Exposure to PCB and p, p'-DDE in European and Inuit populations: impact on human sperm chromatin integrity

BACKGROUND: Persistent organochlorine pollutants (POP), such as polychlorinated biphenyls (PCB) and dichlorodiphenyldichloroethylene (p, p'-DDE), are widely found in the environment and considered potential endocrine-disrupting compounds (EDC). Their impact on male fertility is still unknown. METHODS: To explore the hypothesis that POP is associated with altered sperm chromatin integrity, a cross-sectional study involving 707 adult males (193 Inuits from Greenland, 178 Swedish fishermen, 141 men from Warsaw, Poland, and 195 men from Kharkiv, Ukraine) was carried out. Serum levels of 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153), as a proxy of the total PCB burden, and of p,p'-DDE were determined. Sperm chromatin structure assay (SCSA) was used to assess sperm DNA/chromatin integrity. RESULTS: We found a strong and monotonically increasing DNA fragmentation index with increasing serum levels of CB-153 among European but not Inuit men, reaching a 60% higher average level in the highest exposure group. No significant associations were found between SCSA-derived parameters and p, p'-DDE serum concentrations. CONCLUSION: These results suggest that human dietary PCB exposure might have a negative impact on the sperm chromatin integrity of adult males but additional issues, including differences in the genetic background and lifestyle habits, still need to be elucidated.     

DNA damage in human sperm is related to urinary levels of phthalate monoester and oxidative metabolites

BACKGROUND: The ubiquitous use of phthalate esters in plastics, personal care products and food packaging materials results in widespread general population exposure. In this report, we extend our preliminary study on the relationship between urinary concentrations of phthalate metabolites and sperm DNA damage among a larger sample of men and include measurements of mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), two oxidative metabolites of di-(2-ethylhexyl) phthalate (DEHP). METHODS: Among 379 men from an infertility clinic, urinary concentrations of phthalate metabolites were measured using isotope-dilution high-performance liquid chromatography-tandem mass spectrometry. Sperm DNA damage measurements, assessed with the neutral comet assay, included comet extent (CE), percentage of DNA in tail (Tail%) and tail distributed moment (TDM). RESULTS: Monoethyl phthalate (MEP), a metabolite of diethyl phthalate, was associated with increased DNA damage, confirming our previous findings. Mono-(2-ethylhexyl) phthalate (MEHP), a metabolite of DEHP, was associated with DNA damage after adjustment for the oxidative DEHP metabolites. After adjustment for MEHHP, for an interquartile range increase in urinary MEHP, CE increased 17.3% [95% confidence interval (CI) = 8.7-25.7%], TDM increased 14.3% (95% CI = 6.8-21.7%) and Tail% increased 17.5% (95% CI = 3.5-31.5%). CONCLUSIONS: Sperm DNA damage was associated with MEP and with MEHP after adjusting for DEHP oxidative metabolites, which may serve as phenotypic markers of DEHP metabolism to 'less toxic' metabolites. The urinary levels of phthalate metabolites among these men were similar to those reported for the US general population, suggesting that exposure to some phthalates may affect the population distribution of sperm DNA damage.     

Urinary levels of insecticide metabolites and DNA damage in human sperm

BACKGROUND: Members of the general population are exposed to non-persistent insecticides at low levels. The present study explored whether environmental exposures to carbaryl and chlorpyrifos are associated with DNA damage in human sperm. METHODS: Subjects (n=260) were recruited through a Massachusetts infertility clinic. Individual exposures were measured as spot urinary metabolite concentrations of chlorpyrifos [3,5,6-trichloro-2-pyridinol (TCPY)] and carbaryl [1-naphthol (1N)], adjusted using specific gravity. Sperm DNA integrity was assessed by neutral comet assay and reported as comet extent, percentage DNA in comet tail (Tail%) and tail distributed moment (TDM). RESULTS: A statistically significant increase in Tail% was found for an interquartile range (IQR) increase in both 1N [coefficient=4.1; 95% confidence interval (CI) 1.9-6.3] and TCPY (2.8; 0.9-4.6), while a decrease in TDM was associated with IQR changes in 1N (-2.2; -4.9 to 0.5) and TCPY (-2.5; -4.7 to -0.2). A negative correlation between Tail% and TDM was present only when stratified by comet extent, suggesting that Tail% and TDM may measure different types of DNA damage within comet extent strata. CONCLUSIONS: Environmental exposure to carbaryl and chlorpyrifos may be associated with increased DNA damage in human sperm, as indicated by a change in comet assay parameters.     

DNA damage assessment by comet assay of human lymphocytes exposed to jet propulsion fuels

Exposure to jet fuel damages DNA and results in a number of physiological changes in liver, lung, immune, and neurological tissue. In this study the single-cell gel electrophoresis assay or comet assay was used to compare the DNA damage in human peripheral lymphocytes produced by three jet propulsion fuels: JP-8, JP-5, and JP-8+100. These fuels consist of complex mixtures of aliphatic, aromatic, and substituted naphthalene hydrocarbons. Two exposure times were investigated which correspond to estimated occupational exposure times and concentrations of fuels were used that were based on previous fuel toxicity studies. Analysis of samples for the extent of DNA damage as determined by tail moment and percent tail DNA was performed on exposed cells following a brief recovery time. All fuels produced significant increases in DNA damage; however, only JP-8+100 was genotoxic at the lowest exposure concentration (1:500). At the highest exposure concentration (1:75), the mean tail moments for JP-8 and JP-8+100 (32.041 +/- 2.599 and 45.774 +/- 4.743, respectively) were significantly greater than for JP-5 (1.314 +/- 0.474). These results indicate that JP-8+100 is the most potent inducer of DNA damage in human peripheral lymphocytes and that both JP-8+100 and JP-8 are capable of damaging lymphocyte DNA to a greater extent than JP-5.     

In vivo exposure to microcystins induces DNA damage in the haemocytes of the zebra mussel, Dreissena polymorpha, as measured with the comet assay

The Comet assay was used to investigate the potential of the biotoxin microcystin (MC) to induce DNA damage in the freshwater zebra mussel, Dreissena polymorpha. Mussels maintained in the laboratory were fed daily, over a 21-day period, with one of four strains of the cyanobacterium, Microcystis aeruginosa. Three of the strains produced different profiles of MC toxin, while the fourth strain did not produce MCs. The mussels were sampled at 0, 7, 14, and 21 days by withdrawing haemocytes from their adductor muscle. In addition, a positive control was performed by exposing a subsample of the mussels to water containing cadmium chloride (CdCl(2)). Cell viability, measured with the Fluorescein Diacetate/Ethidium Bromide test, indicated that the MC concentrations, to which the mussels were exposed, were not cytotoxic to the haemocytes. The Comet assay performed on the haemocytes indicated that exposure to CdCl(2) produced a dose-responsive increase in DNA damage, demonstrating that mussel haemocytes were sensitive to DNA-damaging agents. DNA damage, measured as percentage tail DNA (%tDNA), was observed in mussels exposed to the three toxic Microcystis strains, but not in mussels exposed to the nontoxic strain. Toxin analysis of the cyanobacterial cultures confirmed that the three MC-producing strains exhibit different toxin profiles, with the two MC variants detected being MC-LF and MC-LR. Furthermore, the DNA damage that was observed appeared to be strain-specific, with high doses of MC-LF being associated with a higher level of genotoxicity than low concentrations of MC-LR. High levels of MC-LF also seemed to induce relatively more persistent DNA damage than small quantities of MC-LR. This study is the first to demonstrate that in vivo exposure to MC-producing strains of cyanobacteria induces DNA damage in the haemocytes of zebra mussels and confirms the sublethal toxicity of these toxins.     

The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development

BACKGROUND: The integrity of sperm DNA is important for the success of natural or assisted fertilization, as well as normal development of the embryo, fetus and child. ICSI, by bypassing sperm selection mechanisms, increases the risk of transmitting damaged DNA and the significance of this requires investigation. METHODS: DNA damage in sperm from an unselected group of 60 men undergoing IVF treatment was measured by single cell gel electrophoresis (Comet assay) and correlated with semen and treatment cycle parameters. RESULTS: Wide spectra of sperm DNA damage were found both within and between men but no specific subgroups were identified. Semen and treatment cycle parameters were not different in men grouped according to high or low sperm DNA damage. However, regression analysis showed that DNA damage was positively associated with age (29-44 years), abnormal sperm and motility and negatively associated with sperm concentration. In ICSI cycles DNA damage was positively associated with impairment of post-fertilization embryo cleavage. CONCLUSIONS: This study contributes to the evidence of DNA damage within sperm. High loads of DNA damage measured by the Comet assay were predictive of failure of embryo development after ICSI. As it is likely that sperm with DNA damage contributed to successful fertilization and in-vitro development, potential adverse effects remain to be clarified.     

Alkaline comet assay study with breast cancer patients: evaluation of baseline and chemotherapy-induced DNA damage in non-target cells

Abstract The sensitivity of the alkaline comet assay for the evaluation of baseline and treatment-induced DNA damage in white blood cells of breast cancer patients receiving adjuvant chemotherapy according to three conventional anthracycline- and cyclophosphamide-containing protocols was investigated. Additionally, baseline DNA damage in cancer patients was compared with the levels of DNA damage recorded in healthy women. Altogether 30 patients with diagnosed breast cancer and 30 female blood donors with no known familial history of breast cancer participated in the study. Alkaline comet assay was performed according to standard protocol and DNA migration in peripheral blood leukocytes was measured by a computer-based image analysis system. For each subject the frequency of “damaged” cells, i.e., long-tailed nuclei (LTN) with tail length exceeding 95th percentile for the considered parameter among controls, is also reported. Breast cancer patients had significantly increased background levels of DNA damage in their peripheral blood leukocytes as compared to healthy women. Prior to the chemotherapy, a majority of patients showed a statistically significant increase in the number of LTN compared to healthy blood donors. Marked interindividual variations in baseline DNA damage among patients were recorded, some of them related to the disease stage and status. The present study confirmed the alkaline comet assay as a sensitive technique able to detect significantly elevated DNA migration in blood cells of patients already one hour after completion of the first cycle of chemotherapy. Administration of antineoplastic drugs in three chemotherapy protocols studied induced a similar increase of primary DNA damage in nontarget cells. The evaluation of the LTN frequencies indicates the best response to the protocol containing cyclophosphamide, methotrexate and 5-fluorouracil (CMF). Our results point to the significance of simultaneous evaluation of DNA migration and frequency of LTN in the same subject and approved the use of alkaline comet assay as a suitable method for the routine detection of critical DNA lesions produced after administration of antineoplastic drugs in the clinical settings.     

DNA damage and metabolic activity in the preimplantation embryo

BACKGROUND: Embryos with greater viability have a lower or ‘quieter’amino acid metabolism than those which arrest. We have hypothesizedthis is due to non-viable embryos possessing greater cellular/moleculardamage and consuming more nutrients, such as amino acids forrepair processes. We have tested this proposition by measuringphysical damage to DNA in bovine, porcine and human embryosat the blastocyst stage and relating the data to amino acidprofiles during embryo development. METHODS: Amino acid profiles of in vitro-derived porcine and bovine blastocystswere measured by high-performance liquid chromatography andthe data related retrospectively to DNA damage in each individualblastomere using a modified alkaline comet assay. Amino acidprofiles of spare human embryos on Day 2–3 were relatedto DNA damage at the blastocyst stage. RESULTS: A positive correlation between amino acid turnover and DNA damagewas apparent when each embryo was examined individually; a relationshipexhibited by all three species. There was no relationship betweenDNA damage and embryo grade. CONCLUSIONS: Amino acid profiling of single embryos can provide a non-invasivemarker of DNA damage at the blastocyst stage. The data are consistentwith the quiet embryo hypothesis with viable embryos (lowestDNA damage) having the lowest amino acid turnover. Moreover,these data support the notion that metabolic profiling, in termsof amino acids, might be used to select single embryos for transferin clinical IVF.     

Vitamin C modulates DNA damage induced by hydrogen peroxide in human colorectal adenocarcinoma cell lines (HT29) estimated by comet assay in vitro

Introduction

Cancer cells, compared to normal cells, are under increased oxidative stress associated with oncogenic transformation, alterations in metabolic activity, and increased generation of reactive oxygen species.

Material and methods

We investigated the ability of vitamin C to reduce the damage induced by hydrogen peroxide, in human colorectal adenocarcinoma cells in vitro by the comet assay. Additionally, we measured the kinetics and efficacy of the repair of DNA damage after incubation with vitamin C in the presence of H₂O₂.

Results

The obtained results showed that 1 h pre-incubation with vitamin C and exposure to H₂O₂ for the last 10 min of incubation caused a statistically significant (p < 0.05) increase in DNA migration in comet tails in all experimental series. For the 10 µM, 25 µM, 50 µM, 100 µM vitamin C concentrations the levels of DNA damage were as follows: 18.6%, 21.1%, 25.3% and 27.2%, respectively, as compared to the untreated cells (3.26%). However, in comparison with H₂O₂ alone (29.1%), we observed a statistically significant (p < 0.05) decrease of the genotoxic effect in HT29 cells induced by H₂O₂ for the two lowest of concentrations of vitamin C: 10 µM and 25 µM. The HT29 cells were able to achieve effective repair of the damaged DNA within 60 and 120 min after incubation with the tested compounds. All the values obtained in the test were statistically significant (p < 0.05).

Conclusions

Vitamin C caused a weaker DNA damaging effect of hydrogen peroxide and positively influences the level of oxidative DNA damage in HT29 cells (decrease ∼ 30%). We noted that DNA damage was effectively repaired during 120 min postincubation in the tested cells and that oxidative damage was the major type of damage.     

DNA damage in lymphocytes of Indian rickshaw pullers as measured by the alkaline Comet assay

Rickshaw pullers (RPs) engage in strenuous physical activity and are exposed to the air pollutants found in urban environments. Air pollutants and the reactive oxygen species generated by the physical activity both potentially can damage DNA. In the present study, the Comet assay, a sensitive tool for measuring DNA damage in single cells, was used to study genomic DNA damage in lymphocytes of Indian RPs. The study evaluated DNA damage in 118 healthy male volunteers, including 63 RPs whose work demanded high levels of physical activity for 7-9 hr/day, and 55 controls matched for age, habits, socio-economic status, and exposure to air pollution. A significant increase was found for the mean Olive tail moment (arbitrary units) among the RPs (4.13 +/- 0.11; P < 0.001) in comparison with the controls (3.21 +/- 0.10). Likewise, comet tail length (microm) (RPs: 58.98 +/- 1.01 vs. controls: 52.38 +/- 1.24) and tail DNA (%) (RPs: 13.52 +/- 0.31 vs. controls: 10.04 +/- 0.24) were also significantly higher for RPs compared with those of their matched controls (both, P < 0.001). To our knowledge, this is the first demonstration that physical activity due to occupation can produce DNA damage in peripheral lymphocytes.     

Comet assay of cumulus cell DNA status and the relationship to oocyte fertilization via intracytoplasmic sperm injection

This paper postulates that in the ovary, the close association between the cumulus cells and the oocytes permits the fertilizing ability of the oocytes to be determined indirectly through cumulus cell DNA status. The objective was to use a modified comet assay to analyse cumulus cell DNA and relate the data to oocyte fertilization after intracytoplasmic sperm injection (ICSI) procedures. Oocytes were retrieved (n = 15 cases) and free-floating cumulus cells were pooled and smeared on clear glass slides to dry. Meanwhile, the denuded oocytes were injected with spermatozoa and fertilization was assessed, followed by embryo transfer. The fixed cumulus cells were stained in acridine orange, coated with a mini-gel agarose layer, lysed in alkaline buffer and electrophoresis performed. Analyses of fluorescent cell images (n = 449) showed that the tail moment was positively correlated to the percentage of fertilization after ICSI (r = 0.567, P < 0.05). In contrast, there was no correlation between tail moment and number of oocytes retrieved, total ampoules used, endometrial thickness and age of patient. The results suggested that the competence of the oocytes was associated with the cumulus cell DNA status. A unique feature here was the comet assay for archived material with obvious advantages.     

Influence of DNA repair gene polymorphisms on the initial repair of MMS-induced DNA damage in human lymphocytes as measured by the alkaline comet assay

We have applied the alkaline comet assay to study the functional impact of gene polymorphisms in base excision repair (APEX1 Asp148Glu, XRCC1 Arg194Trp, XRCC1 Arg399Gln) and homologous recombination repair (XRCC3 Thr241Met, NBS1 Glu185Gln), two pathways that play crucial roles in the repair of DNA damage induced by methylmethane sulphonate (MMS). We also examined the effect of polymorphisms in mismatch repair (MLH1 -93 A/G) and nucleotide excision repair (XPD Lys751Gln) as putative negative controls based on the limited roles of these pathways in MMS-induced repair. Phytohemagglutinin-stimulated peripheral lymphocytes from 52 healthy individuals were treated with MMS and allowed to repair for 0, 15, 40, or 120 min after a 6-min washing step. DNA damage was measured as a pseudo-percentage score (comparable to % tail DNA) converted from a total visual score calculated from the distribution of cells with different degrees of damage (normal, mild, moderate and severe). The repair was faster at the beginning of the observation period than towards the end, and was not complete after 2 hr. Presence of the APEX1 148Asp, XRCC3 241Met or NBS1 185Gln alleles were significantly associated with a high pseudo-percentage score (above median) at early time points, with the APEX1 effect being most prolonged (up to 40 min after washing, odds ratio 5.6, 95% confidence interval 2.0-15.5). No significant effects were seen with the XRCC1 Arg194Trp, XRCC1 Arg399Gln, MLH1 -93A/G and XPD Lys751Gln polymorphisms. Our results provide evidence for the functional nature of the variant alleles studied in the APEX1, XRCC3, and NBS1 genes.     

DNA damage in peripheral blood leukocytes of physically active individuals as measured by the alkaline single cell gel electrophoresis assay

DNA damage induced by physical activity and/or exercise has been reported under different conditions but not for individuals maintaining physical fitness by regular strenuous exercise. Therefore, we compared levels of DNA damage in blood leukocytes of 40 healthy individuals (35 males, 5 females) who regularly exercised in gymnasiums/health clubs and 15 healthy sedentary controls who had never exercised. The former group was selected (after informed consent) on the basis of how long they had been exercising on a regular basis as well as their exercise schedule and regimen. The length of time since starting a regular exercise regimen ranged from 2 months to 9 years, whereas the daily exercise duration ranged from 40 min to 3 hrs and warm‐up sessions ranged from none to 90 min. The length of DNA migration (44.66 ± 2.68 μm in males, 29.62 ± 1.69 μm in females) and the percentage of cells with tails (79.86 ±1.27% in males, 67.20 ± 0.96% in females) in peripheral blood leukocytes of physically active individuals were increased significantly (P < 0.001) with respect to corresponding values in control males and females (18.85 ± 1.79 μm, 23.37 ± 3.94 μm; 24.50 ± 1.98%, 33.00 ± 4.44%, respectively). Highly significant differences for DNA damage were also observed between physically active males and females. These observations, in the absence of any other exposures, indicate a correlation between strenuous exercise to keep fit and increased levels of DNA damage. This finding may have relevance in terms of the ageing process, with diseases associated with aging, and with carcinogenesis. Environ. Mal. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

Nereis virens (Annelida: Polychaeta) is not an adequate sentinel species to assess the genotoxic risk (comet assay) of PAH exposure to the environment

Polychaetes, because of their bioturbation capacity, play an important role in the distribution of anthropogenic contaminants (including polycyclic aromatic hydrocarbons [PAHs]) throughout the sediments. In this work the use of Nereis virens (Annelida: Polychaeta) as a bioindicator to assess the genotoxic risk of PAH exposure for the environment was evaluated. For this purpose the alkaline single cell gel electrophoresis (comet) assay was applied on the coelomocytes of in vivo exposed Nereis virens. Benzo[a]pyrene (B[a]P) was chosen because it is classified by the IARC (International Agency for Research on Cancer) as “probably carcinogenic to humans” and because its mechanisms of action are well-known. Nereis virens was exposed to B[a]P in concentrations of 0.3, 0.6, 10, 20, 35 and 45 mg/ml by an intracoelomic injection of B[a]P (20 μl) dissolved in dimethyl sulphoxide (DMSO). A solvent control with DMSO, a positive control with ethyl methane sulphonate (EMS) (12.1 mg/ml) and a negative control were included in each experiment. For each treatment four animals were analysed. After 1 hr treatment coelomocytes were harvested by puncturing the coelomic cavity with a sharpened Pasteur pipette, mixed with 0.5% low melting point agarose and sandwiched between two other gel layers. Ethidium bromide stained nuclei were analysed for tail length and tail moment. 12.1 mg/ml EMS, pure DMSO (98.9%) and B[a]P in all tested concentrations induced a statistically significant increase of DNA single strand breaks in the comet assay. The effect of B[a]P, however, was only at the highest concentration (45 mg/ml) significantly stronger than the effect of DMSO alone. Although a relatively large heterogeneity in the results could be observed, these experiments clearly showed that Nereis virens is not suited as a sentinel species for the assessment of the genotoxic risk of PAH exposure because this species seems to be very resistant to benzo[a]pyrene. Environ. Mol. Mutagen. 30:82–90, 1997 © 1997 Wiley-Liss, Inc.     

Radiation-induced DNA damage in canine hemopoietic cells and stromal cells as measured by the comet assay

Stromal cell progenitors (fibroblastoid colony-forming unit; CFU-Fs) are representative of the progenitor cell population of the hemopoietic microenvironment in bone marrow (BM). Previous studies of the radiation dose—effect relationships for colony formation have shown that canine CFU-Fs are relatively radioresistant as characterized by a D₀ value of about 2.4 Gy. In contrast, hemopoietic progenitors are particularly radiosensitive (D₀ values = 0.12-0.60 Gy). In the present study, the alkaline single-cell gel electrophoresis technique for the in situ quantitation of DNA strand breaks and alkalilabile sites was employed. Canine buffy coat cells from BM aspirates and cells harvested from CFU-F colonies or from mixed populations of adherent BM stromal cell (SC) layers were exposed to increasing doses of X-rays, embedded in agarose gel on slides, lysed with detergents, and placed in an electric field. DNA migrating from single cells in the gel was made visible as “comets” by ethidium bromide staining. Immediate DNA damage was much less in cultured stromal cells than in hemopoietic cells in BM aspirates. These results suggest that the observed differences in clonogenic survival could be partly due to differences in the type of the initial DNA damage between stromal cells and hemopoietic cells. © 1996 Wiley-Liss, Inc.     

Assessment of DNA damage in lymphocytes of workers exposed to X-radiation using the micronucleus test and the comet assay.

The mutagenic and carcinogenic effects of genotoxic agents on exposed people have constituted an increasing concern. Therefore, the objective of this work was to assess DNA damage in lymphocytes of workers exposed to X-radiation using the cytokinesis-blocked micronucleus test and the comet assay (single-cell gel electrophoresis), and to compare these two techniques in the monitoring of exposed populations. The cytokinesis-blocked micronucleus test and the comet assay were employed in the monitoring of 22 workers occupationally exposed to X-radiation in a hospital in southern Brazil. The frequency of dicentric bridges was also measured. The results of both assays and the frequency of dicentric bridges revealed a significant increase in genetic effects on the cells of exposed individuals. Age was significantly correlated with micronucleus frequency and damage index in the comet assay. The concomitant analysis of dicentric bridges when determining micronucleus frequency does not require much extra work, and may serve as a reference to the type of mutagenic effect (clastogenic or aneugenic). The combination of the alkaline comet assay with the cytokinesis-blocked micronucleus test appears to be very informative for the monitoring of populations chronically exposed to genotoxic agents.     

Comparison of sensitivity to methyl methanesulphonate among tadpole developmental stages using the alkaline single-cell gel electrophoresis (comet) assay

In a previous study, we demonstrated that tadpoles are suitable organisms for monitoring small bodies of water (e.g., creeks, ponds, and drainage ditches) for genotoxicity using the alkaline single-cell gel DNA electrophoresis (SCG) or “comet” assay [Ralph and Petras, 1997]. This approach involves detection, under alkaline conditions, of cell DNA fragments which on electrophoresis migrate from the nuclear core, resulting in a “comet with tail” formation. In this initial study, most of the tadpoles collected were in the early stages of larval development, but this is not always possible. The present study evaluated the sensitivity of tadpoles, at different stages of larval development, to a range of concentrations of the genotoxicant methyl methane-sulphonate (MMS). Four specific phases of Rana clamitans (green frog) larval development were examined: first-year limbless tadpoles (Stage I as defined by Taylor and Kollros [1946]), second-year limbless tadpoles (Stages II–III), second-year tadpoles with only hindlimbs (Stages X–XVIII), and second-year tadpoles with all four limbs evident and a tail undergoing resorption (Stages XXII–XXIII). Twenty-four hour exposures to MMS of tadpoles in the three earliest phases produced a significant (P < 0.01) added variance component among tadpoles for DNA damage and there were significant increases (P < 0.05) in the length:width ratios of the DNA patterns at concentrations as low as 1.56 mg/l. However, tadpoles in the last phase studied (both pairs of limbs present) showed no significant (P > 0.05) added variance component and no significant increases (P > 0.05) in DNA damage upon exposure to any of the MMS doses tested. A nested ANOVA indicated that, for each of the tested concentrations of MMS, but not the dechlorinated water control, there was significant heterogeneity (P < 0.05) in DNA damage when tadpoles of all four phases studied were compared. However, when tadpoles of the last phase of development were removed from the comparison, there was no significant heterogeneity (P > 0.05) among tadpoles of the remaining three phases. Possible reasons for this insensitivity to MMS as animals enter the metamorphic climax were considered. The results indicate that pooling of the early tadpole phases of R. clamitans for SCG environmental genotoxicity biomonitoring is acceptable. Environ. Mol. Mutagen. 31:374–382, 1998. © 1998 Wiley-Liss, Inc.     

New measure of DNA repair in the single-cell gel electrophoresis (comet) assay

Since its introduction by Ostling and Johanson [1984; Biochem Biophys Res Commun 123:291-298] and independent modifications by Singh et al. [1990; Exp Cell Res 175:184-191] and Olive et al. [1988; Radiat Res 112:86-94], the comet assay has been widely used in genetic toxicology, environmental biomonitoring, molecular and human epidemiology, and clinical investigations. There are still several issues to be resolved before the comet assay is accepted as a standard assay for detecting DNA damage and repair in a single cell. One of the major issues is the proper quantification of DNA damage/repair. The aim of this article is to develop a new quantitative measure of DNA damage/repair which is represented in the dose-time-response surface. We propose to use the second derivative (2D) of the dose-time-response surface for measuring DNA repair activity. This approach enables us to represent the DNA repair activity of cells exposed to a DNA-damaging agent with a single number by combining all the information of a dose-time-response experiment. The computation procedure includes the application of linear regression. An SAS/AF-based program, "Comet Assay," was developed for this computation and is freely available on the Internet. We considered the response of each of four DNA damage parameters: tail moment, tail length, tail DNA, and tail inertia for constructing the dose-time-response surface. Using data from 25 patients, we observed that 2Ds based on tail moment and tail DNA were highly correlated and that tail inertia might provide information on a somewhat different aspect of DNA damage/repair.     

The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI

INTRODUCTION: Sperm chromatin integrity assessment has been suggested as a fertility predictor. The aim of this study was to examine the relationship between the results of sperm chromatin structure assay (SCSA) and the outcome of IVF, ICSI and intrauterine insemination (IUI). METHODS: A total of 306 consecutive couples undergoing assisted reproduction were included. IUI was performed in 131, IVF in 109 and ICSI in 66. SCSA results were expressed as DNA fractionation index (DFI) and highly DNA stainable (HDS) cell fractions. Reproductive outcome parameters were biochemical pregnancy (BP), clinical pregnancy (CP) and delivery (D). RESULTS: For IUI, the chance of pregnancy/delivery was significantly higher in the group with DFI 27% or HDS >10%. The odds ratios (ORs) (95% confidence intervals) were 20 (2.3-117), 16 (1.9-137) and 14 (1.6-110) for BP, CP and D, respectively. No statistical difference between the outcomes of IVF versus ICSI was observed in the group with DFI 27% group, however, the results of ICSI were significantly better than those of IVF. Comparing ICSI with IVF, the OR (95% CI) for BP was 26 (1.9-350). CONCLUSIONS: SCSA is a useful method for prediction of the outcome of assisted reproduction.