Mutagenesis - current issue

Confounding experimental considerations in nanogenotoxicology

2009-06-25

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

2009-06-25

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. — 2009-06-25

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

2009-06-25

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. — 2009-06-25

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. — 2009-06-25

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

2009-06-25

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

2009-06-25

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. — 2009-06-25

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. — 2009-06-25

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. — 2009-06-25

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. — 2009-06-25

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.