Ricardo Marcos

Automated image analysis of cytokinesis-blocked micronuclei : an adapted protocol and a validated scoring procedure for biomonitoring

Micronuclei (MN) frequencies in peripheral blood lymphocytes have been used worldwide as a biomarker of chromosomal damage for genotoxicity testing and biomonitoring studies. Automation of MN analysis would provide faster and more reliable results with minimizing subjective MN identification. We developed an automated facility for the scoring of the in vitro MN cytokinesis-block assay for biomonitoring on Giemsa-stained slides, fulfilling the following criteria: applicable to the cytokinesis-block micronucleus methodology, discriminating between mono-, bi- and polynucleated cells, MN scoring according to HUMN scoring criteria, false-negative MN rate <10% and false-positive (FP) MN rate <1%. We first adapted the slide preparation protocol to obtain an optimal cell density and dispersion, which is important for image analysis. We developed specific algorithms starting from the cell as a detection unit. The whole detection and scoring process was separated into two distinct steps: in the first step, the cells and nuclei are detected; then, in the second step, the MN are searched for in the detected cells. Since the rate of FP MN obtained by the automatic analysis was in the range of 0.5-1.5%, an interactive visual validation step was introduced, which is not time consuming and allows quality control. Validation of the automated scoring procedure was undertaken by comparing the results of visual and automated scoring of micronucleated mono- and binucleated cells in human lymphocytes induced by two clastogens (ionizing radiation and methyl methane-sulphonate), two aneugens (nocodazole and carbendazim) and one apoptogen (staurosporine). Although the absolute MN frequencies obtained with automated scoring were lower as compared to those detected by visual scoring, a clear dose response for MNBN frequencies was observed with the automated scoring system, indicating that it is able to produce biologically relevant and reliable results. These observations, together with its ability to detect cells, nuclei and MN in accordance with the HUMN scoring criteria, confirm the usability of the automated MN analysis system for biomonitoring.

The Effects of GSTM1 and GSTT1 Polymorphisms on Micronucleus Frequencies in Human Lymphocytes In vivo

The influence of genetic polymorphisms in GSTM1 and GSTT1 genes on micronucleus frequencies in human peripheral blood lymphocytes was assessed through a pooled analysis of data from seven laboratories that did biomonitoring studies using the in vivo cytokinesis-block micronucleus assay. A total of 301 nonoccupationally exposed individuals (207 males and 94 females) and 343 workers (237 males and 106 females) occupationally exposed to known or suspected genotoxic substances were analyzed by Poisson regression. The results of the pooled analysis indicate that the GSTT1 null subjects had lower micronucleus frequencies than their positive counterparts in the total population (frequency ratio, 0.55; 95% confidence interval, 0.33-0.89). The protective effect of this genotype is reversed with increasing age, with a frequency ratio of 1.33 (95% confidence interval, 1.06-1.68) in subjects aged 60 years. A significant overall increase in micronucleus frequency with age and gender (P < 0.001 and P = 0.024, respectively) was observed, females having higher micronucleus frequencies than males, when occupationally exposed (P = 0.002). Nonoccupationally exposed smokers had lower micronucleus frequencies than nonsmokers (P = 0.001), whereas no significant difference in micronucleus level was observed between smokers and nonsmokers in the occupationally exposed group (P = 0.79). This study confirms that pooled analyses, by increasing the statistical power, are adequate for assessing the involvement of genetic variants on genome stability and for resolving discrepancies among individual studies. (Cancer Epidemiol Biomarkers Prev 2006;15(5):1038–42)

Genotoxic analysis of silver nanoparticles in Drosophila

Abstract Health risk assessment of nanomaterials is an emergent field, genotoxicity being an important endpoint to be tested. Since in vivo studies offer many advantages, such as the study of the bioavailability of nanomaterials to sensitive target cells, we propose Drosophila as a useful model for the study of the toxic and genotoxic risks associated with nanoparticle exposure. In this work we have carried out a genotoxic evaluation of silver nanoparticles in Drosophila by using the wing somatic mutation and recombination test. This test is based on the principle that loss of heterozygosis and the corresponding expression of the suitable recessive markers, multiple wing hairs and flare-3, can lead to the formation of mutant clones in larval cells, which are expressed as mutant spots on the wings of adult flies. Silver nanoparticles were supplied to third instar larvae at concentrations ranging from 0.1–10 mM. The results showed that small but significant increases in the frequency of total spots were observed, thus indicating that silver nanoparticles were able to induce genotoxic activity in the wing spot assay of D. melanogaster, mainly via the induction of somatic recombination. These positive results obtained with silver nanoparticles contrast with the negative findings obtained when silver nitrate was tested.

Arsenic induces DNA damage in environmentally exposed Mexican children and adults. Influence of GSTO1 and AS3MT polymorphisms

Inorganic arsenic (i-As) is an environmental carcinogen to which millions of people are chronically exposed mainly via drinking water. In this study, we used the comet assay to evaluate DNA damage in i-As-exposed inhabitants of the north of Mexico. The environmental monitoring and the exposure assessment were done by measuring both drinking water arsenic (As) content and total urinary As. In addition, the studied population was genetically characterized for four different glutathione S-transferase omega1 (GSTO1) polymorphisms (Ala140Asp, Glu155del, Glu208Lys, and Ala236Val) and the As (+3 oxidation state) methyltransferase (AS3MT) Met287Thr polymorphism to determine whether such variants influence As-related genotoxicity. As content in the drinking water of the population was found to range between 1 and 187 microg/l, with a mean concentration value of 16 microg/l. The total urinary As content of the exposed individuals was found to be correlated with the As content in drinking water, and subjects were classified as low (< 30 microg As/g creatinine), medium (31-60 microg As/g creatinine), and highly exposed (> 61 microg As/g creatinine). A positive association was found between the level of exposure and the genetic damage measured as percentage of DNA in tail (p < 0.001), and AS3MT Met287Thr was found to significantly influence the effect (p < 0.034) among children carrying the 287Thr variant allele. Altogether, our results evidenced that people living in As-contaminated areas are at risk and that AS3MT genetic variation may play an important role modulating such risk in northern Mexico, especially among children.

High arsenic metabolic efficiency in AS3MT287Thr allele carriers.

Objectives Epidemiological data indicate the existence of wide interindividual differences in arsenic metabolism. It has recently been shown that arsenic(III)methyltransferase (AS3MT) enzyme catalyses the methylation of arsenite and monomethylarsonous acid (MMA). Thus, genetic variations in the AS3MT gene could explain, at least partly, the interindividual variation in the response to arsenic exposure. In an earlier study, we have demonstrated that the AS3MT Met287Thr (C/T) polymorphism affected the urinary arsenic profile in a Chilean group of men (n=50) occupationally exposed to arsenic. Methods To confirm, the influence of the Met287Thr polymorphism in the metabolism of arsenic, a total of 207 Chilean men working at the copper industry were genotyped and their urinary profiles determined. Results The results confirm that Met287Thr polymorphism does influence arsenic metabolism in this population. Those carriers of the variant (287Thr) had a higher methylation efficiency, excreting 4.63% more MMA in urine (P=0.0007) and presenting a 2.98 times higher odd of excreting levels of MMA over the standard (P=0.011) than the participants homozygous for the normal allele. Conclusion We can conclude that individuals with the 287Thr variant display increased arsenic methylation; thus, those participants might be at increased risk for the toxic and genotoxic effects of arsenic exposure.

Effects in the comet assay of storage conditions on human blood

The Comet assay is a rapid and sensitive method for analyzing single cells for DNA damage. Using human lymphocytes, the assay is particularly useful for human monitoring studies, as well as for in vitro genotoxicity testing of chemicals. In such studies, it is not always possible to collect and process matched samples on the same day as the blood is taken. It would be useful if some samples could be stored and examined at a different time, without loss of viability or other factors affecting responses. It is thus important to understand the effects of storage conditions on blood to be used in such studies and how exposure or treatment might modify such responses. In a joint study in two laboratories, blood was taken from various donors and stored under different conditions. It was examined on day 1 (day on which sample was taken) and days 2, 3, 4, 5, or 8 at room temperature, 4 degrees C, or -20 degrees C. Cells were treated after storage (from day 2 onward) with bleomycin (BLM) and ethylnitrosourea (ENU). The data were analyzed either by eye (classifying cells with different categories of damage) or by using a computerized image analysis system (Kinetic Imaging Ltd., Liverpool UK. Software Package Comet 3.0) where the tail moment, which is considered to be a sensitive measurement, has been analyzed. There was no loss of cell viability at 4 degrees C or room temperature up to 8 days when measured by trypan blue dye exclusion. Findings suggest that on days 1-4 for the untreated samples at room temperature or 4 degrees C there were no biologically meaningful changes in both the different categories of cell damage and tail moment data. In treated cultures up to day 4, either at room temperature or at 4 degrees C, responses were only minimally affected and changes were considered not to be of biological significance. However, there was slightly less variability between samples at 4 degrees C than at room temperature in one laboratory. The reverse was true in the other. This would suggest that samples can probably be stored up to day 4 at 4 degrees C or room temperature without any untoward effects. Provided samples can be processed within this 4-day time frame, it would not seem necessary to cryopreserve samples at -196 degrees C.

Cytogenetic damage after 131-iodine treatment for hyperthyroidism and thyroid cancer

Abstract. To detect the incidence and persistence of potential chromosome damage induced by iodine-131 therapy, we applied the cytokinesis-block micronucleus assay to peripheral blood lymphocytes from hyperthyroidism and thyroid cancer patients treated with 131I. Two groups of patients were evaluated in a longitudinal study; one group was composed of 47 hyperthyroid patients and the other of 39 thyroid cancer patients. In the hyperthyroidism group, the micronuclei frequency was determined before 131I therapy and 1 week, 1 month and 3 months after it. Furthermore, an additional sample was taken from a subgroup of 17 hyperthyroidism patients 6 months after treatment. In the thyroid cancer group, the analysis was also conducted over time, and four samples were studied: before treatment and 1 week, 6 months and 1 year later. Simultaneously, a cross-sectional study was performed with 70 control subjects and 54 thyroid cancer patients who had received the last therapeutic dose 1–6 years before the present study. In the hyperthyroidism group a significant increase in the micronuclei average was found over time. In the sample obtained 6 months after therapy, the micronuclei mean frequency was practically the same as in the sample taken 3 months before. In the thyroid cancer group a twofold increase in the frequency of micronuclei was seen 1 week after therapy. Although this value decreased across time, the micronuclei frequency obtained 1 year after 131I therapy remained higher than the value found before it. Concerning the data from the cross-sectional study, a significant increase in the frequency of micronuclei was detected in the subgroup of thyroid cancer patients treated between 1 and 3 years before the current study. These results indicate that exposure to 131I therapy induces chromosome damage in peripheral lymphocytes and that the cytokinesis-block micronucleus assay is sensitive enough to detect the genetic damage by exposure to sufficiently high levels of radiation from internal radioactive sources.

Metabolic profile in workers occupationally exposed to arsenic: role of GST polymorphisms.

Arsenic is a well-known human carcinogen with a ubiquitous distribution in the natural environment. Chronic exposure to inorganic arsenic involves a biotransformation process that leds to the main excretion of organic methylated metabolites, such as monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), as well as the parental inorganic species. Interindividual variation in arsenic metabolism has been extensively reported, and polymorphisms in genes involved in such process could be related to changes in the arsenic excretion profile and the response to chronic exposures. Our analysis of the metabolic profiles in three groups of workers exposed to different arsenic exposure levels showed high amounts of inorganic arsenic and MMA in the most-exposed workers versus the least-exposed workers, in whom high amounts of DMA were observed. With respect to the role of different genetic polymorphisms in the glutathione S-transferase (GST) genes in the modulation of the urinary profiles, for the overall population only a tendency was just observed between GSTM1 null and MMA excretion as well as between GSTP1 val/val and DMA excretion.

Use of the drosophila wing spot test in the genotoxicity testing of different herbicides.

Four herbicides, namely propanil, maleic hydrazide, glyphosate, and 2,4,5‐trichlorophenoxyacetic acid (2,4,5‐T), were investigated for genotoxicity in the wing spot test of Drosophila melanogaster. The herbicides were administered by chronic feeding to 3‐day‐old larvae. Two different crosses, a standard (ST) and a high‐bioactivation (HB) cross, involving the flare‐3 (flr3) and the multiple wing hairs (mwh) markers, were used. The HB cross uses flies characterized by an increased cytochrome P‐450–dependent bioactivation capacity, which permits a more efficient biotransformation of promutagens and procarcinogens. In both crosses, the wings of the two types of progeny, which are inversion‐free marker heterozygotes and balancer heterozygotes, were analyzed. Maleic hydrazide and glyphosate proved to be more genotoxic in the ST cross, whereas propanil appeared to be slightly more genotoxic in the HB cross. On the other hand, the herbicide 2,4,5‐T increased the mutation frequency for only the small single spots in the ST cross. Environ. Mol. Mutagen. 36:40–46, 2000.

Proposal of an in vivo comet assay using haemocytes of Drosophila melanogaster

This study presents the first application of an in vivo alkaline comet assay using haemocytes of Drosophila melanogaster larvae. These cells, which play a role similar to that of mammalian blood, can be easily obtained and represent an overall exposure of the treated larvae. To validate the assay, we evaluated the response of these cells to three well‐known mutagenic agents: ethyl methanesulfonate (EMS), potassium dichromate (PD), and gamma radiation (γ‐irradiation). Third‐instar Drosophila larvae were exposed to different concentrations of EMS (1, 2, and 4 mM) and PD (0.5, 1, and 2.5 mM) and to different doses of γ‐irradiation (2, 4, and 8 Gγ). Subsequently, haemolymph was extracted from the larvae, and haemocytes were isolated by centrifugation and used in the comet assay. Haemocytes exhibited a significant dose‐related increase in DNA damage, indicating that these cells are clearly sensitive to the treatments. These results suggest that the proposed in vivo comet test, using larvae haemocytes of D. melanogaster, may be a useful in vivo assay for genotoxicity assessment. Environ. Mol. Mutagen., 2011.