Gender-related differences in basal DNA damage in lymphocytes of a healthy Indian population using the alkaline Comet assay

The Comet assay, a sensitive, rapid and non-invasive technique, measures DNA damage in individual cells and has found wide acceptance in epidemiological and biomonitoring studies to determine the DNA damage resulting from lifestyle, occupational and environmental exposure. The present study was undertaken to measure the basal level of DNA damage in a normal, healthy Indian male and female population. Out of the 230 volunteers included in this study, 124 were male and 106 were female. All the individuals belonged to a comparable socio-economic background and aged between 20 and 30 years. They were also matched for their smoking and dietary habits. The period of sample collection was also matched. The results revealed a statistically significant higher level of DNA damage in males when compared to females as evident by an increase in the Olive tail moment [3.76+/-1.21 (arbitrary units) for males as compared to 3.37+/-1.47 for females (P<0.05)], tail DNA (%) [10.2+/-2.96 for males as compared to 9.40+/-2.83 for females (P<0.05)] and tail length (microm) [59.65+/-9.23 for males and 49.57+/-14.68 for females (P<0.001)]. To our knowledge, this report has, for the first time demonstrated significant differences in the basal level of DNA damage between males and females in a normal healthy Indian population.     

Influence of environmental exposure to PAHs on the susceptibility of lymphocytes to DNA-damage induction and on their repair capacity

The influence of occupational exposure to environmental carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) on DNA damage detected in lymphocytes of exposed people (city policemen) was studied. The cellular susceptibility to the induction of the DNA damage and the repair capacity of exposed donors are presented in comparison with matched controls. Monitoring was performed and blood samples (164 donors) were collected in Prague, Czech Republic, during the winter and summer seasons. The single-cell gel electrophoresis (SCGE) assay with an internal standard was applied to evaluate the DNA damage. A challenging dose of 2 Gy of X-rays was used to study cellular capacities. In the results of studies of the DNA damage induced in vivo or as an immediate response to the challenging treatment no significant difference was found between exposed and unexposed subgroups. The percentage of non-repaired X-ray-induced DNA damage (residual damage, RD) overall in both seasons was significantly higher in lymphocytes of policemen exposed to c-PAHs than in matched controls (RD_T-DNA, %DNA in the comet tail: winter 36.4 ± 22.1 versus 22.7 ± 10.8, p < 0.001; summer 47.7 ± 22.9 versus 34.7 ± 15.2, p < 0.001). The results suggest that occupational exposure to environmental c-PAHs significantly reduces the cellular capacity to repair the DNA damage induced by a challenging treatment. A significant decrease of repair efficiency in donors occupationally exposed to environmental c-PAHs was also observed when subgroups were stratified according to smoking history. In conclusion, our results suggest that environmental exposure to c-PAHs affects the cellular repair processes and can lead to harmful effects hazardous to human health.     

Comet assay responses in human lymphocytes are not influenced by the menstrual cycle: a study in healthy Indian females

The single-cell gel electrophoresis or Comet assay measures qualitative and quantitative DNA damage in single cells. Its simplicity and non-invasive nature has made it widely accepted for the monitoring of human genotoxicity, employing peripheral blood lymphocytes. Factors, such as gender, age, and dietary and smoking habits are known to affect the Comet assay responses in lymphocytes. However, there is no information regarding the influence of the menstrual cycle on the results of the assay in lymphocytes of females.A study was therefore undertaken among 18 healthy Indian female volunteers to assess the effect of the menstrual cycle on Comet assay responses. During a complete menstrual cycle, only minor changes were observed in the basal levels of DNA damage in the lymphocytes as evident by Comet parameters, such as tail length (μm), tail DNA (%) and Olive tail moment (arbitrary units).To assess the effect of the estrogen 17β-estradiol (at physiological concentrations of 0.5, 1.0 and 2.0 nM) on the Comet assay responses, an in vitro study was conducted in the human lymphocyte cell line JM-1 and the breast cancer cell line MCF-7. As was evident from the Comet parameters, a significant (p < 0.01) concentration-dependent increase in the level of DNA damage was observed in the MCF-7 cells while no significant change was found in the JM-1 cells.The results indicate that the menstrual cycle does not influence the Comet assay responses in lymphocytes; hence, these can serve as a model for monitoring genotoxicity in females.     

Age-related increases of 8-hydroxy-2′-deoxyguanosine and DNA–protein crosslinks in mouse organs

Experimental data suggest a possible role of DNA damage in aging, mainly related to oxidative lesions. With the objective of evaluating DNA lesions as molecular biomarkers of aging, we measured 8-hydroxy-2′-deoxyguanosine (8-OH-dG) and DNA–protein crosslinks (DPXL) levels in different organs of mice aged 12 and 24 months. 8-OH-dG was detected by ³²P postlabelling after removing unmodified dG by trifluoracetic acid, which prevented the artificial formation of 8-OH-dG during ³²P labelling procedures. Appreciable 8-OH-dG amounts were detected in 12-month-old mice in liver (1.8±0.7 8-OH-dG/10⁵ normal nucleotides), brain (1.6±0.5) and heart (2.3±0.5). In 24-month-old mice these values were higher in all examined organs (liver, 2.7±0.4; brain, 3.6±1.1; heart, 6.8±2.2 8-OH-dG/10⁵ normal nucleotides). This accounted for a 1.5-fold increase in liver (not significant), 2.3-fold increase in brain (P<0.01), and 3.0-fold increase in heart (P<0.001). A similar trend was observed for DPXL levels, which were the 1.8±0.3%, 1.2±0.2%, and 2.2±0.3% of total DNA in liver, brain, and heart of 12-month-old mice and 1.9±0.4%, 2.0±0.4%, and 3.4±0.5% in 24-month-old mice, with ratios of 1.0, 1.7 (P<0.01), and 1.5 (P<0.001), respectively. Highly significant correlations between 8-OH-dG and DPXL levels were recorded in brain (r=0.619, P<0.001) and heart (r=0.800, P<0.0001), but not in liver (r=0.201, not significant). These data suggest that brain and heart are more severely affected by the monitored age-related DNA lesions than liver, which can be ascribed to certain characteristics of these postmitotic organs, including the low detoxifying capacities, the high oxygen consumption, and the impossibility to replace damaged cells by mitosis. The strong correlation between 8-OH-dG and DPXL supports a possible contribution of oxidative mechanisms to formation of DPXL in those organs, such as brain and heart, which play a primary role in the aging of the whole organism.     

Changes in various plasma lipid components, glucose, and insulin in Spermophilus lateralis during hibernation

This study evaluated fat mobilization as related to gluconeogenesis in two age groups of hibernating golden-mantled ground squirrels (Spermophilus lateralis). Our experimental group consisted of a total of 16 male and 15 female squirrels. Plasma samples were collected from selected animals being killed weekly from January to March, and the concentration of triglycerides, glycerol (GY), free fatty acids (FFA), total cholesterol, lipase activity, glucose, and insulin, were determined by biochemical assays and radioimmunoassay. Our results showed a mean FFA/GY ratio of five, which was higher than the predicted value of three, suggesting a significant depletion of GY and an enhanced rate of gluconeogenesis via GY to maintain glucose homeostasis in the hibernating animals. The factor of age did not significantly affect plasma lipid components. However, in the male group, plasma glucose levels were significantly higher for adults than for juveniles. Overall, females had significantly higher plasma glucose levels than males (150 ± 11 vs. 110 ± 8 mg%, P < 0.05). In the adult group, a gender influence was also seen on plasma insulin levels, with females' being higher than males' (66 ± 13 vs. 25 ± 3 μIU/ml, P < 0.01). We suggest that during hibernation, female squirrels may have a higher rate of lipolysis and gluconeogenesis along with a lower glucose utilization than their male counterparts. Additionally, adult females may exhibit a higher peripheral insulin resistance during hibernation than adult males, a possibility which merits further study.     

Increased lymphocyte DNA strand breaks in rubber workers

Objective: To study the effect of occupational exposure to rubber processing, smoking, and alcohol drinking on lymphocyte DNA damage. Subjects and Methods: Of 371 employees (197 men and 174 women) from a rubber factory in Guangzhou, 281 were rubber processing workers from five production sections and 90 were managerial workers. Information on occupational exposure, smoking, and drinking was collected by interviews. Blood samples were taken in the morning by venipuncture. DNA damages were measured by the Comet assay. Possible DNA-protein crosslinks were broken down by proteinase K. Tail moment, measured by Komet 4.0 image analysis software, was the measure of DNA damage. Results: The rubber processing workers had larger tail moment than the managerial workers (Geometric mean, 95%CI) [1.77 μm (1.64–1.90) versus 1.52 μm (1.36–1.71), P=0.04]. Both smoking [1.93 μm (1.74–2.13) versus 1.59 μm (1.47–1.71), P=0.003] and alcohol drinking [2.21 μm (1.87–2.62) versus 1.63 μm (1.53–1.74), P<0.001] increased tail moment. Tail moment differed significantly among job categories (F=3.21, P=0.008), the largest was observed in mixers. In the non-smoking and non-drinking workers, rubber processing workers had larger tail moment than managerial workers after adjusting for age (P=0.033). General linear model analysis showed that after adjusting for each other, occupational exposure (P=0.027), smoking (P=0.012), and alcohol drinking (P=0.013) was associated with larger tail moment, whereas age and gender had no effect. Conclusions: Occupational exposure to rubber processing, smoking, and alcohol drinking can cause DNA damage.     

Nutritional supplementation improves ovulation and pregnancy rates in female goats managed under natural grazing conditions and exposed to the male effect

Two experiments were conducted to determine if nutritional supplementation improved ovulation and pregnancy rates in female goats managed under grazing conditions and submitted to the male effect. In Experiment 1, one group of does did not receive nutritional supplementation, while the other group was supplemented daily for 7 days starting at the time when the males were introduced to the females. The ovulation rate at the second male-induced ovulation was greater (P < 0.05) in supplemented (2.0 ± 0.1) than in non-supplemented (1.6 ± 0.1) does. For Experiment 2, female goats were supplemented for 0, 7, 14 or 28 days, starting 9 days following buck introduction. The proportion of does that were pregnant in the group supplemented for 28 days was greater (P < 0.05) than in the non-supplemented group, but did not differ from 14-day and the 7-day supplemented groups. The proportion of pregnant does was greater (P < 0.05) in the group supplemented for 14 days compared to the group supplemented for 7 days and the non-supplemented group. These latter two groups did not differ (P > 0.05). In conclusion, feed supplementation for 7 days, starting at the time when males were introduced increased ovulation rate and feed supplementation for 14 or 28 days starting 9 days after males were introduced improved pregnancy rates in goats managed under grazing conditions and exposed to males.     

Lactate Oxidation for the Detection of Mitochondrial Dysfunction in Human Skin Fibroblasts

To screen fibroblasts for defects in lactate/pyruvate oxidation, cells were grown to confluence in 25-cm² flasks, rinsed, and incubated in glucose-free media containing 25 μM L-lactate and 0.1 μCi [D,L-1-¹⁴C]lactate. Lactate oxidation was measured as the amount of lactate oxidized in nmol of ¹⁴CO₂ generated /mg protein/min. Fibroblasts from patients with mitochondrial or peroxisomal disorders had decreased lactate oxidation compared to the control (CON): CON, 1.9 ± 0.13 nmol/mg/min; neonatal adrenoleukodystrophy (NALD), 0.45 ± 0.01 (P < 0.001); rhizomelic chondrodysplasia punctata (RCDP), 0.13 ± 0.002 (P < 0.001); mitochondrial defect of unknown etiology (MIT), 0.77 ± 0.003 (P <0.001); pyruvate dehydrogenase (PDH) deficiency, 0.98 ± 0.02 (P < 0.001). This method is useful for screening fibroblasts for defects in lactate oxidation in patients with mitochondrial or peroxisomal disorders. Confirmation of the site of the defect may then be investigated with specific assays, e.g., PDH, in cellular homogenates: CON, 0.93 ± 0.02 nmol/mg/min; NALD, 0.55 ± 0.02; RCDP, 0.44 ± 0.02; MIT, 0.53 ± 0.03; PDH deficiency, 0.19 ± 0.02.     

Investigating DNA damage in tannery workers occupationally exposed to trivalent chromium using comet assay

DNA damage of peripheral lymphocytes in 60 workers occupationally exposed to trivalent chromium [Cr(III)] in a tannery was studied using comet assay. The urinary and blood chromium levels were detected as a biomarker of internal exposure. The 90 subjects were divided into three groups: (i) exposure group I included 30 tannery workers highly exposed to chromium from tanning department; (ii) exposure group II included 30 tannery workers with moderate chromium exposure from finishing department; (iii) control group included 30 individuals without exposure to physical or chemical genotoxic agents. No significant difference was found among the three groups for age and smoking. The results showed that the medians of blood and urinary Cr of two exposure groups were significantly higher than those of control group (P < 0.01). And the medians of blood and urinary Cr of exposure group I were significantly higher than those of exposure group II (P < 0.05 or P < 0.01). The medians of mean tail length (MTL) of the three groups were 5.33 (2.90–8.50), 3.43 (2.31–8.29) and 2.04 (0.09–3.83) μm, respectively; The medians of mean tail moment (MTM) of the three groups were 6.28 (2.14–11.81), 3.41 (1.25–11.07) and 0.53 (0.13–3.29), respectively. The MTL and MTM of two exposure groups were significantly higher than those of control group (P < 0.01). The MTL and MTM of exposure group I were significantly higher than those of exposure group II (P < 0.01). The results of the present investigation suggest that occupational exposure to trivalent chromium can lead to a detectable DNA damage of human peripheral lymphocytes. Moreover, DNA damage was associated with chromium levels in blood. DNA damage may serve as a valuable effective biomarker and total chromium in blood may serve as a useful internal exposure biomarker in the population occupationally exposed to trivalent chromium.     

Fasting metabolism in Antarctic fur seal (Arctocephalus gazella) pups

The metabolism of 52–73-day old Antarctic fur seal pups from Bird Island, South Georgia, was investigated during fasting periods of normal duration while their mothers were at sea foraging. Body mass decreased exponentially with pups losing 3.5–3.8% of body mass per day. Resting metabolic rate also decreased exponentially from 172–197 ml (O₂)·min⁻¹ at the beginning of the fast and scaled to M_b^0.74 at 2.3 times the level predicted for adult terrestrial mammals of similar size. While there was no significant sex difference in RMR, female pups had significantly higher (F_1,18=6.614, P<0.019) mass-specific RMR than male pups throughout the fasting period. Fasting FMR was also significantly (t₁₅=2.37, P<0.035) greater in females (823 kJ·kg⁻¹·d⁻¹) than males (686 kJ·kg⁻¹·d⁻¹). Average protein turnover during the study period was 19.3 g·d⁻¹ and contributed to 5.4% of total energy expenditure, indicating the adoption of a protein-sparing strategy with a reliance on primarily lipid catabolism for metabolic energy. This is supported by observed decreases in plasma BUN, U/C, glucose and triglyceride concentrations, and an increase in β-HBA concentration, indicating that Antarctic fur seals pups adopt this strategy within 2–3 days of fasting. Mean RQ also decreased from 0.77 to 0.72 within 3 days of fasting, further supporting a rapid commencement of protein-sparing. However, RQ gradually increased thereafter to 0.77, suggesting a resumption of protein catabolism which was not substantiated by changes in plasma metabolites. Female pups had higher TBL (%) than males for any given mass, which is consistent with previous findings in this and other fur seal species, and suggests sex differences in metabolic fuel use. The observed changes in plasma metabolites and protein turnover, however, do not support this.     

Assessment of cytogenetic damage in lymphocytes and in exfoliated nasal cells of dental laboratory technicians exposed to chromium, cobalt, and nickel

Dental laboratory technicians may be exposed to metal alloys that are used in the production of crowns, bridges and removable partial dentures. These alloys consist of 35–65% cobalt, 20–30% chromium, 0–30% nickel, and small amounts of molybdenum, silica, beryllium, boron and carbon. The aim of this study was to assess whether dental technicians are occupationally exposed to chromium, cobalt and nickel, by analyzing urinary excretion levels of these metals and to investigate the genotoxic effects of occupational exposure associated with dental prostheses production operations by analyzing cytokinesis-blocked micronucleus (CB-MN) frequencies in peripheral lymphocytes and micronucleus (MN) frequencies in exfoliated nasal cells from 27 dental laboratory technicians and 15 control subjects. The differences in the urinary excretion of metals between technicians and controls were statistically significant. The mean (±S.D.) CB-MN frequencies (‰) in peripheral lymphocytes were 4.00 (±2.98) among the dental technicians and 1.40 (±1.30) among the controls, a statistically significant difference (P<0.005). The mean (±S.D.) MN frequencies (‰) in nasal cells were 3.50 (±1.80) among the dental technicians and 1.19 (±0.53) among the controls, which was also a statistically significant difference (P<0.005). There was a significant correlation between duration of exposure and MN frequencies in lymphocytes (r=0.642, P<0.01), but not in nasal cells of technicians. Our data reveal that in vivo exposure to chromium, nickel and cobalt metals is evident and that this occupational exposure may contribute to the observed genotoxic damage in two types of cells, e.g. lymphocytes and exfoliated nasal cells. However, it cannot be determined which compound(s) are responsible for the genotoxic damage observed in this study.     

Smoking-related DNA adducts in anal epithelium

Several studies have identified tobacco smoking as a risk factor for anal cancer in both women and men. Samples of anal epithelium from haemorrhoidectomy specimens from current smokers (n=20) and age-matched life-long non-smokers (n=16) were analysed for DNA adducts by the nuclease P₁ digestion enhancement procedure of ³²P-postlabelling analysis. The study included 14 men and 22 women. Both qualitative and quantitative differences in the adduct profiles were observed between the smokers and non-smokers. The mean adduct level was significantly higher in the smokers than in the non-smokers (1.88±0.71 (S.D.) versus 1.36±0.60 adducts per 10⁸ nucleotides, P=0.02, two-tailed unpaired t-test with Welch’s correction); furthermore, the adduct pattern seen in two-dimensional chromatograms revealed the smoking-related diagonal radioactive zone in 17/20 smokers, but not in any of the non-smokers (P<0.00001, Fisher’s exact test). These results indicate that components of tobacco smoke inflict genotoxic damage in the anal epithelium of smokers and provide a plausible mechanism for a causal association between smoking and anal cancer.     

DNA damage in storage cells of anhydrobiotic tardigrades

In order to recover without any apparent damage, tardigrades have evolved effective adaptations to preserve the integrity of cells and tissues in the anhydrobiotic state. Despite those adaptations and the fact that the process of biological ageing comes to a stop during anhydrobiosis, the time animals can persist in this state is limited; after exceedingly long anhydrobiotic periods tardigrades fail to recover. Using the single cell gel electrophoresis (comet assay) technique to study the effect of anhydrobiosis on the integrity of deoxyribonucleic acid, we showed that the DNA in storage cells of the tardigrade Milnesium tardigradum was well protected during transition from the active into the anhydrobiotic state. Specimens of M. tardigradum that had been desiccated for two days had only accumulated minor DNA damage (2.09 ± 1.98% DNA in tail, compared to 0.44 ± 0.74% DNA in tail for the negative control with active, hydrated animals). Yet the longer the anhydrobiotic phase lasted, the more damage was inflicted on the DNA. After six weeks in anhydrobiosis, 13.63 ± 6.41% of DNA was found in the comet tail. After ten months, 23.66 ± 7.56% of DNA was detected in the comet tail. The cause for this deterioration is unknown, but oxidative processes mediated by reactive oxygen species are a possible explanation.     

Gamma ray induced DNA damage in human and mouse leucocytes measured by SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay

The studies reported in this communication had two major objectives: first to validate the in-house developed SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay using human peripheral blood leucocytes exposed to radiation doses, viz. 2, 4 and 8 Gy, which are known to produce DNA/chromosome damage using alkaline Comet assay. The second objective was to investigate the effect of gamma radiation on DNA damage in mouse peripheral blood leucocytes using identical doses and experimental conditions, e.g. lyses, electrophoretic conditions and duration of electrophoresis which are known to affect tail moment (TM) and tail length (TL) of comets. Human and mouse whole blood samples were irradiated with different doses of gamma rays, e.g. 2, 4 and 8 Gy at a dose rate of 0.668 Gy/min between 0 and 4°C in air. After lyses, cells were electrophorased under alkaline conditions at pH 13, washed and stained with propidium iodide. Images of the cells were acquired and analyzed using in-house developed imaging software, SCGE-Pro, for Comet assay. For each comet, total fluorescence, tail fluorescence and tail length were measured. Increase in TM and TL was considered as the criteria of DNA damage. Analysis of data revealed heterogeneity in the response of leucocytes to gamma ray induced DNA damage both in human as well as in mouse. A wide variation in TM and TL was observed in control and irradiated groups of all the three donors. Data were analyzed for statistical significance using one-way ANOVA. Though a small variation in basal level of TM and TL was observed amongst human and mouse controls, the differences were not statistically significant. A dose-dependent increase in TM (P<0.001) and TL (P<0.001) was obtained at all the radiation doses (2–8 Gy) both in human and mouse leucocytes. However, there was a difference in the nature of dose response curves for human and mouse leucocytes. In human leucocytes, a linear increase in TM and TL was observed up to the highest radiation dose of 8 Gy. However, in case of mouse leucocytes, a sharp increase in TM and TL was observed only up to 4 Gy, and there after saturation ensued. In human samples, the dose response of both TM and TL showed best fits with linear model (r_TM=0.999 and r_TL=0.999), where as in mouse, the best fit was obtained with Sigmoid (Boltzman) model. From the present data on leucocytes with increase in TM and TL as the criteria of DNA damage, it appears that mouse is relatively more sensitive to radiation damage than humans.     

Prednisolone enhances aminopeptidase turnover in adult rat small intestine

In adult male rats, fed prednisolone (0.75 mg/kg/day) for 7 days, brush border aminopeptidase activity was increased (P < 0.001) by 106% compared to pair-fed controls. [¹⁴C]Tyrosine was injected intraperitoneally 16 h and [³H]tyrosine 6 h before death. The ³H/¹⁴C ratio was 1.79 ± 0.21 (S.D.) in purified microvillus membranes from treated rats compared to 1.30 ± 0.16 (P < 0.01) in controls. Polyacrylamide gel electrophoresis of brush border membranes under denaturing conditions showed that the increased double-isotope ratio in membranes from treated rats was mainly in the high molecular weight protein subunits (> 80 kDa) Detergent-solubilized aminopeptidase was purified after in vivo labeling by protein A-Sepharose-antiaminopeptidase affinity chromatography. The ³H/¹⁴C ratio in aminopeptidase was 2.42 ± 0.15 (P < 0.05) in treated rats compared to 1.63 ± 0.13 in controls. Over the experimental period steady-state isotope reutilization and protein labeling was demonstrated and there was no isotope metabolism. Total microvillus membrane lipid content was unaffected by prednisolone. We conclude that prednisolone increases brush border aminopeptidase activity by increasing enzyme turnover. Other high molecular weight brush border proteins were similarly affected.     

Inter and intra-species-related differences in the regulation of the cardiac autonomic system

The heart rate response to isoproterenol (HR-Iso), density and affinity (k_d) of β-adrenergic (β-AR) and muscarinic (M₂) receptors were compared among three rodents with different generation-life histories of confinement and of high altitude exposure. The European guinea pig (Cavia porcellus) (EGp), a laboratory animal that arrived in Europe after the Spanish Conquest of South America and the Peruvian guinea pig (C. porcellus) (PGp), a semi-wild animal that came from the altiplano to sea level at least 25 generations ago, were used for intra-species comparison. Wistar rats (WR) were used for inter-species comparison as representative of a typical sea level laboratory animal. The HR-Iso was lower in EGp than in the PGp. The PGp showed the highest β-AR density (P<0.0005) and the highest β-AR k_d values (P<0.0005) when compared to both EGp and WR groups (β-AR B_max (fmol mg⁻¹ prot), WR, 19±4; Egp, 34±10; PGp, 74±15. β-AR k_d (pM), WR, 24±10; Egp, 17±7; PGp, 39±14). In contrast, PGp showed lower M₂ receptor density values than the EGp (P<0.0005). The WR had the highest M₂ receptor densities (M₂ B_max (fmol mg⁻¹ prot), WR, 188±15; Egp, 147±9; PGp, 118±6 and M₂ k_d (pM), WR, 65±12; Egp, 67±6; PGp, 92±2). The inter and intra-species differences found may be related to their respective history of confinement rather than to their history of exposure to high altitude.     

Early changes in adenosine A1 receptors in cerebral cortex slices submitted to in vitro ischemia

The effects of brain ischemia on the maximum binding capacity (B_max) and affinity (K_d) of A₁ receptors were studied in the rat cerebral cortex, with an in vitro approach. The results were correlated with changes in ³H-adenosine release, studied under identical experimental conditions. Fifteen minutes of in vitro ‘ischemia’ (hypoxic, glucose-free medium) induced a significant increase in both B_max (2398±132 fmol/mg protein, 151% of the control, P<0.05) and in K_d (2.43±0.12 nM, 161% of the control, P<0.01). At the same time, an increase in tritium efflux from [³H]-adenosine labeled cerebral cortex slices to 324% of the control was observed. A trend toward normalization was evident 5–15 min after ‘reoxygenation’ (restoring normal medium), but the binding parameters were still altered after 60 min (B_max 2110±82 fmol/mg protein, K_d 2.26±0.14 nM, P<0.01 vs the corresponding control) as was adenosine release (196% of the control). These findings suggest that the increased availability of adenosine and its receptors may be a defense mechanism against ischemic injury, while the reduced affinity of A₁ receptors, possibly due to desensitization, may be a sign of ischemia-induced cellular damage.     

Cytogenetic monitoring of industrial radiographers using the micronucleus assay

Industrial radiography is the process of using either gamma-emitting radionuclide sources or X-ray machines to examine the safety of industrial materials. Industrial radiographers are among the radiation workers who receive the highest individual occupational radiation doses. To assess occupationally induced chromosomal damage, we performed the cytokinesis-block micronucleus (CBMN) assay in peripheral lymphocytes of 29 male industrial radiographers, exposed to ionizing radiation for 12.8 years±11.2, in comparison with 24 gender-, age-, and smoking habits-matched controls. The CBMN assay was combined with fluorescent in situ hybridization with a pan-centromeric DNA probe in 17 exposed subjects and 17 controls randomized from the initial populations. The mean cumulative equivalent dose, recorded by film dosimeters, was 67.2 mSv±49.8 over the past 5 years. The mean micronucleated binucleated cell rate (MCR) was significantly higher in the industrial radiographers than in the controls (10.7‰±5.2 versus 6.6‰±3.1, P=0.009); this difference was due to a significantly higher frequency of centromere-negative micronuclei (C−MN) in exposed subjects than in controls (8.5‰±4.9 versus 2.2‰±1.6, P<0.001). The two populations did not significantly differ in centromere-positive micronuclei (C+MN) frequency. These findings demonstrate a clastogenic effect in lymphocytes of industrial radiographers. MCR significantly positively correlated with age in the two groups. After correction for the age effect, MCR did not correlate with duration of occupational exposure. No correlation between radiation doses and MCR, C−MN, and C+MN frequencies was observed. In addition to physical dosimetry records, the enhanced chromosomal damage in lymphocytes of industrial radiographers emphasizes the importance of radiation safety programs.     

DNA damage induced by copper deficiency in cattle assessed by the Comet assay

Cattle hypocuprosis is a well-known endemic disease in several parts of the world. In a previous paper, the clastogenic effect of copper deficiency in cattle has been described although the occurrence of DNA damage was not directly tested. For this reason, the relation between DNA damage assessed by the Comet assay and Cu plasma concentration was studied in Aberdeen Angus cattle.Blood samples were obtained in heparinized Vacutainer^® tubes from 28 female Aberdeen Angus cows during pregnancy or immediately after to give birth. Each sample was divided into two aliquots for Comet assay and Cu plasma determination, respectively. From the 28 cattle sampled, 17 were normocupremic and 11 were hypocupremic.Results obtained showed that whereas the average plasma Cu level in normocupremic cattle was 67.6 μg/dl, in hypocupremic cattle it was 32.1 μg/dl. The increase of DNA damage was mostly evidenced by the decrease of comet degree 1 cells and an increase of comet degree 2 cells. Correlation analysis comparing plasma Cu levels and degree 1 cells showed a correlation coefficient 0.72 (P<0.01). The comparison between plasma Cu levels and comet degree 2 cells was −0.65 (P<0.01). The comparison between plasma Cu levels and the comet length-head diameter medians determined in 23 out of 28 animals showed a correlation coefficient of −0.54 (P<0.01).The induction of DNA damage was clearly supported by the fact that the decrease of plasma Cu levels was correlated with the increase of comet length-head diameter. These findings could be considered as a contribution to the hypothesis that DNA and chromosome damage are a consequence of the higher oxidative stress suffered by hypocupremic animals.     

Effects of cigarette smoking, XRCC1 genetic polymorphisms, and age on basal DNA damage in human blood mononuclear cells

The aim of this study was to investigate the effects of smoking, polymorphisms of XRCC1 codons 194 and 399, and age on levels of basal DNA damage (as measured by an alkaline comet assay) on mononuclear cells in 122 healthy Japanese workers. In the whole group of 122 individuals, the tail moment (TM) values of current smokers (P < 0.001) or former smokers (P = 0.03) were significantly higher than those of nonsmokers. Individuals bearing the XRCC1 399Gln variant allele showed significant increases in TM values in all subjects or in referent subgroups stratified by age or smoking status except in the current smokers group; in contrast, the TM values of individuals bearing the XRCC1 194Trp variant allele were significantly lower than those of individuals bearing wild-type Arg/Arg genotypes. Furthermore, older subjects (≥47 years old) had significantly higher TM values than younger subjects (<47 years old) in all subjects (P = 0.008). Multiple regression analysis indicated that smoking habits, polymorphisms of XRCC1 codons 194 and 399, and age were important variables affecting individuals basal DNA damage.