Mean Tail Intensity Research Articles

DNA Damage Estimation after Chronic and Combined Exposure to Endocrine Disruptors: An In Vivo Real-Life Risk Simulation Approach.

Exposure to chemical substances has always been a matter of concern for the scientific community. During the last few years, researchers have been focusing on studying the effects resulting from combined exposure to different substances. In this study, we aimed to determine the DNA damage caused after chronic and combined exposure to substances characterized as endocrine disruptors using comet and micronuclei assays, specifically glyphosate (pure and commercial form), bisphenol A, parabens (methyl-, propyl- and butylparaben), triclosan and bis (2-ethylhexyl) phthalate. The highest mean tail intensity was observed in the group exposed to a high-dose (10 × ADI) mixture of substances (Group 3), with a mean value of 11.97 (11.26-13.90), while statistically significant differences were noticed between the groups exposed to low-dose (1 × ADI) (Group 2) and high-dose (10 × ADI) (Group 3) mixtures of substances (p = 0.003), and between Group 3 and both groups exposed to high doses (10 × ADI) of the pure and commercial forms of glyphosate (Groups 4 (p = 0.014) and 5 (p = 0.007)). The micronuclei assay results were moderately correlated with the exposure period. Group 5 was the most impacted exposure group at all sampling times, with mean MN counts ranging between 28.75 ± 1.71 and 60.75 ± 1.71, followed by Group 3 (18.25 ± 1.50-45.75 ± 1.71), showing that commercial forms of glyphosate additives as well as mixtures of endocrine disruptors can enhance MN formation. All exposure groups showed statistically significant differences in micronuclei counts with an increasing time trend.

Age-related DNA damage in stallions: an ongoing investigation

Foals sired by younger stallions record significantly higher racing speeds (Sharman.et.al. Royal Society Open Science. 2022;9:220691), and more race wins than foals sired by older stallions (Brazil.et.al. Honours Dissertation. University of Limerick. 2016). We hypothesize that this may be due to an age-associated increase in sperm DNA damage, as has been demonstrated in other species. Therefore, this study aimed to investigate the relationship between stallion age and sperm DNA damage. Post-coital dismount semen samples (n=75) were collected weekly from 46 commercial Thoroughbred stallions in the Hunter Valley (Australia). Samples were immediately diluted (2:1, EquiPlus:semen), and the high-density spermatozoa were isolated using EquiPure centrifugation to reduce contaminants (somatic cells and other debris). Any samples that were contaminated with urine were excluded from the study. Sperm count and motilities were recorded onsite (iSperm™), and samples were snap frozen for DNA damage analyses. Management information, including pregnancy scan results, were also collected. For this study, stallions aged ≤9 years were categorized as “young” while those aged ≥12 were categorized as “old”. Stallions between 9 and 12 years were excluded to improve the likelihood of observing an age-related difference. Mare age and fertility status were similar across both groups. All data were checked for normality (Shapiro-Wilk) and analyzed using unpaired t-tests. Sperm count and motility did not differsignificantly between age cohorts. DNA strand breaks were significantly higher in spermatozoa from older stallions compared to younger stallions (alkaline comet assay: 19.7±0.55% vs 15.4±0.57% mean tail intensity; P≤0.05, and sperm chromatin dispersion or ‘Halo’ assay; 0.48±0.045in vs 0.70±0.074in ‘halo’ area; P≤0.05, respectively). These breaks may be attributed to poor chromatin packaging, as older stallions had a concomitant deficiency of sperm protamines compared to young stallions (11.6±1.53 vs 6.9±1.07 chromomycin A3 positive cells; P≤0.05, respectively). Although younger stallions had lower levels of DNA strand breakage, they had more oxidized DNA adducts (8-hydroxy-2′-deoxyguanosine (8-OHdG) assay; P≤0.05). Interestingly, regardless of age, positive correlations between the proportion of oxidized DNA adducts and fertility were observed (R2=0.269 for younger, R2=0.483 for older stallions; P≤0.05). This is likely a reflection of the increased use of oxidative phosphorylation, and subsequent ROS production, in higher fertility ejaculates (Gibb.et.al. Biology of Reproduction. 2014;91(3):77). In conclusion, while stallion sperm DNA damage rises with age, it does not appear to influence fertility outcomes. However, further research is needed to determine if age-related sperm DNA damage compromises the next generation's genome. In addition, we must identify the most vulnerable genomic loci for sperm DNA damage and determine how these DNA lesions may contribute to reduced racing performance and health in offspring.

Acetamiprid-induced Cyto- and Genotoxicity in the AR42J Pancreatic Cell Line.

Neonicotinoid insecticides, 30% of insecticides marketed worldwide, have selective toxicity on insects through α4p2 nicotinic acetylcholine receptors. Although it is known that acetamiprid exerts toxicity on several organ systems, its toxic effects on the pancreas and its mechanism of action have not been clarified yet. Therefore, in the present study, the cytotoxic and genotoxic potentials of acetamiprid on the AR42J pancreatic cell line were evaluated. The (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT) assay and comet assay were conducted for the cyto- and genotoxicity evaluations, respectively. Reactive oxygen species (ROS) production was assessed by flow cytometry and glutathione (GSH) levels were determined by ELISA for oxidative damage potential, which is thought to be an underlying mechanism of cyto-/genotoxic effects. To reveal the dose-response relationship the concentration range of 1-6 mM was selected for the assays. Cell viability decreased in a dose-dependent manner and the inhibitory concentration 50 value was calculated as 12.61 mM by the MTT assay. Acetamiprid induced DNA damage in all concentrations tested in a dose-depending manner. The mean tail intensity values were 3.84 and ≤32.96 for the control and exposure groups, respectively. There was no significant difference for ROS production; however, the GSH level was reduced at the highest concentration. It is thought that the present study will contribute to the literature due to the lack of data on the potential toxic effects of acetamiprid on the pancreas. To better understand acetamiprid toxicity, further studies including a wide range of mechanistic parameters are needed.

DNA damage in BALB/c mice infected with Lacazia loboi and its relation to nutritional status.

BackgroundJorge Lobo’s disease, also known as lacaziosis, is a cutaneous-subcutaneous mycosis with chronic evolution. It is caused by the fungus Lacazia loboi. Herein we report a study that relates the genotoxicity caused by L. loboi in isogenic mice with nutritional status, through a normal or restricted diet.MethodsDNA damage was assessed in the peripheral blood by the comet assay (tail intensity).ResultsThe results for leukocytes showed increases in the mean tail intensity in mice under dietary restriction, in infected mice under dietary restriction and in infected mice ingesting a normal diet.ConclusionThese results indicate that dietary restriction and L. loboi infection may increase DNA damage levels in mice, as detected by the comet assay.

DNA damage in human lymphocytes exposed to four food additives in vitro

In vitro genotoxic effects of antioxidant additives, such as citric acid (CA) and phosphoric acid (PA) and their combination, as well as antimicrobial additives, such as benzoic acid (BA) and calcium propionate (CP), on human lymphocytes were determined using alkaline single-cell gel electrophoresis. There was a significant increase in the DNA damage in human lymphocytes after 1h of in vitro exposure to CA, PA, BA and CP (200, 25-200, 50-500, 50-1000μg/mL, respectively). The combination of CA and PA significantly increased the mean tail intensity at all the concentrations used (25-200μg/mL) and significantly increased the mean tail length mainly after higher concentrations (100and 200μg/mL). Data in this study showed that the concentrations of food additives used induce DNA damage and PA was the most genotoxic and CA was less genotoxic additives among them.

Assessment of cytogenetic damage and oxidative stress in personnel occupationally exposed to the pulsed microwave radiation of marine radar equipment

Due to increased usage of microwave radiation, there are concerns of its adverse effect in today's society. Keeping this in view, study was aimed at workers occupationally exposed to pulsed microwave radiation, originating from marine radars. Electromagnetic field strength was measured at assigned marine radar frequencies (3 GHz, 5.5 GHz and 9.4 GHz) and corresponding specific absorption rate values were determined. Parameters of the comet assay and micronucleus test were studied both in the exposed workers and in corresponding unexposed subjects. Differences between mean tail intensity (0.67 vs. 1.22) and moment (0.08 vs. 0.16) as comet assay parameters and micronucleus test parameters (micronuclei, nucleoplasmic bridges and nuclear buds) were statistically significant between the two examined groups, suggesting that cytogenetic alterations occurred after microwave exposure. Concentrations of glutathione and malondialdehyde were measured spectrophotometrically and using high performance liquid chromatography. The glutathione concentration in exposed group was significantly lower than in controls (1.24 vs. 0.53) whereas the concentration of malondialdehyde was significantly higher (1.74 vs. 3.17), indicating oxidative stress. Results suggests that pulsed microwaves from working environment can be the cause of genetic and cell alterations and that oxidative stress can be one of the possible mechanisms of DNA and cell damage.

RE: DNA damage detected by the alkaline comet assay in the liver of mice after oral administration of tetrachloroethylene. (Mutagenesis, 25, 133-138, 2010)

In their Letter to the Editor, Lillford et al. have questioned the analyses and conclusions of the results presented in our recently published paper (1). In this study, mice were exposed to tetrachloroethylene at the doses of 0 (vehicle control), 1000 and 2000 mg/kg/day. From the results obtained, we concluded that tetrachloroethylene is weakly genotoxic in the liver of mice. This conclusion was not shared by Lillford et al. as it, in their view, was heavily dependent on statistical analysis and did not take biological relevance of the results into account. We do not agree with this opinion and, therefore, the criteria and arguments we have taken into account in the evaluation of the results are further elaborated below. In toxicity testing, it is generally held that the finding of a dose-related effect of a test substance provides a very strong argument for concluding that the observed effect is biologically relevant. In the study we have published, it was noticed that the group mean values for tail intensity in the liver increased with the dose, suggesting a biologically relevant effect of tetrachloroethylene, i.e. induction of DNA damage. This positive trend was then analysed with the Jonckheere–Terpstra test and found to be statistically significant. Thereby, the statistical analysis has been used as an aid in evaluating the results, and the outcome of the analysis showed that there was a low probability for the observed positive trend to occur by chance only. We disagree with the comment by Lillford et al. stating that our conclusion was heavily dependent on statistical analysis and that statistical analysis was the sole criterion for a positive effect. The biological relevance of the effect, demonstrated by the dose-related increase in tail intensity in the liver, was considered first and has been taken into account for determining the positive response. Lillford et al. argue that groups of mice treated with tetrachloroethylene exhibited tail moments and tail intensities that were similar to those of the concurrent vehicle control group. Given the flexible meaning of the word similar and that the DNA-damaging effect of tetrachloroethylene was weak, we understand the basis for such a statement. However, 8 of the 12 tetrachloroethylene-treated animals had higher tail intensity values in the liver than the animal with the highest tail intensity value in the concurrent vehicle control group and, furthermore, the dose-related increase in tail intensity established in the liver implies that the mean tail intensity values for the three experimental groups were indeed different. We note that Lillford et al. comment that the maximum group mean increase was only 1.43-fold (tail intensity in the liver, 2000 mg/kg group) above the vehicle control group and that the increase in tail moment in this group was only 1.36-fold. In our opinion, the crucial result of the study was that a weak DNA-damaging effect of tetrachloroethylene was established. Whether the magnitude of this weak effect was 1.43-fold or 1.36-fold is of less importance. Furthermore, the reference to tail moment is of limited value since according to international expert panel recommendations for conducting the alkaline in vivo comet assay (2), the use of tail intensity (i.e. per cent DNA in tail) is recommended, as indicated by the following statement:

Analysis of Pesticide Exposure and DNA Damage in Immigrant Farmworkers

ABSTRACT In the last decade, the Comet assay has been used increasingly in studies of workers potentially exposed to genotoxic substances in the workplace or environment. Significant increases in DNA damage measured with the Comet assay has been reported in lymphocytes of agricultural workers; however, less intrusive means of biomonitoring are needed in epidemiological investigations. This study was designed to use the Comet assay to describe the association of markers of DNA damage in oral leukocytes with biomarkers of pesticide exposure in 134 farmworkers working in berry crops in Oregon compared to control populations. The authors also examined the extent of DNA damage in young workers compared to adults and the effect of work histories, lifestyle factors, and diet on markers of DNA damage. Urinary levels of organophosphate pesticides were low at the time of sampling; however, mean levels of the Captan metabolite tetrahydrophthalimide (THPI) were found to be shifted significantly higher in the farmworkers (0.14 μg/ml) compared to controls (0.078 μg/ml) (one-sided p value = .01). Likewise, the combined molar equivalent of all dialkylphosphate metabolites was marginally higher in farmworkers (p value = .05). The mean tail intensity was significantly greater for agricultural workers compared to controls (one-sided p value <.001), indicating more DNA damage in the oral leukocytes. On average, the mean tail intensity was 10.9 units greater for agricultural workers (95% CI: 6–16 units greater). Tail moment was also significantly greater for agricultural workers compared to nonagricultural workers (one-sided p value <.001). No Comet parameter was significantly associated with years spent working in agriculture, age, sex, body mass index, diet, and alcohol or tobacco use. The results of this study demonstrate the feasibility for using the Comet assay in biomonitoring studies of farmworkers. Additional studies are needed to examine the effects of different pesticide types on DNA damage and to capture the temporal relationship between exposure to agricultural chemicals and changes in Comet parameters.

Cloudy apple juice decreases DNA damage, hyperproliferation and aberrant crypt foci development in the distal colon of DMH-initiated rats

Clear (CleA) and cloudy (CloA) apple juices containing different amounts of analyzed procyanidins and pectin were investigated for preventive effects of colon cancer and underlying molecular mechanisms in F344 rats given intraperitoneal injections of 1,2-dimethylhydrazine (DMH; 20 mg/kg body wt) once a week for 4 weeks. Rats received either water (Cont), CleA or CloA (ad libitum) for 7 weeks starting 1 week before the first DMH injection. CloA inhibited DMH induced genotoxic damage in mucosa cells of the distal colon compared with Cont as investigated by single-cell microgel electrophoresis assay. The mean tail intensity in mucosa cells of DMH-treated controls (Cont/DMH: 6.1+/-0.9%) was significantly reduced by CloA (2.4+/-0.8%; P<0.01) but not by CleA intervention (4.1+/-1.2%; P>0.05). The crypt cell proliferation index induced by DMH (Cont/NaCl: 10.0+/-0.7%; Cont/DMH: 19.9+/-1.0%; P<0.001) was significantly decreased by CleA (15.7+/-0.7%; P<0.001) and CloA intervention (11.9+/-0.4%; P<0.001). CloA but not CleA significantly reduced the number of large aberrant crypt foci (ACF) consisting of more than four aberrant crypts (AC) (Cont/DMH: 37.4+/-5.4; CleA/DMH: 32.8+/-4.4, P>0.05; CloA/DMH: 18.8+/-2.5 ACF; P<0.05) and the overall mean ACF size in the distal colon (Cont/DMH: 2.31+/-0.09; CleA/DMH: 2.27+/-0.05; CloA/DMH: 2.04+/-0.03 AC/ACF; P<0.05). After treatment with DMH and/or apple juices there were no changes in transcript levels of colonic cyclooxygenase isoforms (COX-1, COX-2) or glutathione-associated enzymes (GST-M2, gamma-GCS, GST-P), the splenocyte natural killer cell activity and plasma antioxidant status. However, CloA but not CleA prevented the DMH-induced reduction of splenocyte CD4/CD8 (T-helper cells to cytotoxic lymphocytes) ratio. Since both formulations contained comparable concentrations and types of monomeric polyphenols, complex polyphenols or non-polyphenolic compounds, such as pectin might be responsible for the stronger cancer-preventive effect by CloA.