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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.

DNA damage in the liver and kidney cells of adult male Wistar rats was studied using the comet assay after a 28-day oral administration of tembotrione at doses of 0.0007, 0.0013 and 0.7 mg/kg b.w./day [AOEL (acceptable operator exposure level), REL (residual exposure level) and 1000× AOEL]. As a descriptor of DNA damage, tail intensity was used. Antioxidant status was assessed by activity of glutathione peroxidase (GPx). Significant DNA damage was recorded in the kidney cells at all three doses as compared to negative control. In parenchymal liver cells, significant DNA damage was observed in AOEL and 1000× AOEL doses, while in non-parenchymal liver cells, only AOEL-treated group was significantly different compared to negative control. In both types of liver cells, REL and 1000× AOEL doses were significantly different from the AOEL dose. No significant changes in GPx activity compared to control were observed at any exposure level. The results of the present study suggest that repeated in vivo exposure to tembotrione led to low-level DNA instability in kidney and liver cells. Exposure to the highest tembotrione dose showed a relatively weak response with the alkaline comet assay. Further research should focus on the effects of this herbicide in other models along with different exposure scenarios.

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.

The potential of low doses of the chloro-triazine herbicide terbuthylazine to induce DNA damage and impair activity of glutathione peroxidase (GPx) was evaluated in kidney and parenchymal and non-parenchymal liver cells of adult male rats. In a 28-day study, terbuthylazine was applied daily by oral gavage at doses: 0.004, 0.4 and 2.29 mg/kg bw/day. Tail Intensity (T Int) and Tail Length (TL) were used as descriptors of DNA damage. In the kidney, Tail Int was significantly different in all treated groups, while TL was different in 0.4 and 2.29 mg/kg bw/day groups, compared to controls. Significant differences in TL were recorded in parenchymal and non-parenchymal liver cells of all treated groups. Tail Int was significantly different from controls in non-parenchymal liver cells at all applied doses and in parenchymal cells at terbuthylazine doses of 0.004 and 2.29 mg/kg bw/day. A significant increase in GPx activity was observed only in the kidney at doses 0.4 and 2.29 mg/kg bw/day compared to the controls indicating its possible role in the protection of kidney from free radicals. It appears that repeated exposure to low doses of terbuthylazine could cause DNA instability in kidney cells and in parenchymal and non-parenchymal liver cells in rats.

Tembotrione is a rather novel pesticide, usually used for post-emergence weed control. Even though its use is rapidly growing, it is not followed by an adequate flow of scientific evidence regarding its toxicity towards non-target organisms. We evaluated the potential of low doses of tembotrione to induce oxidative stress and cytogenetic damage in blood and brain cells of adult male Wistar rats. Parameters of lipid peroxidation, glutathione levels, activities of antioxidant enzymes and primary DNA damage were assessed following 28-day repeated oral exposure to doses comparable with the currently proposed health-based reference values. The results of the alkaline comet assay showed that such low doses of tembotrione have the potency to inflict primary DNA damage in both peripheral blood leukocytes and brain of treated rats, even with only slight changes in the oxidative biomarker levels. The DNA damage in blood and brain cells of Wistar rats significantly increased at all applied doses, suggesting that tembotrione genotoxicity is mainly a result of direct interaction with DNA while the induction of oxidative stress responses contributes to DNA instability in a lesser extent. The findings of the present study call for further research using other sensitive biomarkers of effect and different exposure scenarios.

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.

We studied the toxic effects of glyphosate in vitro on HepG2 cells exposed for 4 and 24 h to low glyphosate concentrations likely to be encountered in occupational and residential exposures [the acceptable daily intake (ADI ; 0.5 μg/mL), residential exposure level (REL ; 2.91 μg/mL) and occupational exposure level (OEL ; 3.5 μg/mL)]. The assessments were performed using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell proliferation, alkaline comet assay and cytokinesis-block micronucleus (CBMN) cytome assay. The results obtained indicated effects on cell proliferation, both at 4 and 24 h. The levels of primary DNA damage after 4-h exposure were lower in treated vs. control samples, but were not significantly changed after 24 h. Using the CBMN assay, we found a significantly higher number ofMNand nuclear buds at ADI and REL after 4 h and a lower number of MN after 24 h. The obtained results revealed significant oxidative damage. Four-hour exposure resulted in significant decrease at ADI [lipid peroxidation and glutathione peroxidase (GSH-Px)] and OEL [lipid peroxidation and level of total antioxidant capacity (TAC)], and 24-h exposure in significant decrease at OEL (TAC and GSH-Px). No significant effects were observed for the level of reactive oxygen species (ROS) and glutathione (GSH) for both treatment, and for 24 h for lipid peroxidation. Taken together, the elevated levels of cytogenetic damage found by the CBMN assay and the mechanisms of primary DNA damage should be further clarified, considering that the comet assay results indicate possible cross-linking or DNA adduct formation.

In this study, the influence of anthropogenic pollution on the aquatic environment of Plitvice Lakes National Park (PLNP) was investigated during 2011-2012 using a combination of chemical and cytogenetic analyses. Four groups of major contaminants [(volatile organic compounds: benzene, toluene, ethylbenzene, and xylenes (BTEX) ; persistent organochlorine pollutants: organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) ; major and trace elements ; anthropogenic radionuclides (90Sr, 134Cs, and 137Cs)] were determined in three aquatic compartments (water, sediment, fish). Mass fractions of inorganic constituents in different compartments reflected the geological background of the area, indicating their origin from predominantly natural sources. Levels of volatile and persistent organic compounds in water and fish, respectively, were very low, at levels typical for remote pristine areas. Analysis of anthropogenic radionuclides in water and sediment revealed elevated activity concentrations of 137Cs in water, and measurable 134Cs in the upper sediment layers from April 2011, possibly as a consequence of the Fukushima nuclear accident in March 2011. The potential genotoxicity of river and lake water and lake sediment was assessed under laboratory conditions using the alkaline comet assay on human peripheral blood lymphocytes, and measured levels of primary DNA damage were within acceptable boundaries. The results showed that despite the protected status of the park, anthropogenic impact exists in both its terrestrial and aquatic components. Although contaminant levels were low, further monitoring is recommended to make sure that they will not rise and cause potentially hazardous anthropogenic impacts.

Terbuthylazine belongs to the chloro-s-triazine group of herbicides and acts primarily as a photosynthesis inhibitor. The mechanisms of action related to its exposure, relevant both in animals and humans, are still insufficiently investigated. This comprehensive study focused on the outcomes of terbuthylazine exposure at cell level in vitro, and a mice model in vivo. Experiments in vitro were conducted on whole human peripheral blood, isolated lymphocytes, and HepG2 cells exposed for 4 h to terbuthylazine at 8.00, 0.80, and 0.58 ng/mL, which is comparable with current reference values set by the European Commission in 2011. Terbuthylazine cytotoxicity was evaluated using dual fluorescent staining with ethidium bromide and acridine orange on lymphocytes, and CCK-8 colorimetric assay on HepG2 cells. The levels of DNA damage were measured using alkaline and hOGG1-modified comet assays. The potency of terbuthlyazine regarding induction of oxidative stress in vitro was studied using a battery of standard oxidative stress biomarkers. The in vivo experiment was conducted on Swiss albino mice exposed to terbuthlyazine in the form of an active substance and its formulated commercial product Radazin TZ-50 at a daily dose of 0.0035 mg/kg bw for 14 days. Following exposure, the DNA damage levels in leukocytes, bone marrow, liver, and kidney cells of the treated mice were measured using an alkaline comet assay. In vitro results suggested low terbuthylazine cytotoxicity in non-target cells. The highest tested concentration (8.00 ng/mL) reduced lymphocyte viability by 15%, mostly due to apoptosis, while cytotoxic effects in HepG2 cells at the same concentration were negligible. Acute in vitro exposure of human lymphocytes and HepG2 cells to terbuthylazine resulted in low-level DNA instability, as detected by the alkaline comet assay. Further characterization of the mechanisms behind the DNA damage obtained using the hOGG1-modified comet assay indicated that oxidative DNA damage did not prevail in the overall damage. This was further confirmed by the measured levels of oxidative stress markers, which were mostly comparable to control. Results obtained in mice indicate that both the active substance and formulated commercial product of terbuthylazine produced DNA instability in all of the studied cell types. We found that DNA in liver and kidney cells was more prone to direct toxic effects of the parent compound and its metabolites than DNA in leukocytes and bone marrow cells. The overall findings suggest the formation of reactive terbuthylazine metabolites capable of inducing DNA cross-links, which hinder DNA migration. These effects were most pronounced in liver cells in vivo and HepG2 cells in vitro. To provide a more accurate explanation of the observed effects, additional research is needed. Nevertheless, the present study provides evidence that terbuthylazine at concentrations comparable with current reference values possesses toxicological risk because it caused low-level DNA instability, both at cellular and animal organism level, which should be further established in forthcoming studies.

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.

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., Individualna osjetljivost na ionizirajuće zračenje rezultat je međudjelovanja samog izlaganja zračenju, oštećenja DNA nastalog prilikom tog izlaganja te samog popravka nastalog oštećenja. Veliki doprinos razlikama čine i polimorfizmi u genima za popravak DNA. U ovom radu istražili smo povezanost nastalih oštećenja DNA u obliku mikronukleusa (MN), jezgrinih pupova (NB) i nukleoplazmatskih mostova (NPB) s polimorfizmima jednog nukleotida (SNP) u genima za popravak DNK (APE1, hOGG1, XRCC1, XRCC3, XPD, PARP1, MGMT) koji sudjeluju u različitim mehanizmima popravka. Rezultati skupine od 77 medicinskih radnika kronično izloženih niskim dozama ionizirajućeg zračenja uspoređeni su s rezultatima skupine od 70 odgovarajućih kontrola. Izložena skupina imala je značajno veću učestalost MN-a (16,2±10,4 vs. 11.5±9.4; P=0,003), a sama pojavnost oštećenja bila je neovisna o medijatornoj varijabli (kovarijati). Značajno više učestalosti MN nađene su u izloženoj skupini u homozigotnih nositelja divljeg tipa gena hOGG1, XRCC1, PARP1 i MGMT i u heterozigotnih nositelja gena APEX1 i XPD (rs13181) u odnosu na kontrolnu skupinu istoga genotipa. Analiza genskih polimorfizama i citogenetička dozimetrija važna su dopuna osobnom dozimetrijskom nadzoru izloženih radnika.

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.

Adult male Wistar rats were exposed for 2 h a day, 7 days a week for up to 30 days to continuous 2,450 MHz radiofrequency microwave (rf/MW) radiation at a power density of 5-10 mW/cm(2). Sham-exposed rats were used as controls. After ether anesthesia, experimental animals were euthanized on the final irradiation day for each treated group. Peripheral blood smears were examined for the extent of genotoxicity, as indicated by the presence of micronuclei in polychromatic erythrocytes (PCEs). The results for the time-course of PCEs indicated significant differences (P0.05) for the 2nd, the 8th and the 15th day between control and treated subgroups of animals. Increased influx of immature erythrocytes into the peripheral circulation at the beginning of the experiment revealed that the proliferation and maturation of nucleated erythropoietic cells were affected by exposure to the 2,450 MHz radiofrequency radiation. Such findings are indicators of radiation effects on bone-marrow erythropoiesis and their subsequent effects in circulating red cells. The incidence of micronuclei/1,000 PCEs in peripheral blood was significantly increased (P0.05) in the subgroup exposed to rf/MW radiation after eight irradiation treatments of 2 h each in comparison with the sham-exposed control group. It is likely that an adaptive mechanism, both in erythrocytopoiesis and genotoxicity appeared in the rat experimental model during the subchronic irradiation treatment.

Cadmium chloride (CdCl2 x H2O) in concentrations 10 -3 to 10 -6 M was tested for genotoxicity in human lymphocytes in vitro. The DNA damage was expressed through the occurrence of micronuclei (MN) and was detected using the cytochalasin-B-blocked MN assay. Human blood was treated in the G0 and S phse of the cell cycle. All except the highest concentration of cadmium chloride of 10 -3M applied in the G0 phase of the cell cycle resulted in the increase in MN cells, but it was not statistically significant. Cadmium chloride added to the cultures in the concentration of 10 -3M affected the cell growth regardless of the phase. Cadmium chloride added to cultures 24 h after their initiation (early S phase) wasfound to significantly increase the MN frequency in 10 -4 to 10 -6 M concentrations (P>0.05).

Peripheral blood lymphocytes were tested in vitro for genotoxic effects of cadmium chloride. Whole blood samples of four healthy, non-smoking subjects were preincubated with CdCl2 in concentrations of 10(-4), 10(-3), and 5 . 10(-3) mol/L for three hours before the cells were assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay) or cultivated for chromosomal aberrations (CA), sister chromatid exchanges (SCE), and the micronucleus (MN) test. The comet assay showed notable interindividual differences. The results of the cytogenetic tests showed an increase in the frequency of CA, MN, and SCE with CdCl2 in the treated cultures, yet none was able to show a correlation between concentrations of cadmium chloride and the frequency of damages. The MN slides were stained with Giemsa and with DNA fluorochrome 4', 6'-diamidino-2-phenylindole (DAPI). The frequency of MN in slides stained with DAPI was significantly higher than in those stained with Giemsa, which might be due to an underestimation of small micronuclei in Giemsa-stained slides.

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 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., Svrha istraživanja bila je procijeniti genotoksični potencijal komponenata koje izlučuju dva konvencionalna samopolimerizirajuća stakleno-ionomerna cementa (Fuji IX i Ketac Molar) te svjetlosno polimerizirajući i smolom modificirani stakleno-ionomerni cementi (Vitrebond, Fuji II LC). Istraživanje je provedeno na ljudskim limfocitima primjenom alkalnog komet testa, komet testa modificiranog hOGG1 enzimom te mikronukleus testa. Svaki materijal, polimerizirani i nepolimerizirani, eluiran je u fiziološkoj otopini (1 cm2 mL-1) tijekom jednog sata, jednog dana i tijekom 5 dana. Kulture limfocita tretirane su eluatima u razrjeđenjima 10-2, 10-3 i 10-4. Alkalnim komet testom nisu zabilježene promjene u migraciji DNA iz tretiranih stanica bez obzira na ispitani ionomer, vrstu polimerizacije i trajanje elucije. Izloženost staklenim ionomerima nije značajno utjecala na učestalost mikronukleusa. Primjenom hOGG1 modificiranog komet testa nije zamijećeno oksidativno oštećenje DNA u tretiranim limfocitima. Dobiveni rezultati upućuju na visoki stupanj biokompatibilnosti svih testiranih materijala koji su se koristili u eksperimentalnim uvjetima.

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).

Long-term exposure to low doses of ionizing radiation may affect cells and tissues and result in various adverse health effects. The purpose of this study was to investigate weather chromosome aberrations and haematological alterations could be used as biomarkers of possible radiation injury in workers expposed to ionizing radiation. Groups totalling 323 medical professionals handling X-ray equipment and 160 control subjects were examined for incidence of chromosome aberrations and changes in leukocyte, lymphocyte and thrombocyte counts. The incidence of all types of chromosome aberrations was higher in the expposed groups than in controls, yet no significant difference was found between the exposed groups. A many-fold increase in chromosome aberration frequency in all exposed groups was not followed by a corresponding haematological depression. This suggests that chromosome aberrations are a significantly more sensitive indicator of changes caused by low doses of ionizing radiation than haematological alterations.

This study emphasizes the problems encountered in obtaining suitable contol levels for comparison with occupational studies of exposure to clastogens. Blood samples taken from 135 individuals (69 females and 66 males) were examined for chromosome aberrations. The data include 15 368 cells scored for chromosome aberrations. The frequencies of chromatid and chromosome breaks, acentric fragments and dicentrics were determined. The frequency of the most common chromosome aberration, chromosome breaks, was 1.1 x 10-2 and for dicentries 0.26 x 103. The values obtained were investigatd in relation to sex, cigarette smoking habits, diagnostic X-ray exposure and use of antibiotics. In all the parameters, no significant differences were found.

During their working and living activities, humans are constantly exposed to different environmental genotoxic agents. Biological consequences of the exposure are accumulation of different mutations and DNA damage that can lead to disruption of genetic material. Numerous endogenous genotoxic agents can also cause DNA damage. Loss of normal cell function can cause cell death or can result in different health disorders, including teratogenic and cancerogenic effects (Jeggo & Lavin, 2009). There is an increasing concern about mutagenic and cancerogenic effects of genotoxic agents and their influence on individuals who are exposed to them by accident, or by living/working lifestyle (Au, 1991; Carrano & Natarajan, 1988; Kassie et al., 2000). Exposure to ionising radiation, as a well known genotoxic agents (Balasem & Ali, 1991; Erexon et al., 1991; Fenech et al., 1990; He et al., 2000; Jeggo & Lavin, 2009), can leed to different DNA damage, such as oxidative damage, base and sugar modification in DNA, apurinic/apirimidinic places, single/double strand chromosomal breaks, adduct creation, inter/intra DNA cross linking and other types of damage (Hall & Giaccia, 2006). Numerous studies deal with ionising radiation exposure of individuals who are chronically professionally exposed to low doses (Andreassi, 2009; Au, 1991; Carrano & Natarajan, 1988; Kassie et al., 2000). The results showed the increase in chromosomal damage during chronically low dose exposure (Barquinero et al., 1993; Boutcher, 1985; Cardoso et al., 2001; Jha, 1991; Nowak & Jankowski, 1991), but without confirmed relationship between the received dose and the intensity of DNA damage (Bolus, 2001; Coates et al., 2004; Morgan, 2003; Mothersill et al., 2000, 2001; Seymour & Mothersill, 2000). The influence of chronically low dose exposure has been considered mutagenic and cancerogenic (Fachini et al., 2009). Although it is possible to estimate the influence of absorbed dose and the effect on the

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.

Irinotecan 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 signifi cance 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 signifi cant increase in micronucleated cells in respect to control, no dose-dependent relation was established among the treated cultures. Timedependent effect was also not observed., Irinotekan je citotoksični lijek koji inhibira enzim DNA-topoizomerazu I. U širokoj je primjeni u terapiji metastatskog karcinoma kolona i rektuma. U uvjetima in vitro primjenom komet-testa, analize kromosomskih aberacija i mikronukleus-testa na V79-stanicama istražili smo genotoksični učinak maksimalne pojedinačne doze (18 μg mL-1), preporučene monoterapijske doze (9 μg mL-1) i preporučene doze irinotekana za kombiniranu terapiju (4,5 μg mL-1). Kulture stanica bile su tretirane irinotekanom 2 h i 24 h. Statistička značajnost određivana je jednosmjernim ANOVA-testom i neparametrijskim Mann Whitneyevim U-testom. Komet-testom nije utvrđen učinak koncentracije i/ili vremena izloženosti. Analiza kromosomskih aberacija pokazala je prisutnost izmjena kromatida, tj. porast broja triradijusa i tetraradijusa. Iako je u kulturama stanica izloženi irinotekanu opažen značajan porast broja mikronukleusa u odnosu na kontrolu, nije uočena ovisnost o dozi lijeka ni o vremenu izloženosti u opisanim eksperimentalnim uvjetima. Dobiveni rezultati upućuju na genotoksičnost irinotekana za V79-stanice. Nijednom od primijenjenih metoda nije utvrđena ovisnost učinka irinotekana o vremenu ili dozi.

Cilj ovog istraživanja bio je procijeniti razinu oštećenja DNA u leukocitima periferne krvi prije, neposredno nakon i 120 min nakon ozračivanja dozom zračenja od 2 Gy te usporediti razine nastalih oštećenja između kontrolne skupine i skupine medicinskih radnika izloženih niskim dozama ionizirajućeg zračenja na svojim radnim mjestima. Istražen je utjecaj polimorfi zma u genima koji sudjeluju u popravku DNA; *hOGG1, XRCC1 i MGMT na razinu izmjerenih oštećenja. Istraživanjem je obuhvaćeno 40 zdravih ispitanika obaju spolova (20 kontrola i 20 izloženih). Razina oštećenja DNA mjerena je komet-testom u alkalnim uvjetima, pri čemu su za svakog ispitanika i svaku vremensku točku analizirane vrijednosti dužine repa, % DNA u repu i repnog momenta kometa. Rezultati pokazuju da su vrijednosti % DNA u repu i repni moment kometa bili statistički značajno viši u izloženoj populaciji prije, neposredno nakon i 120 min nakon ozračivanja. Pokazano je da nosioci polimorfnih alela ovih gena u izloženoj skupini imaju statistički značajno više razine oštećenja DNA, kako naspram homozigota pripadne skupine, tako i naspram cijele kontrolne skupine te da početno oštećenje DNA značajno negativno korelira s ukupnom dozom zračenja koju su primili tijekom radnog vijeka. Dobiveni rezultati upućuju na vrijednost kombiniranja alkalnoga komet-testa i genotipizacije izloženih pojedinaca u genima za popravak DNA, što bi moglo pridonijeti prepoznavanju subpopulacija sklonijih nakupljanju oštećenja DNA, a time i sklonijih riziku od razvoja tumorskih bolesti., Increasing exposure to ionising radiation raises a great concern about potential DNA damage in occupationally exposed individuals. Polymorphisms of DNA repair genes can determine individual sensitivity and DNA damage response to low doses of ionising radiation. The objective of this study was to assess DNA damage in leukocytes at baseline, immediately after and 120 min after exposure to gamma-radiation of 2 Gy, to compare DNA damage between the control group of subjects and subjects occupationally exposed to low-dose gamma-radiation, and to determine the relationship between hOGG1 (8-oxoG specifi c DNA glycosylase/AP-Lyase, Ser326Cys), XRCC1 (X-ray repair cross-complementing protein-group 1, Arg194Trp), and MGMT(O6-methylguanine-DNA methyltransferase, Leu84Phe) gene and DNA damage. The study enrolled 40 healthy subjects (20 controls and 20 occupationally exposed subjects), whose leukocytes were exposed to ionising radiation and tested for DNA damage (tail length, percentage od DNA in comet tail, and tail moment) using the alkaline version of the comet assay. Our results show that tail DNA percentage and tail moment were signifi cantly higher in the exposed group at baseline, immediately after, and 120 min after exposure to 2 Gy. The exposed subjects carrying polymorphic alleles had signifi cantly higher DNA damage than homozygous carriers of the same gene and controls. Combined use of the alkaline comet assay and genotyping of DNA repair genes could help discover sensitive occupationally exposed individuals who can accumulate higher DNA damage and are at higher risk of developing tumours.

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.

1. 1. Earthworms possess immunological recognition and memory as well as high regenerative abilities. Coelomic fluid was used as a source of biologically active compounds. 2. 2. A biologically active glycolipoprotein extract from a whole earthworm tissue homogenate was isolated and named G-90. 3. 3. G-90 forms precipitation arcs in gel with different animal and human sera. 4. 4. It alters murine cell growth rate in vitro in serum in a dose-dependent manner and slows murine tumor growth in vivo . 5. 5. G-90 does not contain mutagens or carcinogens.

Present study aimed at an integral assessment of sister chromatid exchange (SCE) frequencies in the health care workers occupationally exposed to cytostatics. The results of 500 individual analyses were evaluated. Drug handling practice was investigated in parallel and the results showed that cytostatics are mostly prepared outside hospital pharmacy (98%) and mainly handled by nurses (96%). Mean frequency of SCE was 5.63 +/- 2.28, while HFC represented 9.65% of the cells analysed. Both values were higher compared to previously established control values for Croatian population. The duration of exposure, profession, age, gender, smoking habit, medical exposures, and simultaneous exposure to other occupational mutagens significantly contributed to SCE and HFC values. The usefulness both biomarkers in the assessment of cytogenetic damage is confirmed. Since current practice in Croatian hospitals does not include regular monitoring of workplaces, to ensure maximal occupational safety, a surveillance on exposed health care workers, including periodic biomonitoring, is recommended.

To evaluate the effect of leakage on differences in genotoxicity of root canal sealers ex vivo according to their main components using two different cytogenetic assays.Six materials of different composition (GuttaFlow, Epiphany, Diaket, IRM, SuperEBA and Hermetic) were tested on human peripheral blood lymphocytes using the comet assay and chromosomal aberration analysis. Prepared materials were eluted in physiological solution for 1 h, 1 day, 5 and 30 days. Thereafter cultures were treated with 8 microg, 4 microg and 2 microg of each sealer. Frequencies of chromatide and chromosome breaks and accentric fragments were determined. Comet assay was used to evaluate primary DNA damage by measuring tail length and tail intensity. Chi-square, Fisher's PLSD (Protected Least Significant Difference) and Kruskall-Wallis non parametric tests were used for statistical analysis.After 1-h elution only the highest dose of Diaket, Hermetic and SuperEBA significantly (P = 0.035, P = 0.048, P = 0.037 respectively) affected the measured cytogenetic parameters. The migration ability of DNA was more strongly affected than induction of chromosomal aberrations. After elutions longer than 24 h none of the tested sealers exhibited a genotoxic effect.Under the conditions used in the study all sealers had acceptable biocompatibility in terms of genotoxicity.