen in women?

Brown, K. — Fri, 28 Aug 2009 10:40:08 PDT

The anti-oestrogen tamoxifen, which is widely used in the treatment of breast cancer and is also approved for the prevention of this disease, causes an increased incidence of endometrial cancer in women. The ability of tamoxifen to induce endometrial tumours and the underlying carcinogenic mechanisms have been a subject of intense interest over the last ~20 years. They are central to the assessment of risks versus benefits for the drug, especially in a chemopreventive context. This review outlines the clinical justification for using tamoxifen as a chemopreventive agent and describes the genotoxic mechanisms considered responsible for tamoxifen-induced tumours in rat liver and how these might relate to women. In rat hepatic tissue, tamoxifen is metabolically activated via -hydroxylation and sulphate conjugation to give a reactive species that binds to DNA predominantly at the N²-position of guanine, producing pro-mutagenic lesions. Whether tamoxifen–DNA adducts contribute similarly to the development of cancers in women depends on whether they can be formed in human tissues and the type of specific molecular and cellular responses they induce, if present. This review discusses the current data relating to these issues and highlights areas where further research is needed.

Flavonoids inhibit the genotoxicity of hydrogen peroxide (H2O2) and of the food mutagen 2-amino-3-methylimadazo[4,5-f]-quinoline (IQ) in lymphocytes from patients with inflammatory bowel disease (IBD)

Najafzadeh, M., Reynolds, P. D., Baumgartner, A., Anderson, D. — Fri, 28 Aug 2009 10:40:08 PDT

Inflammatory bowel disease (IBD) including Crohn's disease (CD) and ulcerative colitis (UC) is a chronic inflammatory gastrointestinal autoimmune condition with an inappropriate immune response. We investigated DNA damage induced in vitro in lymphocytes from IBD patients caused by oxidative stress through H₂O₂ and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and whether the plant flavonoids, quercetin and epicatechin, found in fruits, tea and soybeans could effectively reduce such stress. Lymphocytes from IBD patients and healthy volunteers were treated with 50 µg/ml H₂O₂ or IQ in the presence of quercetin (0–250 µg/ml) or epicatechin (0–100 µg/ml). Flavonoid supplementation (250 µM quercetin or 100 µM epicatechin) caused an overall significant decrease of induced DNA damage resulting in a 48.6% (P < 0.001) reduction of H₂O₂-induced and a 43% (P < 0.001) reduction of IQ-induced DNA damage within the patient groups; for the control groups, reductions in DNA damage were 35.2 and 57.1%, respectively (both, P < 0.001). There was less induced DNA damage within lymphocytes from UC patients compared to CD patients for both series of experiments (H₂O₂ and quercetin, IQ and epicatechin). In conclusion, flavonoids dramatically reduced oxidative stress in vitro in lymphocytes from IBD patients and healthy individuals. Thus, flavonoids could be very effective in the treatment of oxidative stress and encouraged in the diet of IBD patients.

Plumbagin induces cell death through a copper-redox cycle mechanism in human cancer cells

Fri, 28 Aug 2009 10:40:08 PDT

Plumbagin, a naphthoquinone derived from the medicinal plant Plumbago zeylanica has been shown to exert anticancer and anti-proliferative activities in cells in culture as well as animal tumor models. In our previous paper, we have reported the cytotoxic action of plumbagin in plasmid pBR322 DNA as well as human peripheral blood lymphocytes through a redox mechanism involving copper. Copper has been shown to be capable of mediating the action of several plant-derived compounds through production of reactive oxygen species (ROS). The objective of the present study was to determine whether plumbagin induces apoptosis in human cancer cells through the same mechanism which we proposed earlier. Using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay, 3-(4,5-B-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for cell growth inhibition, histone/DNA ELISA, homogeneous caspase-3/7 assay for apoptosis as well as alkaline comet assay for DNA single-strand breaks detection in this report, we confirm that plumbagin causes effective cell growth inhibition, induces apoptosis and generates single-strand breaks in cancer cells. Incubation of cancer cells with scavengers of ROS and neocuproine inhibited the cytotoxic action of plumbagin proving that generation of ROS and Cu(I) are the critical mediators in plumbagin-induced cell growth inhibition. This study is the first to investigate the copper-mediated anticancer mechanism of plumbagin in human cancer cells and these properties of plumbagin could be further explored for the development of anticancer agents with higher therapeutic indices, especially for skin cancer.

The rat bone marrow micronucleus test--study design and statistical power

Hayes, J., Doherty, A. T., Adkins, D. J., Oldman, K., O'Donovan, M. R. — Fri, 28 Aug 2009 10:40:08 PDT

Although the rodent bone marrow micronucleus test has been in routine use for over 20 years, little work has been published to support its experimental design and all this has used the mouse rather than the rat. When it was decided to change the strain of rat routinely used in this laboratory to the Han Wistar, a preliminary study was performed to investigate the possible factors influencing experimental variability and to use statistical tools to examine possible study designs. Subsequently, a historical database comprising of vehicle controls accumulated from 65 studies was used to establish test acceptance criteria and a strategy for analysing equivocal results. The following conclusions were made: (i) no statistically significant differences were observed in experimental variability within or between control animals; although not statistically significant, the majority of experimental variability seen was found to be between separate counts on the same slide, with minimal differences found between duplicate slides from the same rat or between individual rats; (ii) power analyses showed that, if an equivocal result is obtained after scoring 2000 immature erythrocytes (IE), it is appropriate to re-code the slides and score an additional 4000 IE, i.e. analysing a total of 6000 IE; no meaningful increase in statistical power is gained by scoring >6000 IE; this is consistent with the variability observed between separate counts on the same slide; (iii) there was no significant difference between the control micronucleated immature erythrocyte (MIE) values at 24 and 48 h after dosing or between males and females; therefore, if an unusually low control value at either time point results in apparent small increases in MIE in a treated group, it is valid to pool control values from both time points for clarification and (iv) similar statistical power can be achieved by scoring 2000 IE from seven rats or 4000 IE from five rats, respectively. However, this is based only on control animals and does not consider possible differences in responses between animals to treatment with a potential genotoxin. In order to minimize the possible influence of responders and non-responders, the preferred study design in this laboratory is to score 2000 IE from groups of seven rats. Study data obtained over time confirmed observations made in the control study. Also from an ethical viewpoint, clarifying equivocal responses by combining control data from the 24- and 48-h time points and/or increasing the number of IE scored per animal has minimized the numbers of repeat studies necessary to determine the genotoxic status of a novel compound. However, before any laboratory can use these procedures, experimental data must be generated to demonstrate their validity.

RUNX2 mutations in Chinese patients with cleidocranial dysplasia

Fri, 28 Aug 2009 10:40:08 PDT

Cleidocranial dysplasia (CCD) is an autosomal dominant bone disease in humans caused by haploinsufficiency of the RUNX2 gene. The RUNX2 has two major isoforms derived from P1 and P2 promoters. Over 90 mutations of RUNX2 have been reported associated with CCD. In our study, DNA samples of nine individuals from three unrelated CCD families were collected and screened for all exons of RUNX2 and 2 kb of P1 and P2 promoters. We identified two point mutations in the RUNX2 gene in Case 1, including a nonsense mutation (c.577C>T) that has been reported previously and a silent substitution (c.240G>A). In vitro studies demonstrated that c.577C>T mutation led to truncated RUNX2 protein production and diminished stimulating effects on mouse osteocalcin promoter activity when compared with full-length Runx2-II and Runx2-I isoforms. These results confirm that loss of function RUNX2 mutation (c.577C>T) in Case 1 family is responsible for its CCD phenotype.

Inter-laboratory evaluation of the bioluminescent Salmonella reverse mutation assay using 10 model chemicals

Fri, 28 Aug 2009 10:40:08 PDT

We have developed the bioluminescent Salmonella reverse mutation assay as a tool for detecting mutagenicity applicable for high-throughput screening of new chemicals. In this study, we report the inter-laboratory evaluation of the assay using 10 model chemicals in five independent laboratories located in the USA (Groton, CT; Cambridge, MA and La Jolla, CA), Europe (Sandwich, Kent, UK) and Asia (Nagoya, Japan). The studies were performed in blinded fashion in all sites except for Groton and Cambridge laboratories. The chemicals were tested in at least three independent experiments using strains TA98-lux and TA100-lux in the presence and absence of metabolic activation. The results were statistically evaluated and compared to published results. Seven of the 10 compounds were positive in either TA98-lux and/or TA100-lux in the presence or absence of metabolic activation. The positive compound set included: nitrofurazone, 3-3'-dimethoxybenzidine, benzo[a]pyrene, 1,4-benzoquinone dioxime, 2-amino-5-nitrophenol, 2-bromo-4,6-dinitroaniline and busulfan. The remaining three compounds, namely, anthracene, crystal violet and benzyl chloride were negative in both Salmonella strains. Final results for individual compounds yielded 100% agreement among the laboratories and published data. Detailed comparison of all 40 individual test conditions yielded 93% (37 of 40) agreement among participating laboratories. We conclude that the bioluminescent Salmonella reverse mutation assay is a robust, accurate and economical higher throughput assay applicable for the mutagenicity screening of chemicals.

Genotoxicity and morphological transformation induced by cobalt nanoparticles and cobalt chloride: an in vitro study in Balb/3T3 mouse fibroblasts

Fri, 28 Aug 2009 10:40:08 PDT

Nanotechnology is an emerging field that involves the development, manufacture and measurement of materials and systems in the submicron to nanometer range. Its development is expected to have a large socio-economical impact in practically all fields of industrial activity. However, there is still a lack of information about the potential risks of manufactured nanoparticles for the environment and for human health. In this work, we studied the cytotoxicity, genotoxicity and morphological transforming activity of cobalt nanoparticles (Co-nano) and cobalt ions (Co²⁺) in Balb/3T3 cells. We also evaluated Co-nano dissolution in culture medium and cellular uptake of both Co-nano and Co²⁺. Our results indicated dose-dependent cytotoxicity, assessed by colony-forming efficiency test, for both compounds. The toxicity was higher for Co-nano than for Co² after 2 and 24 h of exposure, while dose–effect relationships were overlapping after 72 h. Statistically significant results were observed for Co-nano with the micronucleus test and the comet assay, while for Co²⁺ positive results were observed only with the latter. In addition, even when Co-nano was genotoxic (at >1 µM), no evident dose-dependent effect was observed. Concerning morphological transformation, we found a statistically significant increase in the formation of type III foci (morphologically transformed colonies) only for Co-nano. Furthermore, we observed a higher cellular uptake of Co-nano compared with Co²⁺.

FOXL2 gene mutations and blepharophimosis-ptosis-epicanthus inversus syndrome (BPES): a novel mutation detected in a Chinese family and a statistic model for summarizing previous reported records

Xu, Y., Lei, H., Dong, H., Zhang, L., Qin, Q., Gao, J., Zou, Y., Yan, X. — Fri, 28 Aug 2009 10:40:08 PDT

Previous studies found that the forkhead transcription factor 2 (FOXL2) gene mutations are responsible for both types of blepharophimosis–ptosis–epicanthus inversus syndrome (BPES) but have not established any systematic statistic model for the complex and even contradictory results about genotype–phenotype correlations between them. This study is aimed to find possible mutations of FOXL2 gene in a Chinese family with type II BPES by using DNA sequencing and to further clarify genotype–phenotype correlations between FOXL2 mutations and BPES by using a systematic statistical method, namely Multifactor Dimensionality Reduction (MDR). A novel mutation (g.933_965dup) which could result in an expansion of the polyalanine (polyAla) tract was detected in all patients of this family. MDR analysis for intragenic mutations of FOXL2 gene reported in previous BPES studies indicated that the mutations which led to much stronger disturbance of amino acid sequence were responsible for more type I BPES, while other kinds of mutation were responsible for more type II BPES. In conclusion, the present study found a novel FOXL2 gene mutation in a Chinese BPES family and a new general genotype–phenotype correlation tendency between FOXL2 intragenic mutations and BPES, both of which expanded the knowledge about FOXL2 gene and BPES.

Analysis of 75 marketed pharmaceuticals using the GADD45a-GFP 'GreenScreen HC' genotoxicity assay

Fri, 28 Aug 2009 10:40:08 PDT

The GADD45a-GFP (GreenScreen HC) reporter assay detects genotoxic damage in the human lymphoblastoid TK6 cell line and gives positive results for all classes of genotoxin, including mutagens, aneugens and clastogens. In this study, a collection of 75 marketed pharmaceuticals were tested in the assay. Compounds in the collection represent a broad range of chemical structures, pharmacologies and therapeutic indications, including neoplasia and viral infection where positive genotoxicity results are often associated with the pharmacological activity. Based on the results of this study, two main conclusions can be drawn: (i) the GreenScreen HC is more predictive of in vivo genotoxicity (88%) and genotoxic carcinogenicity (93%) data than the any of the other regulatory in vitro genotoxicity assay and (ii) no compounds were uniquely positive in the GADD45a-GFP assay. This analysis therefore provides additional evidence to support the use of the GADD45a-GFP assay as an effective tool either in early genotoxic liability identification or non-clinical safety assessment of candidate pharmaceuticals during development.

Confounding experimental considerations in nanogenotoxicology

Thu, 25 Jun 2009 20:58:36 PDT

The development of novel nanomaterials with unique physico-chemical properties is increasing at a rapid rate, with potential applications across a broad range of manufacturing industries and consumer products. Nanomaterial safety is therefore becoming an increasingly contentious issue that has intensified over the past 4 years, and in response, a steady stream of studies focusing on nanotoxicology are emerging. However, it is becoming increasingly evident that nanomaterials cannot be treated in the same manner as chemical compounds with regards to their safety assessment, as their unique physico-chemical properties are also responsible for unexpected interactions with experimental components that generate misleading data-sets. In this report, we focus on nanomaterial interactions with colorimetric and fluorometric dyes, components of cell culture growth medium and genotoxicity assay components, and the resultant consequences on test systems are demonstrated. Thus, highlighting some of the potential confounding factors that need to be considered in order to ensure that in vitro genotoxicity assays report true biological impacts in response to nanomaterial exposure.

State of the art survey of the buccal micronucleus assay--a first stage in the HUMNXL project initiative

Thu, 25 Jun 2009 20:58:36 PDT

The study of DNA damage in exfoliated buccal cells is a minimally invasive method for monitoring populations for exposure to genotoxic agents. The presence of micronuclei (MN) and other nuclear anomalies within these cells has been shown to be associated with genetic defects in genome maintenance, accelerated ageing, genotoxic damage and some degenerative diseases. To identify important information gaps regarding these biomarkers, a new initiative was launched within the framework of the HUman MicroNucleus (HUMN) collaborative programme, the HUMN_XL project (‘XL’ designating eXfoLiated cell). An invitation to join the project was sent out together with a questionnaire to all laboratories that have published on the buccal micronucleus assay. Overall, 188 messages were delivered and 58 laboratories from 25 countries agreed to participate (43 contributing data). The questionnaire was designed to collect methodological information regarding the laboratory's performance of the assay and to assess the extent and type of epidemiological data that are routinely collected. The results provide an overview of the most commonly used methods for buccal cell collection and preparation, slide preparation, staining, scoring criteria and an evaluation of epidemiological data, including demographics, genetic background, gender, health status, occupation, exposure, lifestyle and dietary habit. According to this survey, a potential base of 15 103 subjects can be included in future pooled analyses. A number of protocol discrepancies emerged, implying that method standardization is a major priority. The results of this survey will contribute to (i) identify technical and epidemiological key variables that impact on buccal MN frequency in human populations, (ii) drive the design of future intra- and interlaboratory validation studies and (iii) determine the role of MN frequency and other biomarkers, in monitoring genomic damage and predicting cancer and other degenerative diseases.

Is habitual alcohol drinking associated with reduced electrophoretic DNA migration in peripheral blood leukocytes from ALDH2-deficient male Japanese?

Lu, Y., Morimoto, K. — Thu, 25 Jun 2009 20:58:36 PDT

Alcohol drinking-derived acetaldehyde is believed to cross-link DNA and induce sister chromatid exchanges in peripheral blood lymphocytes. However, little population data are available to illustrate effects of alcohol-derived acetaldehyde on DNA migration as assayed by the comet assay in peripheral lymphocytes. In the present study, we investigated lifestyle behaviours, including alcohol consumption, in 150 Japanese males by questionnaire, determined their aldehyde dehydrogenase 2 (ALDH2) family genotypes by polymerase chain reaction and measured the DNA migration in peripheral blood leukocytes by the alkaline comet assay. The results showed that habitual alcohol drinking is significantly negatively associated with DNA migration in peripheral blood leukocytes (r = –0.321, P = 0.005) of ALDH2-deficient, but not of ALDH2-proficient genotypes (r = 0.048, P = 0.683). The amount of pure alcohol consumed per time by the subjects showed a similar phenomenon (r = –0.257, P = 0.025 for the ALDH2-deficient, but r = –0.061, P = 0.606 for the ALDH2-proficient genotype). Further stepwise multiple regression analysis showed that alcohol drinking frequency was a significant predictor of DNA migration for subjects with ALDH2-deficient genotype, but not for subjects with ALDH2-proficient genotype. In summary, the present result suggests that frequent alcohol drinking is significantly associated with a reduced electrophoretic DNA migration in peripheral blood leukocytes from ALDH2-deficient male Japanese subjects.

The Xpc gene markedly affects cell survival in mouse bone marrow

Thu, 25 Jun 2009 20:58:36 PDT

The XPC protein (encoded by the xeroderma pigmentosum Xpc gene) is a key DNA damage recognition factor that is required for global genomic nucleotide excision repair (G-NER). In contrast to transcription-coupled nucleotide excision repair (TC-NER), XPC and G-NER have been reported to contribute only modestly to cell survival after DNA damage. Previous studies were conducted using fibroblasts of human or mouse origin. Since the advent of Xpc–/– mice, no study has focused on the bone marrow of these mice. We used carboplatin to induce DNA damage in Xpc–/– and strain-matched wild-type mice. Using several independent methods, Xpc–/– bone marrow was ~10-fold more sensitive to carboplatin than the wild type. Importantly, 12/20 Xpc–/– mice died while 0/20 wild-type mice died. We conclude that G-NER, and XPC specifically, can contribute substantially to cell survival. The data are important in the context of cancer chemotherapy, where Xpc gene status and G-NER may be determinants of response to DNA-damaging agents including carboplatin. Additionally, altered cell cycles and altered DNA damage signalling may contribute to the cell survival end point.

Enhancement of the recombinagenic and mutagenic activities of bleomycin in yeast by intercalation of acridine compounds into DNA

Hoffmann, G. R., Ronan, M. V., Sylvia, K. E., Tartaglione, J. P. — Thu, 25 Jun 2009 20:58:36 PDT

Strain D7 of Saccharomyces cerevisiae was used to measure the induction by bleomycin (BLM) of mitotic recombination at the trp5 locus and point mutations at ilv1 in the presence and absence of acridine compounds. BLM is a potent mutagen and recombinagen in the D7 assay. The acridines vary, some being mutagenic or recombinagenic and others not. Combined treatments were used to distinguish whether a genetically inactive acridine has no effect on the genetic activity of BLM or modulates its action. When an acridine is itself genetically active, combined treatments were used to determine whether its effects are additive with those of BLM or whether there is interaction between the two compounds. Acridine compounds that share the ability to intercalate between the base pairs of DNA but differ in their mutagenic specificity owing to the presence of different substituent groups were analysed. Clear potentiation and synergistic interactions were detected in combined treatments with BLM and aminoacridines, nitroacridines or an acridine mustard. Potentiation and synergy were also observed in sequential exposures in which the yeast were grown in the presence of acridine compounds and then treated with BLM in the absence of free acridine. The results are consistent with an increase in BLM susceptibility conferred by acridine intercalation. It is likely that the intercalating agents increase the access of BLM to the minor groove of DNA, where it abstracts a hydrogen from the 4' position of deoxyribose, creating a free radical that is processed into strand breaks.

XRCC1 Arg399Gln, Arg194Trp and Arg280His polymorphisms in breast cancer risk: a meta-analysis

Huang, Y., Li, L., Yu, L. — Thu, 25 Jun 2009 20:58:36 PDT

X-ray repair cross-complementing group 1 (XRCC1) plays an important role in base excision and single-strand break repair, as a scaffold protein that brings together proteins of the DNA repair complex, and appears to be a candidate for cancer risk. However, studies on the association between polymorphisms in this protein and cancer have yielded conflicting results. We performed a meta-analysis to investigate the association between the breast cancer and the XRCC1 polymorphisms Arg194Trp (9411 cases and 9783 controls), Arg399Gln (22 481 cases and 23 905 controls) and Arg280His (6062 cases and 5864 controls) in different inheritance models. Our analysis suggested that Arg399Gln was associated with a trend of increased breast cancer risk when using both dominant [odds ratio (OR) = 1.06, 95% confidence interval (CI): 1.00–1.13] and recessive models (OR = 1.12, 95% CI: 1.02–1.23) to analyse the data. In ethnic subgroups and using recessive model analysis: Arg399Gln increased breast cancer risk in Asians (OR = 1.26, 95% CI: 0.96–1.64) and Africans (OR = 1.80, 95% CI: 0.97–3.32), and also while only slightly increasing the breast cancer risk in Caucasians (OR = 1.08, 95% CI: 0.95–1.22). However, Arg194Trp (recessive model, OR = 0.95, 95% CI: 0.75–1.20) and Arg280His (recessive model, OR = 1.28, 95% CI: 0.64–2.55) did not appear to be risk factors for breast cancer. Larger scale primary studies are required to further evaluate the interaction of XRCC1 polymorphisms and breast cancer risk in specific populations.

Mutagenicity testing for chemical risk assessment: update of the WHO/IPCS Harmonized Scheme

Thu, 25 Jun 2009 20:58:36 PDT

Since the publication of the International Programme on Chemical Safety (IPCS) Harmonized Scheme for Mutagenicity Testing, there have been a number of publications addressing test strategies for mutagenicity. Safety assessments of substances with regard to genotoxicity are generally based on a combination of tests to assess effects on three major end points of genetic damage associated with human disease: gene mutation, clastogenicity and aneuploidy. It is now clear from the results of international collaborative studies and the large databases that are currently available for the assays evaluated that no single assay can detect all genotoxic substances. The World Health Organization therefore decided to update the IPCS Harmonized Scheme for Mutagenicity Testing as part of the IPCS project on the Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals. The approach presented in this paper focuses on the identification of mutagens and genotoxic carcinogens. Selection of appropriate in vitro and in vivo tests as well as a strategy for germ cell testing are described.

Micronuclei in peripheral blood from patients after cytostatic therapy mainly arise ex vivo from persistent damage

Thu, 25 Jun 2009 20:58:36 PDT

The micronucleus test (MNT) is a well-established assay in genotoxicity testing and human biomonitoring. The cytokinesis-block micronucleus test (CBMNT) is the preferred method for measuring MN in cultured human lymphocytes from human subjects exposed to genotoxins. It is, however, unclear to what extent mutagen exposure either leads to the formation of MN already in vivo or to the formation of MN ex vivo during cell culture as a consequence of persisting DNA damage. To address this question, we investigated peripheral blood of 22 patients who had received cytostatic therapies including drugs with clastogenic and aneugenic effects. We also performed the MNT with blood samples from 13 healthy controls without relevant mutagen exposure. The incidence of MN was studied 24, 48 and 72 h after the start of the culture in mononuclear lymphocytes in cultures without cytochalasin B and also at 72 h in binucleated lymphocytes in the standard CBMNT. The mean frequency of binuclear cells with MN in the CBMNT was clearly increased in blood samples from patients (29.3 versus 10.2 per 1000 in controls). In contrast, mononuclear lymphocytes analysed 24 or 48 h after start of the cultures only revealed a marginal increase in MN frequencies in comparison to controls. These results suggest that mutagen exposure in vivo mainly leads to the formation of MN during ex vivo proliferation of lymphocytes as a consequence of persistent damage. Characterization of MN in binuclear lymphocytes from patients by fluorescence in situ hybridization (FISH) with a pan-centromeric probe indicated that MN arose by clastogenic and aneugenic mechanisms. A high portion of MN was relatively large and exhibited several centromere signals. If the results of this study with patients exposed to cytostatic drugs also apply to other kinds of mutagen exposure, increased MN frequencies in the CBMNT can only be expected for exposures leading to persistent damage in peripheral lymphocytes and MN formation during ex vivo lymphocyte culture.

Comparison of the Ames II and traditional Ames test responses with respect to mutagenicity, strain specificities, need for metabolism and correlation with rodent carcinogenicity

Kamber, M., Fluckiger-Isler, S., Engelhardt, G., Jaeckh, R., Zeiger, E. — Thu, 25 Jun 2009 20:58:36 PDT

The Ames II Salmonella mutagenicity assay procedure was used to test 71 chemicals, and the results were compared with those from the traditional Ames Salmonella test using the NTP database as the reference. All Ames II tests were performed using a fluctuation procedure in microplate format, using TAMix for the detection of base pair substitutions and TA98 to detect frameshift mutations. There was 84% agreement between the two procedures in identifying mutagens and non-mutagens, which is equivalent to the intra- and interlaboratory reproducibility of 87% for the traditional test. The two tests also performed similarly in their predictions of rodent carcinogenicity.

The benzene metabolite, hydroquinone and etoposide both induce endoreduplication in human lymphoblastoid TK6 cells

Ji, Z., Zhang, L., Guo, W., McHale, C. M., Smith, M. T. — Thu, 25 Jun 2009 20:58:36 PDT

Both occupational exposure to the leukemogen benzene and in vitro exposure to its metabolite hydroquinone (HQ) lead to the induction of numerical and structural chromosome changes. Several studies have shown that HQ can form DNA adducts, disrupt microtubule assembly and inhibit DNA topoisomerase II (topo II) activity. As these are potential mechanisms underlying endoreduplication (END), a phenomenon that involves DNA amplification without corresponding cell division, we hypothesized that HQ could cause END. We measured END in the human lymphoblastoid cell line, TK6, treated with HQ (0–20 µM) and etoposide (0–0.2 µM) for 48 h. Etoposide was used as a positive control as it is a topo II poison and established human leukemogen that has previously been shown to induce END in Chinese hamster ovary cells. Both HQ and etoposide significantly induced END in a dose-dependent manner (P_trend < 0.0001 and P_trend = 0.0003, respectively). Since END may underlie the acquisition of high chromosome numbers by tumour cells, it may play a role in inducing genomic instability and subsequent carcinogenesis from HQ and etoposide. In order to further explore the cytogenetic effects of HQ and etoposide, we also examined specific structural changes. HQ did not induce translocations of chromosome 11 [t(11;?)] but significantly induced translocations of chromosome 21 [t(21;?)] and structural chromosome aberrations (SCA) (P_trend = 0.0415 and P_trend < 0.0001, respectively). Etoposide potently induced all these structural changes (P_trend < 0.0001). The lack of an effect of HQ on t(11;?) and the reduced ability of HQ to induce t(21;?) and SCA, compared with etoposide, further suggests that HQ acts primarily as a topo II catalytic inhibitor rather than as a topo II poison in intact human cells.

Systematic random sampling of the comet assay

McArt, D. G., Wasson, G. R., McKerr, G., Saetzler, K., Reed, M., Howard, C. V. — Thu, 25 Jun 2009 20:58:36 PDT

The comet assay is a technique used to quantify DNA damage and repair at a cellular level. In the assay, cells are embedded in agarose and the cellular content is stripped away leaving only the DNA trapped in an agarose cavity which can then be electrophoresed. The damaged DNA can enter the agarose and migrate while the undamaged DNA cannot and is retained. DNA damage is measured as the proportion of the migratory ‘tail’ DNA compared to the total DNA in the cell. The fundamental basis of these arbitrary values is obtained in the comet acquisition phase using fluorescence microscopy with a stoichiometric stain in tandem with image analysis software. Current methods deployed in such an acquisition are expected to be both objectively and randomly obtained. In this paper we examine the ‘randomness’ of the acquisition phase and suggest an alternative method that offers both objective and unbiased comet selection. In order to achieve this, we have adopted a survey sampling approach widely used in stereology, which offers a method of systematic random sampling (SRS). This is desirable as it offers an impartial and reproducible method of comet analysis that can be used both manually or automated. By making use of an unbiased sampling frame and using microscope verniers, we are able to increase the precision of estimates of DNA damage. Results obtained from a multiple-user pooled variation experiment showed that the SRS technique attained a lower variability than that of the traditional approach. The analysis of a single user with repetition experiment showed greater individual variances while not being detrimental to overall averages. This would suggest that the SRS method offers a better reflection of DNA damage for a given slide and also offers better user reproducibility.

In vitro comet assay for DNA repair: a warning concerning application to cultured cells

Azqueta, A., Lorenzo, Y., Collins, A. R. — Thu, 25 Jun 2009 20:58:36 PDT

The comet assay (single-cell gel electrophoresis) is a sensitive and simple method for measuring DNA damage. An early modification of the assay involved the application of specific repair endonucleases to convert lesions to breaks; thus, for example, endonuclease III was used to measure oxidized pyrimidines. This concept has now been extended to produce an in vitro assay for DNA repair activity in a cell-free extract, for example from lymphocytes. The extract is incubated with substrate DNA containing specific base damage, and repair incision is detected as breaks in this DNA. We have recently been studying effects of phytochemicals in cultured cells, whether as antioxidants or as potential modulators of DNA repair. We realized that there is a need to check that observed effects that appear as an enhancement of repair (i.e. increased breaks in substrate DNA) are not simply due to a direct damaging effect of the phytochemical or to induction of non-specific nucleases. Here, we describe a rigorous approach to testing for this possibility, which we recommend to anyone carrying out similar experiments.