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To contribute new information to the pyrethroid pesticide α-cypermethrin toxicity profile, we evaluated its effects after oral administration to Wistar rats at daily doses of 2.186, 0.015, 0.157, and 0.786 mg/kg bw for 28 days. Evaluations were performed using markers of oxidative stress, cholinesterase (ChE) activities, and levels of primary DNA damage in plasma/whole blood and liver, kidney, and brain tissue. Consecutive exposure to α-cypermethrin affected the kidney, liver, and brain weight of rats. A significant increase in concentration of the thiobarbituric acid reactive species was observed in the brain, accompanied by a significant increase in glutathione peroxidase (GPx) activity. An increase in GPx activity was also observed in the liver of all α-cypermethrin-treated groups, while GPx activity in the blood was significantly lower than in controls. A decrease in ChE activities was observed in the kidney and liver. Treatment with α-cypermethrin induced DNA damage in the studied cell types at almost all of the applied doses, indicating the highest susceptibility in the brain. The present study showed that, even at very low doses, exposure to α-cypermethrin exerts genotoxic effects and sets in motion the antioxidative mechanisms of cell defense, indicating the potential hazards posed by this insecticide.

Manufacture of lead-containing products has long been associated with various health risks. To get an insight into the related genotoxic risks, we conducted a biomonitoring study in 50 exposed workers and 48 matched controls using a battery of endpoints that sensitively detect the extent of genome instability in peripheral blood lymphocytes. The levels of primary DNA damage were estimated with the alkaline comet assay, while cytogenetic abnormalities were determined with the cytokinesis-block micronucleus (CBMN) cytome assay. Additionally, CBMN slides of 20 exposed and 16 control participants were subjected to fluorescence in situ hybridisation (FISH), coupled with pancentromeric probes to establish the incidence of centromere-positive micronuclei, nuclear buds, and nucleoplasmic bridges. Blood lead levels (B-Pb) were measured with atomic absorption spectrometry. To further characterise cumulative effects of occupational exposure, we measured erythrocyte protoporphyrin (EP) concentrations and delta-aminolevulinic acid dehydratase (ALAD) activity in blood. We also assessed the influence of serum folate (S-folate) and vitamin B12 (S-B12) on genome stability. Compared to controls, occupationally exposed workers demonstrated significantly higher B-Pb (298.36±162.07 vs 41.58±23.02), MN frequency (18.71±11.06 vs 8.98±7.50), centromere positive MN (C+ MN) (8.15±1.8 vs 3.69±0.47), and centromere negative MN (C- MN) (14.55±1.80 vs 4.56±0.89). Exposed women had significantly higher comet tail intensity (TI) and length (TL) than control women. Furthermore, workers showed a positive correlation between age and nuclear buds and MN, between MN and years of exposure, and between S-B12 levels and TI and ALAD activity, while a negative correlation was found between TI and B-Pb. These findings suggest that occupational settings in the manufacture of lead-containing products pose significant genotoxic risks, which calls for developing more effective work safety programmes, including periodical monitoring of B-Pb and genetic endpoints., Profesionalna izloženost u industriji olova štetno utječe na zdravlje radnika. Ovo je istraživanje provedeno na 50 radnika izloženih olovu te 48 ispitanika kontrolne skupine primjenom testova za procjenu genomske nestabilnosti u limfocitima periferne krvi. Razine primarnoga oštećenja DNA procijenjene su alkalnim komet-testom, a citogenetičke abnormalnosti utvrđene su citohalazin blokiranim mikronukleus-testom (CBMN). Na podskupini od 20 izloženih i 16 kontrolnih ispitanika dodatno je proveden mikronukleus-test u kombinaciji s fluorescencijskom in situ hibridizacijom (MN-FISH). Primjenom pancentromernih sonda istražili smo učestalost mikronukleusa pozitivnih na centromere, nuklearnih pupova I nukleoplazmatskih mostova. Razine olova u krvi (B-Pb) izmjerene su atomskom apsorpcijskom spektrometrijom. Kako bismo utvrdili kumulativne učinke profesionalne izloženosti, izmjerili smo koncentracije eritrocitnoga protoporfirina (EP) i aktivnost dehidrataze delta-aminolevulinske kiseline (ALAD) u krvi. Također smo procijenili utjecaj serumskoga folata (S-folata) i vitamina B12 (S-B12) na stabilnost genoma. U usporedbi s podudarnim kontrolama, profesionalno izloženi radnici imali su značajno višu razinu B-Pb (298,36±162,07 vs. 41,58±23,02), učestalost MN-a (18,71±11,06 vs. 8,98±7,50), mikronukleusa pozitivnih na centromere (C+ MN) (8,15±1,8 vs. 3,69±0,47) i mikronukleusa negativnih na centromere (C- MN) (14,55±1,80 vs. 4,56±0,89). Izložene radnice imale su značajno veći intenzitet (TI) i duljinu (TL) repa u komettestu od ženskih kontrola. Nadalje, u radnika je utvrđena pozitivna korelacija između učestalosti jezgrinih pupova i MN-a s dobi, između MN-a i godina izloženosti te između TI-ja i aktivnosti ALAD-a i razina S-B12. Negativna korelacija utvrđena je između TI-ja i B-Pb. Dobiveni rezultati upućuju na to da profesionalna izloženost olovu predstavlja značajan genotoksični rizik, što zahtijeva razvoj učinkovitijih programa zaštite na radu, uključujući povremeno praćenje B-Pb i genetičkih markera.

This paper assessed the potential of trans-placental and -lactational genotoxicity and oxidative stress induction of tembotrione, a naturally derived allelopathic herbicide. Several treatment protocols were applied to measure primary DNA damage by alkaline comet assay in leucocytes and liver. To address the oxidative stress induction, TBARS, ROS, SOD, CA, GSH-Px activity were recorded. The dams were treated from the first gestation day and pups sacrificed after birth. The second treatment protocol comprised treating the dams during gestation and lactation and sacrificing the pups at weaning. The third group of pups comprised offspring of dams that were treated in gestation and lactation and sacrificed in puberty. To address translactational genotoxicity, dams were treated in lactation only. Dams treated in gestation and lactation were sacrificed after reentering the estrous cycle and analyzed for DNA damage and oxidative stress. Tembotrione doses encountered in everyday human exposure, as estimated by the EFSA, were applied in dam treatment in consecutive days (ADI: 0.0004 mg/kg b.w./day, AOEL: 0.0007 mg/kg b.w./day, 1/500 LD50 4.0 mg/kg b.w./day). Although we observed mitigated DNA integrity at the dose of 4.0 mg/kg/b.w./day in female pubertal rats, we can conclude that at the conditions employed in the study low doses of tembotrione do not pose a risk for DNA damage of the offspring of treated dams. Contrary to this, the highest dose significantly affected all the oxidative stress parameters in the liver and plasma of pubertal females, CAT and GSH-Px in the liver of males and ROS and CAT of dams.

Imidacloprid is a neonicotinoid insecticide that acts selectively as an agonist on insect nicotinic acetylcholine receptors. It is used for crop protection worldwide, as well as for non-agricultural uses. Imidacloprid systemic accumulation in food is an important source of imidacloprid exposure. Due to the undisputable need for investigations of imidacloprid toxicity in non-target species, we evaluated the effects of a 28-day oral exposure to low doses of imidacloprid (0.06 mg/kg b. w./day, 0.8 mg/kg b. w./day and 2.25 mg/kg b. w./day) on cholinesterase activity, oxidative stress responses and primary DNA damage in the blood and brain tissue of male Wistar rats. Exposure to imidacloprid did not cause significant changes in total cholinesterase, acetylcholinesterase and butyrylcholinesterase activities in plasma and brain tissue. Reactive oxygen species levels and lipid peroxidation increased significantly in the plasma of rats treated with the lowest dose of imidacloprid. Activities of glutathione-peroxidase in plasma and brain and superoxide dismutase in erythrocytes increased significantly at the highest applied dose. High performance liquid chromatography with UV diode array detector revealed the presence of imidacloprid in the plasma of all the treated animals and in the brain of the animals treated with the two higher doses. The alkaline comet assay results showed significant peripheral blood leukocyte damage at the lowest dose of imidacloprid and dose-dependent brain cell DNA damage. Oral 28-day exposure to low doses of imidacloprid in rats resulted in detectable levels of imidacloprid in plasma and brain tissue that directly induced DNA damage, particularly in brain tissue, with slight changes in plasma oxidative stress parameters.

In agricultural activities, pest control is essential, and the most effective method is the use of chemical agents that also represent an important source of exposure to potentially toxic compounds. Pesticides constitute a heterogeneous group of compounds designed specifically to control different pests. Besides measuring their levels or that of their metabolites in air, plasma, serum, blood, urine, etc., some studies reported increased DNA damage levels after occupational or environmental pesticides exposure, evidenced by several cytogenetic biomarkers such as chromosomal aberrations (CA), sister chromatid exchanges (SCE), micronuclei frequency (MN) together with other nuclear abnormalities (NA), alkaline comet assay, but also changes in oxidative stress parameters and miRNA levels. Single or combined, these techniques have also been used in genotoxic biomonitoring studies of workers occupationally exposed to pesticides in Mexico. Despite being a country with great agricultural activity and reported excessive pesticide use, genotoxic studies have been relatively few and, in some cases, contradictory. A review was made of the studies available (published until the end of 2020 on PubMed, Web of Science, Redalyc and Scielo, both in English and Spanish) in the scientific literature that evaluated occupational exposure of human samples to pesticides assessed with DNA damage and related biomarkers in Mexico.

In parallel with the continuous use of conventional insecticides, introduction of more environmentally friendly substances continues to grow in modern agriculture. In the present study, we evaluated chlorpyrifos, and imidacloprid and a-cypermethrin as two representatives of green insecticides for their genotoxic activity.We conducted a 14-day treatment in extended human lymphocytes cultures using real life exposure relevant concentrations. An alkaline comet assay was used to detect primary DNA damage. Simultaneously, the effect on the specific action towards the TP 53 and c-Myc genes in terms of fragmentation and copy number were determined. Both genes are responsible for cell cycle regulation ; thus playing an active role in carcinogenesis. Contrary to what was expected, imidacloprid showed the highest genotoxicity potential, irrespective of the fact that none of the insecticides induced a significant level of primary DNA damage at all tested concentrations. Similar, no significant effect towards the TP 53 and c- Myc gene was recorded. The present study indicates that low level use of chlorpyrifos as a conventional insecticide and imidacloprid and a-cypermethrin as green insecticides does not pose a risk to DNA in general, nor to the TP 53 and c-Myc gene structural integrity.

In this 28 day-study, we evaluated the effects of herbicide glyphosate administered by gavage to Wistar rats at daily doses equivalent to 0.1 of the acceptable operator exposure level (AOEL), 0.5 of the consumer acceptable daily intake (ADI), 1.75 (corresponding to the chronic population-adjusted dose, cPAD), and 10 mg kg-1 body weight (bw) (corresponding to 100 times the AOEL). At the end of each treatment, the body and liver weights were measured and compared with their baseline values. DNA damage in leukocytes and liver tissue was estimated with the alkaline comet assay. Oxidative stress was evaluated using a battery of endpoints to establish lipid peroxidation via thiobarbituric reactive substances (TBARS) level, level of reactive oxygen species (ROS), glutathione (GSH) level, and the activity of glutathione peroxidase (GSH-Px). Total cholinesterase activity and the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were also measured. The exposed animals gained less weight than control. Treatment resulted in significantly higher primary DNA damage in the liver cells and leukocytes. Glyphosate exposure significantly lowered TBARS in the liver of the AOEL, ADI, and cPAD groups, and in plasma in the AOEL and cPAD group. AChE was inhibited with all treatments, but the AOEL and ADI groups significantly differed from control. Total ChE and plasma/liver ROS/GSH levels did not significantly differ from control, except for the 35 % decrease in ChE in the AOEL and ADI groups and a significant drop in liver GSH in the cPAD and 100xAOEL groups. AOEL and ADI blood GSH-Px activity dropped significantly, but in the liver it significantly increased in the ADI, cPAD, and 100xAOEL groups vs. control. All these findings show that even exposure to low glyphosate levels can have serious adverse effects and points to a need to change the approach to risk assessment of low-level chronic/sub-chronic glyphosate exposure, where oxidative stress is not necessarily related to the genetic damage and AChE inhibition., U okviru 28-dnevnog pokusa istražili smo učinke herbicida glifosata na modelu odraslih mužjaka Wistar štakora koji su oralno dobivali testirani spoj u subletalnim dnevnim dozama: 0,1 od prihvatljive razine izloženosti operatera (0,1xAOEL), 0,5 od prihvatljivog dnevnog unosa za potrošače (0,5xADI), 1,75 (odgovara kroničnoj populacijskoj prilagođenoj dozi, cPAD) i 10 mg kg-1 tjelesne težine na dan (odgovara 100xAOEL). Tijekom pokusa praćeni su sistemski toksični učinci. Nakon završetka svih tretmana svakoj je pokusnoj životinji izmjerena tjelesna težina i težina jetre te su uspoređene s polazišnim vrijednostima. Alkalnim komet-testom izmjerena je razina primarnih oštećenja DNA u leukocitima i jetrenim stanicama. Primjenom metoda za procjenu oksidacijskog stresa izmjerene su razine lipidne peroksidacije (TBARs), reaktivnih kisikovih vrsta (ROS) i glutationa (GSH) te aktivnost enzima glutation peroksidaze (GSH-Px). Izmjerene su i aktivnosti ukupnih kolinesteraza (ChE), acetilkolinesteraze (AChE) i butirilkolinesteraze (BChE). Izloženi štakori imali su manje priraste težine od kontrolnih. Izloženost glifosatu uzrokovala je značajne poraste razine primarnih oštećenja DNA u jetrenim stanicama te malo manje u leukocitima. U svim izloženim skupinama izmjerene su niže vrijednosti TBARs u odnosu na kontrolu, sa značajno nižim vrijednostima u AOEL, ADI i cPAD skupinama u uzorcima jetre te u AOEL i cPAD skupinama u uzorcima plazme. Aktivnost AChE bila je smanjena u svim tretmanima, s najnižom stopom nakon izlaganja dozi ADI. Aktivnost BChE blago je smanjena nakon izlaganja ADI, a povećana nakon izlaganja dozama cPAD i 100xAOEL. Ukupna aktivnost ChE te razine ROS/GSH u plazmi / jetri nisu se značajno razlikovale od kontrole, osim značajnog smanjenja jetrenog GSH nakon izlaganja dozama cPAD i 100xAOEL te 35-postotnog smanjenja aktivnosti ChE nakon izlaganja dozama AOEL i ADI. Aktivnost GSH-Px u krvi značajno je smanjena u AOEL i ADI tretmanu, a aktivnost GSH-Px u uzorcima jetre značajno je povećana u skupinama ADI, cPAD i 100xAOEL prema kontroli. Dobiveni rezultati pokazuju da čak i izloženost vrlo niskim dozama glifosata može izazvati mjerljive toksične učinke te upućuje na potrebu za promjenom pristupa procjeni rizika zbog kronične/subkronične izloženosti niskim dozama glifosata gdje oksidacijski stres ne mora nužno korelirati s razinom oštećenja DNA i inhibicijom acetilkolinesteraze

Individual sensitivity to ionising radiation (IR) is the result of interaction between exposure, DNA damage, and its repair, which is why polymorphisms in DNA repair genes could play an important role. We examined the association between DNA damage, expressed as micronuclei (MNi), nuclear buds (NBs), and nucleoplasmic bridges (NPBs) and single nucleotide polymorphisms in selected DNA repair genes (APE1, hOGG1, XRCC1, XRCC3, XPD, PARP1, MGMT genes; representative of the different DNA repair pathways operating in mammals) in 77 hospital workers chronically exposed to low doses of IR, and 70 matched controls. A significantly higher MNi frequency was found in the exposed group (16.2±10.4 vs. 11.5±9.4; P=0.003) and the effect appeared to be independent from the principal confounding factor. Exposed individuals with hOGG1, XRCC1, PARP1, and MGMT wild-type alleles or APEX1, as well as XPD (rs13181) heterozygous showed a significantly higher MNi frequency than controls with the same genotypes. Genetic polymorphism analysis and cytogenetic dosimetry have proven to be a powerful tool complementary to physical dosimetry in regular health surveillance programmes.

In this 28 day-study, we evaluated the effects of the insecticide chlorpyrifos orally administered to Wistar rats at doses 0.160, 0.015, and 0.010 mg/kg b. w./day. Following treatment, total cholinesterase activity and activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were measured. Oxidative stress responses were evaluated using a battery of endpoints to establish lipid peroxidation, changes in total antioxidant capacity, level of reactive oxygen species (ROS), glutathione (GSH) level and activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase. Using HPLC-UV DAD analysis, levels of the parent compound and its main metabolite 3,5,6-trichloro-2-pyridinol in plasma and brain tissue were measured. The genotoxic effect was estimated using alkaline comet assay in leukocytes and brain tissue. The exposure did not result in significant effects on total cholinesterase, AChE and BChE activity in plasma and brain tissue. Lipid peroxidation slightly increased both in plasma and brain tissue. Total antioxidant capacity, ROS and GSH levels were marginally influenced by the exposure. Treatment led to significant increases of GSH-Px activity in blood, SOD activity in erythrocytes and a slight increase of catalase activity in plasma. HPLC-UV DAD analysis revealed the presence of both the parent compound and its main metabolite in the plasma of all of the experimental animals and brain tissue of the animals treated at the two higher doses. All of the tested doses of chlorpyrifos were slightly genotoxic, both to leukocytes and brain tissue. Our results call for further research using other sensitive biomarkers of effect, along with different exposure scenarios.

Terbuthylazine is a selective pre- and post- emergency chloro-triazine herbicide used for a broad spectrum of weed control. We evaluated the potential of low doses of terbuthylazine to induce oxidative stress and cytogenetic damage in peripheral blood samples of adult male Wistar rats. Following 28-day repeated oral exposure at 0.004 mg/kg b.w./day, 0.4 mg/kg b.w./day and 2.29 mg/kg b.w./day, parameters of lipid peroxidation, total antioxidant capacity, and activities of antioxidant enzymes were measured in blood samples. Alkaline comet assay on leukocytes and erythrocyte micronucleus assay were used to measure DNA damage. In addition, the concentration of terbuthylazine and its metabolite in urine and plasma were determined using high performance liquid chromatography with UV diode-array detector (HPLC-UV-DAD). The fraction of terbuthylazine excreted in urine was negligible and was not found in plasma. Deethylterbuthylazine was only compound detected in plasma samples. Exposure to terbuthylazine did not induce significant lipid peroxidation products. The significant changes in antioxidant enzyme activities and the elevated total antioxidant capacity indicated that terbuthylazine at experimental conditions applied has potential to disturb oxidative/antioxidant balance. Results regarding the alkaline comet assay as well as micronucleated reticulocyte frequency indicated that treatment led to low-level DNA instability. Our results call for further research using other sensitive biomarkers of effect, along with different exposure scenarios.

Tembotrione is a triketone herbicide, usually used for post-emergence weed control in corn. Currently, there is little or no published data on its genotoxicity to human cells either in vitro or in vivo. This study evaluated the impact of acute (4 and 24 h) exposure to low concentrations of tembotrione [corresponding to the acceptable daily intake (0.17 μg/mL), residential exposure level (0.002 μg/mL) and acceptable operator exposure level (0.0012 μg/mL)] on human hepatocellular carcinoma cell line HepG2, using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell viability, alkaline comet assay, and cytokinesis-block micronucleus “cytome” assay. Tembotrione applied at concentrations likely to be encountered in occupational and residential exposures induced cytogenetic outcomes in non-target cells despite non-significant changes in the values of oxidative stress biomarkers. We assume that the observed effects were mainly the consequence of impaired metabolic pathways in HepG2 cells due to the inhibition of the enzyme 4-hydroxyphenyl-pyruvate-dioxygenase by tembotrione, which possibly caused a depletion of folate levels leading to excess formation of nuclear buds in the affected cells. Regardless of the fact that tembotrione was previously reported negative for mutations and chromosome aberrations in vitro, our findings call for more precaution in its use.

This study evaluated the cyto- and genotoxic effects of three pesticides: α-cypermethrin, chlorpyrifos and imidacloprid applied in vitro to human lymphocytes and HepG2 cells for exposure times of 4 and 24 h at concentrations corresponding to OEL, ADI and REL. Assessments were made using oxidative stress biomarkers and the alkaline comet, cytokinesis-block micronucleus cytome and cell viability assays. Low doses of all three pesticides displayed DNA damaging potential, both in lymphocytes and HepG2 cells. At the tested concentrations, all three compounds induced lymphocyte apoptosis, though α-cypermethrin and chlorpyrifos were generally more cyto- and genotoxic than imidacloprid. At the tested concentrations, oxidative stress biomarkers were not significantly altered, and the effects mediated indirectly through free radicals may not have a key role in the formation of DNA damage. It is likely that the DNA damaging effects were caused by direct interactions between the tested compounds and/or their metabolites that destabilized the DNA structure. The tested pesticides had the potential for MN, NB and NPB formation and to disturb cell cycle kinetics in both cell types. There were also indications that exposure to α-cypermethrin led to the formation of crosslinks in DNA, though this would require more detailed study in the future.

This study was aimed at investigating the genotoxic potential of single beauvericin (BEA) and ochratoxin A (OTA) as well as their interaction in porcine kidney epithelial PK15 cells and human leukocytes using the alkaline comet assay. IC(50) of BEA (5.0 +/- 0.6) and OTA (15.8 +/- 1.5) estimated by MTT reduction assay shows that BEA is three times more toxic than OTA. BEA (0.1 and 0.5 microM) and OTA (1 and 5 microM) were applied alone or in combination of these concentrations for 1 and 24 h in PK15 cells and human leukocytes. Genotoxicity of these toxins to PK15 cells was time- and concentration dependent. After 1 h, significant increase in tail length, tail intensity, tail moment, and abnormal sized tails (AST) was noted upon exposure to 1 muM of OTA alone and BEA + OTA combinations. Single BEA (0.5 microM) and OTA (1 and 5 microM) and their combinations evoked significant DNA damage in PK15 cells, considering all comet tail parameters measured after 24 h of treatment. Human leukocytes were slightly concentration but not time dependent. After 1 h of exposure, there were no significant changes in the tail length. Tail intensity, tail moment, and/or incidence of AST were significantly higher in cells treated with single OTA or BEA and their combinations than in control cells. DNA damage in leukocytes was significantly higher after 24 h of exposure to single toxins and their combinations, considering all comet tail parameters, but these changes were less pronounced than in PK15 cells. Combined toxins showed additive and synergistic effects in PK15 cells, while only additive effects were observed in human leukocytes. Combined prolonged exposure to BEA and OTA in subcytotoxic concentrations through food consumption could induce DNA damage contributing to the carcinogenicity in animals and humans.

To investigate whether occupational exposure to tobacco dust is genotoxic, a group of employees in a tobacco factory was tested for structural chromosome aberrations (CA), cytokinesis-block micronucleus assay (CBMN) and sister chromatid exchanges (SCE) that are well established as indicators of early biological effects. The study group consisted of 40 tobacco workers and an equal number of matched controls. The results obtained in the exposed group showed a significant increase in chromosome aberrations (R = 0.26), micronucleus frequency (R = 0.56) and in sister chromatid exchanges (R = 0.75), which was additionally influenced by smoking. A significant increase in high frequency cells (HFC) in the exposed group was also observed. Like the SCE frequency, the HFC frequency increased significantly in smokers of the control and exposed smokers. The study indicates that occupational exposure to tobacco dust induces genome damage. A higher risk was observed in women. The micronucleus frequency and sister chromatid exchange tests seem to be more reliable indicators of genome damage than chromosome aberrations in monitoring chronically exposed subjects.

Question: Genotoxic effect of alpha cypermethrin (type II pyrethroid insecticide) and Imidacloprid (neonicotinoid insecticide) were evaluated on human hepatoma HepG2 cell line. Concentrations used corresponded to real-life exposures will be evaluated based on residential exposure levels (REL α cypermethrin= 0.003643 µg/mL ; REL Imidacloprid= 0.314 µg/mL), occupational exposure limits (OEL) for alpha cypermethrin 0.262 µg/mL, and AOEL (acceptable operator exposure level) for Imidacolprid, 0.13 µg/mL. Methods: Using the in vitro model of cell cultures, genotoxicity evaluation was made. In accordance with OECD guidelines, acute treatments (4h and 24h) was performed. Primary DNA damage was assessed by applying the alkaline comet assay. DNA damage was evaluated using two genotoxicity parameters: tail length and tail intensity, as well as LTN (long tail nuclei). Results: Concentration and time dependent genotoxic effects were observed in both compounds. The highest LTN frequencies observed after 24 h treatment with the α-cypermethrin was 35%. Frequencies of observed LTN reflected well actual concentration range, of both compounds tested (LTNREL Imidacloprid > LTNOEL α cypermethrin > LTNAOEL Imidacloprid > LTNREL α cypermethrin). Conclusions: Positive findings obtained by alkaline comet assay will serve to significantly extend the knowledge of both pollutants genotoxicity and might to contribute to the more relevant risk assessment. This work was financially supported by the Project No. 8366 Organic Pollutants in Environment - Markers and Biomarkers of Toxicity (OPENTOX), funded by the Croatian Science Foundation.

Previous reports indicated adverse effect of herbicides on TP53 as a gene which damage promotes carcinogenesis. We evaluated three insecticides, with an emphasis on green agriculture, on their TP53 gene fragmentation and primary DNA damaging potential. Chlorpyrifos, α-cypermethrin as widely used representative of pyrethroids, and imidacloprid as a neonicotinoid, were tested in low concentrations equivalent of acceptable daily intake, residential exposure level, occupational exposure limit for each of insecticides. Common concentration of 3 µg/ml also was tested. Human peripheral blood lymphocytes were treated in extended cultures for 14 days. Comet assay endpoints, rates of TP53 gene fragmentation and deletion were assessed by comet-FISH technique. No increase in tail length or tail intensity compared to control (13.8±1.19 µm and 0.09±0.20% of DNA, respectively) was observed ; only for 3 µg/ml of imidacloprid tail length significantly increased (14.5±1.06 µm). Concerning TP53 structural integrity, no significant increase in rate of gene fragmentation or deletion was detected compared to control (6.7% and 3.3%, respectively). Statistically insignificant increase in TP53 migration in the comet tail was spotted at 3 µg/ml of α-cypermethrin (16.7%) and imidiacloprid (13.3%). Insignificant increase in TP53 deletion rate was observed for α-cypermethrin at 3 µg/ml (10.0%) compared to control (3.3%). Obsereved effects on level of primary DNA damage and TP53 structural integrity and copy number could not be considered biologically significant. At the low concentrations in extended term exposure applied in the study all three insecticides may be considered safe. This work was supported by Croatian Science Foundation under the project 8366.

Tembotrione is a selective, triketone herbicide used for post-emergence weed control in corn. Some pesticides may act as endocrine disrupting chemicals because of their strong potential to bind to oestrogen and androgen receptors and change sex hormone status in males and females. Especially vulnerable groups are foetuses, infants and children in puberty. We investigated the effects of tembotrione exposure during gestation on oestradiol and testosterone levels in female and male neonatal Wistar rats at doses relevant to real human exposure. Pregnant Wistar rats were exposed orally to 0.0004 mg/kg b.w./day and 0.0007 mg/kg b.w./day of tembotrione each day during the entire gestation period. After delivery, blood samples of newborn rats were collected and oestradiol level was measured in female and testosterone level in male serum by enzyme- linked immunosorbent assay (ELISA). We observed significantly increased serum oestradiol levels in female neonatal rats at 0.0007 mg/kg b.w./day. Similarly, significantly increased serum testosterone levels in male neonatal rats at 0.0004 mg/kg b.w./day were recorded. We also found a statistically significant difference between testosterone levels in male neonatal rats exposed to 0.0004 mg/kg b.w./day and 0.0007 mg/kg b.w./day. Our findings suggest that exposure to tembotrione during intrauterine development disturbs sexual hormone levels both in female and male neonatal rats. This may lead to different adverse health consequences later in adulthood. This work was financially supported by Project No. 8366 funded by the Croatian Science Foundation.

The use of iron oxide nanoparticles (Fe2O3 np) in medical diagnostics and therapy is growing in recent years. Concern about the possible adverse effects on living organisms and the environment has prompted research on its toxicity and genotoxicity. The aim of this study was to investigate the possible genotoxic effects of chronic exposure of rats to Fe2O3 np. Twenty-five albino male Wistar rats aged 10 weeks were divided into groups of five animals. Three groups were orally treated with Fe2O3 np (

The purpose of this study was to evaluate the genotoxic potential of components leached from two conventional self-curing glass-ionomer cements (Fuji IX and Ketac Molar), and light-curing, resin modified glass-ionomer cements (Vitrebond, Fuji II LC). Evaluation was performed on human lymphocytes using alkaline and hOGG1 modified comet, and micronucleus assays. Each material, polymerised and unpolymerised, was eluted in extracellular saline (1 cm2 mL-1) for 1 h, 1 day, and 5 days. Cultures were treated with eluates using final dilutions of 10-2, 10-3, and 10-4. Alkaline comet assay did not detect changes in DNA migration of treated cells regardless of the ionomer tested, polymerisation state, and elution duration. Glass ionomers failed to significantly influence micronucleus frequency. No oxidative DNA damage in treated lymphocytes was observed using hOGG1 modified comet assay. Obtained results indicate high biocompatibility of all tested materials used in the study under experimental conditions.

The genotoxic effect of occuaptional exposure of 20 nurses who handled cytostatic drugs in medical oncology and haematology units was evaluated by micronucleus and sister chromatide exchange test. The duration of employment in the units and of exposure to cytostatics ranged from 1 to 31 years. The exposed nurses manifested an increase in cells with micronuclei as compared to the control group (P0.05). The influence of the exposure period proved to be a significant parameter for the micronucleus test. No statistically significant differences were observed in sister chromatid exchange (P>0.05).

Purpose We investigated genotoxic eVects of occupational exposure to lead acetate in pottery-glaze ceramic workers. Methods The study was carried out in 30 exposed workers and 30 matched controls, to whom several biochemical parameters—the blood lead (B-Pb ; range: exposed, 41.68– 404.77 ; controls, 12–52) and cadmium (B-Cd) level, the activity of delta-aminolevulinic acid dehydratase (ALAD), erythrocyte protoporphyrin (EP), the level of vitamin B12 and folate in serum—were measured. The genotoxic eVects were evaluated by the alkaline comet assay, the DNA diVusion assay and micronucleus test in peripheral blood lymphocytes. Results Subjects exposed to lead had signiWcantly higher B-Pb level and, consequently, increased values of tail intensity (TI), frequency of apoptotic and necrotic cells, and frequency of micronuclei (MN). In contrast, their activity of ALAD, the level of vitamin B12 and folate in serum were signiWcantly lower compared to controls. Poisson regression analysis showed a signiWcant correlation of profession, duration of exposure, smoking, level of cadmium in blood, ALAD and EP with primary DNA damage. A majority of primary damage repairs in a short period after exposure to a genotoxic agent. In addition, the inXuence of gender and level of vitamin B12 and folate in serum MN frequency in exposed group was observed. Conclusions In this study, DNA diVusion and micronucleus test showed higher inXuence of tested parameters to DNA damage. The results indicate a need for concomitant use of at least two diVerent biomarkers of exposure when estimating a genetic risk of lead exposure.

Assessment of Cyto/Genotoxicity of Irinotecan in V79 Cells Using the Comet, Micronucleus, and Chromosome Aberration AssayIrinotecan is a topoisomerase I interactive agent, widely used in the treatment of metastatic colorectal cancer. The genotoxic effects of the maximum single dose (18 μg mL-1), recommended monotherapy dose (9 μg mL-1), and recommended combined therapy dose (4.5 μg mL-1) of irinotecan were studied on V79 cells using the comet assay, chromosome aberration assay, and micronucleus test. The cells were treated with irinotecan for 2 h or 24 h. The statistical significance of the results was determined using the one-way ANOVA test and a nonparametric Mann Whitney U test. The comet assay did not show dose-dependent or time-dependent effects. The chromosome aberration analysis showed large DNA rearrangements, i.e., chromosome exchanges. Although the exposed cultures showed a significant increase in micronucleated cells in respect to control, no dose-dependent relation was established among the treated cultures. Time-dependent effect was also not observed.

U članku je prikazana osnovna podjela antineoplastičnih lijekova prema mehanizmima djelovanja na razini stanice. Objašnjeni su mehanizmi genotoksičnosti najvažnijih vrsta lijekova koji se primjenjuju u okviru uobičajenih protokola za liječenje zloćudnih novotvorina. Navedena je važeća klasifi kacija antineoplastika prema kancerogenom potencijalu, podaci o mutagenom potencijalu te je prikazana njihova podjela u skladu s anatomsko-terapijsko-kemijskim sustavom klasifi kacije. Sustavno su prikazani najvažniji rezultati svjetskih i hrvatskih istraživanja na populacijama radnika izloženih antineoplasticima, provedenih u razdoblju 1980.-2009. s pomoću četiri najčešće primjenjivane metode: analize izmjena sestrinskih kromatida, analize kromosomskih aberacija, mikronukleus-testa i komet-testa. Objašnjena su osnovna načela navedenih metoda te raspravljene njihove prednosti i nedostaci. Biološki pokazatelji daju važne podatke o individualnoj osjetljivosti profesionalno izloženih ispitanika koji mogu poslužiti unaprjeđenju postojećih uvjeta rada i upravljanju rizicima pri izloženosti genotoksičnim agensima. Na osnovi prednosti i nedostataka citogenetičkih metoda zaključeno je da je mikronukleus-test, koji podjednako uspješno dokazuje klastogene i aneugene učinke, jedna od najboljih metoda dostupnih za otkrivanje štetnih djelovanja antineoplastičnih lijekova koji su u aktivnoj primjeni., This article brings an overview of the mechanisms of action of antineoplastic drugs used in the clinical setting. It also describes the genotoxic potentials of the most important classes of antineoplastic drugs involved in standard chemotherapy protocols. Classifi cation of antineoplastic drugs according to the IARC monographs on the evaluation of carcinogenic risks to humans is accompanied by data on their mutagenicity and the most recent updates in the Anatomical Therapeutic Chemical (ATC) Classifi cation System. We report the main fi ndings of biomonitoring studies that were conducted in exposed healthcare workers all over the world between 1980 and 2009 using four biomarkers: sister chromatid exchanges, chromosome aberrations, micronuclei. and the comet assay. The methods are briefl y explained and their advantages and disadvantages discussed. Biomarkers provide important information on individual genome sensitivity, which eventually might help to improve current working practices and to manage the risks related with exposure to genotoxic agents. Taking into consideration all known advantages and drawbacks of the existing cytogenetic methods, the micronucleus assay, which is able to detect both clastogenic and aneugenic action, is the most suitable biomarker for assessing harmful effects of antineoplastic drugs currently used in health care.

The influence of exposure to lead on the frequency of micronuclei (MN), nuclear buds and nucleoplasmatic bridges was investigated in peripheral blood lymphocytes in 15 male battery-manufacturing workers and 15 controls matched for age and smoking habits. In addition to MN test, blood lead (B-Pb) and cadmium (B-Cd), delta aminolevulinic acid dehydratase (ALAD) activity, erythrocyte protoporphyrin (EP), serum vitamin B12 (S-Vit B12) and folate (S-folate) were determined in all subjects. Lead-exposed subjects had significantly higher MN frequency and B-Pb concentrations than controls. In control smokers we found a significant negative correlation between B-Pb concentration and frequency of nucleoplasmatic bridges, and nuclear division index. In control non-smokers a significant positive correlation was observed only between age and nuclear buds frequency, and between S-folate and B-Pb level. In lead exposed smokers, significant positive correlations between MN frequency and S-Vit B12, S-folate, and nuclear buds frequency were found. A positive correlation in exposed smokers was also found between nuclear buds frequency and S-Vit B12 concentration. A negative correlation was found between ALAD and EP, and B-Pb in exposed smokers. Exposed non-smokers showed significant negative correlation between MN frequency and B-Cd, and ALAD and EP. The results indicate a genotoxicity of lead, pointing to a micronucleus assay as a relevant test for assessing genotoxic effects resulting from occupational exposure. The other indicators did not necessarily follow the results of MN test. Influence of smoking should be further investigated. Copyright Ó John Wiley & Sons, Ltd.

Mikronukleus (MN) test na limfocitima periferne krvi jedna je od najvažnijih metoda koje se primjenjuju u citogenetičkom nadzoru. Osnovni preduvjet za primjenu nekog testa u svrhu nadzora profesionalno izloženih populacija jest poznavanje normalnih vrijednosti promatranoga biološkog pokazatelja (biomarkera) u kontrolnoj populaciji. Baze podataka na razini opće populacije moraju se redovito obnavljati novim podacima. Cilj ovog istraživanja bio je utvrditi normalne i granične vrijednosti MN-testa na limfocitima periferne krvi 200 zdravih ispitanika obaju spolova iz opće populacije Republike Hrvatske te ispitati koji čimbenici pridonose spontanom nastanku MN. Na razini istražene populacije utvrđeno je prosječno (6,90±3,32) MN (medijan 7 MN), dok je raspon pojedinačnih vrijednosti iznosio 0 do 18 MN u 1000 binuklearnih stanica. Gornja granična vrijednost dobivena izračunavanjem 95. percentila za cjelokupnu promatranu populaciju iznosi 12,5 MN na 1000 limfocita. Utvrđeno je da na spontani nastanak MN utječu spol, dob i navika pušenja. Žene u prosjeku imaju više vrijednosti svih parametara MN-testa od muškaraca, a u njih je bio i naglašeniji porast vrijednosti citogenetičkog nalaza zbog navike pušenja. Kako su literaturni podaci o utjecaju pušenja cigareta na nastanak MN kontradiktorni, planiran je nastavak istraživanja radi razjašnjavanja utjecaja dnevno utrošenog broja cigareta i ukupnog trajanja pušačkog staža na vrijednosti parametara MN-testa. Usporedba rezultata s literaturnim podacima potvrdila je da su dobivene vrijednosti u skladu s vrijednostima MN-testa zabilježenim na općoj populaciji u drugim svjetskim laboratorijima. Normalne i granične vrijednosti MN-testa utvrđene u ovome istraživanju poslužit će kao osnova za usporedbu i tumačenje nalaza MN-testa u ispitanika izloženih populacija te daljnju nadogradnju laboratorijske baze podataka., The cytokinesis-block micronucleus (CBMN) assay on peripheral blood lymphocytes is one of the most important methods employed in cytogenetic biomonitoring. For the purposes of biological dosimetry, it is important to kno the spontaneous frequency of a biomarker and its normal values in general population. These values are used for population databases, which should be updated regularly. In this study, MN levels were investigated in cytokinesis-blocked lymphocytes of 200 healthy male and female blood donors selected at random from the general population of Croatia. The aim was to assess the variability and determine possible infl uences of external and/or internal factors on the background levels of MN and to establish the cut-off value for the CBMN assay. The background frequency of MN was (6.90±3.32) MN (median 7 MN) and the range was 0 to 18 MN per 1000 binuclear lymphocytes. The cut-off value, which corresponds to 95th percentile of the distribution of 200 individual values, was 12.5 MN. Spontaneous formation of MN was infl uenced by sex, age, and smoking. Women had higher MN levels than men. However, only age and smoking signifi cantly increased the values of all parameters evaluated by the CBMN assay. Since the existing literature data on smoking-related formation of MN are contradictory, we will continue these investigations to resolve how the number of cigarettes smoked per day and the duration of smoking in years infl uence the results of the CBMN assay. Our results are consistent with the background MN frequencies reported by other cytogenetic laboratories worldwide. Normal and cut-off values estimated in this study will be used to update the current general population data and as reference for occupationally or accidental exposure.

Genetic damage induced by anaesthetic gases in occupationally exposed populations was investigated using the comet assay and micronucleus test. The study included two groups of subjects: 50 operating theatre medical workers (anaesthesiologists, technicians and nurses) and 50 control subjects corresponding in sex, age and smoking habit. The exposed group revealed an increase in genome damage in both tests. In the comet assay, exposure to anaesthetics was a highly significant predictor of the tail length for technicians, while sex proved to be significant predictor of tail moment for women in exposed group. Micronucleus frequency increased significantly, showing threefold increase in exposed groups (RR>3.029). Univariate analysis showed significant influence of duration of exposure, while multivariate analysis showed age to be significant predictor of micronucleus frequency. The obtained results call for further, targeted investigation of exposure risk.

Chromosome Aberrations in Peripheral Blood Lymphocytes of Croatian Hospital Staff Occupationally Exposed to Low Levels of Ionising RadiationMedical staff is an occupational group exposed to different agents suspected to induce genetic damage. Among them ionising radiation is the most studied. Cytogenetic analysis of human chromosomes in peripheral lymphocytes allows direct detection of mutation in somatic cells. This study investigated the cytogenetic effects of low-level ionising x-radiation in 48-hour peripheral blood lymphocyte cultures sampled from 765 hospital staff occupationally exposed to several agents known or suspected to induce chromosome damage and compared them with 200 control subjects. The exposed subjects were divided in eight (8) groups according to their specialities and job titles. The exposed groups manifested an increase in all types of chromosome aberrations. Acentric fragments were the most frequent chromosome-type aberration. Dicentric chromosomes were statistically significant only in urologists/gynaecologists. Age and smoking significantly influenced the incidence of dicentrics in the exposed groups. The frequency of ring chromosomes was low in all exposed groups (range: 0-2), and none were found in the control group. These findings indicate the importance of periodic medical checkups of hospital staff occupationally exposed to low doses of ionising radiation. The purpose is to create an individual cytogenetic register, where changes could evidence individual risks.

Lead and lead compounds are important pollutants in human living and working environment. Numerous studies have shown that lead adversely affects the nervous, haematopoietic, renal, endocrine and reproductive systems. The genotoxic effects of lead have been studied both in vivo and in vitro, but the published results are inconsistent. The aim of our study was to investigate the level of exposure to lead and its genotoxic effects in lymphocytes of occupationally exposed subjects. The study group consisted of 35 lead workers, 21 men and 14 women, and 35 control subjects matching in age, sex and the smoking habit. Blood lead concentrations were analysed using electrothermal atomic absorption spectrometry. The comet assay was used to evaluate the primary DNA damage. Mean blood lead level in the exposed group was ten times as high as in controls (344 µ ; g/l vs. 36 µ ; g/l), but the mean tail length (TL) and tail moment (TM) did not differ between the studied groups (15.12 vs. 15.48 and 0.28 vs. 0.33, respectively). However, significantly higher values of TL and TM were observed in some individuals in the exposed group. Although men had significantly higher blood lead concentrations, women with significantly higher TL and TM values showed to be more susceptible to lead exposure.

The aim of this study was to evaluate possible genotoxic damage of cadmium chloride exposure in suckling rats by means of the comet assay and the in vivo micronucleus test of rat blood lymphocytes, because no information is available on the genotoxic effect of cadmium in rats at this early age. Pups were receiving cadmium (as CdCl2.H2O) orally in fractions of 0.5 mg for 9 days, totalling 4.5 mg Cd kg(-1) body wt, or were given a single subcutaneous injection of 0.5 mg Cd kg(-1) body wt. Some pups in both exposed groups were receiving calcium supplement (CaHPO4.2H(2)O) in feed to reduce the body load of cadmium. Control pups did not receive either cadmium or calcium supplement. Cadmium in the carcass and organs was measured by atomic absorption spectrometry. The results showed that the cadmium body burden was significantly lower when the animals were receiving calcium supplements along with oral cadmium. The results of the micronucleus and comet assays showed significant differences between the control and exposed groups, regardless of the route of cadmium administration. The only statistically significant difference between the two exposed groups (oral cadmium and oral cadmium + calcium supplements) was in the number of micronuclei. The results of the comet assay showed that tail length differed statistically only between the control and all exposed groups, regardless of the route of cadmium administration. It can be concluded that the applied cadmium doses caused detectable genome damage but it was lower in calcium-treated pups receiving cadmium orally. Copyright (C) 2002 John Wiley Sons, Ltd. [References: 47]

Occupational exposure to ionizing radiation causes chromosomal damage. Some of the damaged cells show a large number of aberrations such as dicentrics, polycentrics, rings and numerous acentric fragments. This paper describes an analysis of the frequency of dicentric chromosomes and acentric fragments in 1260 subjects occupationally exposed to X-rays and 241 controls. Special attention was paid to the incidence of multi-aberrant cells. The 3 year cumulative dose was a significant predictor for all analyzed aberrations. The duration of exposure was a highly significant predictor of the frequency of rogue cells, but not of acentrics and dicentrics. Age and sex were not found to be significant predictors of the analyzed aberrations. [References: 48]

Micronuclei (MN) in blood lymphocytes, blood lead (BPb), activity of erythrocyte delta-aminolevulinic acid dehydratase (ALAD), erythrocyte protoporphyrin (EP), serum copper (SCu) and serum zinc (SZn) were measured in 46 subjects 21-56 years of age, including 34 lead workers and 12 control subjects with no-occupational exposure to lead. The results of BPb, ALAD and EP showed a highly significant (P=0.0000) difference in lead exposure between the groups. Significantly higher MN (P< 0.001) and significantly lower SCu (P< 0.02) were found in lead workers, whereas no significant difference between the groups was found for SZn. The results in all 46 subjects showed an increase in MN with an increase in log BPb (r=0.350, P< 0.02), a decrease in ALAD (r= - 0.293, P< 0.03), and a decrease in SCu (r= - 0.324, P< 0.03). The results of partial correlations confirmed significant negative correlation between MN and SCu after controlling for lead exposure (BPb, ALAD, EP). In contrast to expected pro-oxidative activity of copper through formation of micronuclei, our results indicate possible beneficial role of copper in maintenance of genomic stability. In that sense, participation of copper in antioxidative defense (as a co-factor of antioxidative enzyme copper, zinc-superoxide dismutase) could have an important role. However, our small sample size may have been a potential limitation in making final conclusions. Therefore, further epidemiological research in evaluating the role of trace elements in genomic (in)stability is recommended.

Mercury, one of the most widely diffused and hazardous organ-specific environmental contaminants, exists in a wide variety of physical and chemical states, each of them with unique characteristics of target organ specificity. An important mechanism of mercury genotoxicity is its ability to produce free radicals that can cause DNA damage. In our study we tried to find out if those effects are capable of triggering the apoptotic pathway. We used a relatively new method in the detection of apoptosis - DNA diffusion assay. It is based on the diffusion of nucleosomal-sized DNA fragments into the agarose, which gives a hazy or undefined outline without any clear boundary to the apoptotic cell nuclei. The assay was performed on the human whole blood samples. Samples were incubated in 10, 50, 100 and 200 μ M mercury chloride solution at 370 C for 24 and 48 hours. For each treatment protocol a thousand micrographs were analyzed. A significant increase in the apoptosis induction capacity of mercury chloride was observed already after 24 hours of treatment for all tested concentrations. However, no strong correlation between concentration and number of apoptotic micrographs was observed. The correlation was found after 48 hours of treatment but only for doses of 50, 100, and 200 μ M. Based on these results it could be suggested that apoptosis induction is not primary effect of mercury chloride on human lymphocytes. It also supports the theory of mercury’ s organ specificity.

Lead is still widely used around the world. Non-fitted lead compounds can be partially soluble and be absorbed into the body. It is a cumulative poison that can cause a wide variety of health problems. This study assessed cumulative exposures to the lead by monitoring the blood lead level (Pb-B) and the frequency of cytochalasin B blocked micronuclei in blood lymphocytes (MN). We sampled 40 persons in exposed and 40 persons in control group. Each group included 15 women and 25 men. The Pb-B of each person (control and exposed) was analyzed using electrothermal atomic absorption spectrophotometer Perkin-Elmer 5100/Zeeman. The blood lead level in control group was 35.2 µ ; g/l, and in exposed group 357.33 µ ; g/l. The Pb-B in smokers in control group was significantly higher (p< 0.008091) than in non-smokers. It was also observed that blood lead level increase with age in both groups, and was significantly higher in men in both groups. The average number of micronuclei in exposed group was 19.46 and in the control group 8.37. T-test for dependent variables showed significance in micronuclei frequency between controls and exposed group (p< 0.001). Our results show significant increase in MN frequency with age in both groups, as well as with years of exposure in exposed group. These results indicate a lead as a hazard agent, and therefore point out to the necessity of periodically monitoring of biomarkers (MN and blood lead level) in workers exposed to lead.

Mercury is the most toxic of the heavy metals and exerts a variety of toxic effects in the body. Within the cell it can destroy the various components selectively or in total by releasing lysosomes, damaging DNA and by rupturing the cell membrane. Mercury from mercuric chloride binds to the sulfhydryl groups of the cell membrane and other proteins, causing increased membrane permeability and inhibition of ATPase-dependent transport. The genotoxicity of mercury chloride in this study was assessed by alkaline single-cell gel electrophoresis (the comet assay) in human peripheral blood lymphocytes. Four concentrations of metal salt dissolved in re-destilled water were used, 10, 50, 100 and 200 mM. Mitomicyn C (MMC) (0.5 mg/ml) was used as a positive control. An untreated control sample was also included in the experiment. Whole blood was exposed to HgCl2 for 24 and 48h at+370 C in CO2 incubator. All the samples were in duplicate. Three parameters were measured: tail length, tail moment, and tail intensity. The data were statistically analyzed by one-way ANOVA followed by Tukey post hoc test. P< 0.05 was assumed significant. After 24 h of exposure to mercury chloride tail length values in samples treated with 50 and 100 mM of HgCl2 were statistically different from the control sample. There were no significant differences neither in tail moment neither in tail intensity after 24 hours of exposure. In 48 h of exposure statistically significant differences in tail length were found between the positive control and all other samples, and non-treated control sample and the highest mercury chloride concentration (200 mM). For the tail intensity and tail moment only the positive control sample was significantly different from all other samples.

Mercury is naturally occurring in the biosphere, in addition, it is released into the environment by human activities, such as mining, combustion of fossil fuels and other industrial release. It is highly toxic element, which after inhalation or ingestion absorbs and deposits mainly in the kidney. The genotoxicity of mercury compounds have been investigated with a variety of genetic endpoints in prokaryotic and eukaryotic cells. Results point to an inhibition of DNA synthesis, DNA damage, inhibition of spindle microtubule assembly, reduction in the frequency of mitosis, endoreduplication, chromosomal damages. This study was carried out to evaluate genotoxicity of mercury chloride in human lymphocytes using cytokinesis blocked micronucleus test. After 24 h and 48 h incubation of whole blood samples with mercury chloride at concentrations of 10, 50, 100 and 200 μ M, cultures were initiated and processed according standard protocol. Mitomycin C at concentration of 0, 5 μ g/ml served as positive control. One thousand binucleated cells per dose and per time were analysed. The results were analysed by chi-square. Our initial findings lead us to next conclusions: no significant differences between analysed samples were found after 24 hours of exposure to mercury chloride ; forty eight hours exposure resulted in increased frequency of micronuclei, but not in dose response way. This preliminary study was aimed to evaluate suitability of micronucleus test in mercury genotoxicity at applied doses. Further study with some improvements in design and more donors will provide more reliable results.

Cadmium chloride was tested for the ability to induce genotoxic effects in V79 cell culture. DNA damage induced by different doses of cadmium chloride was observed through the frequency of micronuclei in cytochalasin B-blocked micronucleus assay and through DNA migration in the single cell gel electrophoresis (comet assay). Cells were treated with different doses of cadmium chloride (10-4 to 10-6 M) 24 hours after seeding. The effects of ascorbic acid (conc. 100 microM) on cadmium-induced DNA damage was also evaluated. The results were analyzed by one-way ANOVA. The cytochalasin B-blocked micronucleus assay showed that 10-4 to 10-6 M and vitamin C supplemented 10-6 M frequencies of MN significantly differed from the control sample, and that 10-4, 10-5 M and vitamin C supplemented 10-5 M samples were significantly differed from the vitamin C sample. The Comet assay showed that the tail lengths from un-supplemented and vitamin C supplemented 10-4 M sample significantly differed from the control sample ; and that 10-5 M and vitamin C supplemented 10-4 M sample significantly differed from vitamin C sample. Tail moments from un-supplemented 10-4 M and vitamin C supplemented 10-6 M samples significantly differed from the vitamin C sample. Results indicate that cadmium chloride increases DNA damages as well as frequency of micronuclei. On the other hand, protective role of vitamin C was not confirmed at all tested concentrations of cadmium chloride.