Evaluation of oxidative stress responses in human circulating blood cells after imatinib mesylate treatment - Implications to its mechanism of action.

Imatinib mesylate (IM) is the first developed protein kinase inhibitor and recently it has topped consumption rates among targeted and total anticancer drugs. Although there are indications that IM possesses cyto/genotoxic activities against normal non-target cells as well, there is a lack of information regarding the underlying mechanism involved in those actions. Therefore, we aimed to evaluate the response of human circulating blood cells towards oxidative stress after IM treatment (0.0001-10 µg/mL) in vitro. Based on the results, IM had an influence on all of the oxidative stress parameters tested. Lower concentrations of IM induced an increase of glutathione level, following its decrease at higher IM concentrations indicating impairment in oxidative stress defences. Concomitant to a glutathione decrease, an increase of malondialdehyde and protein carbonyls level was observed indicating oxidative damage of lipids and proteins. The observed effects overlapped with the observed formation of oxidative base damage detected by formamidopyrimidine-DNA glycosylase modified-comet assay indicating that IM managed to induce oxidative DNA damage. Our results provide novelty in their mechanistic approach to IM-induced toxicity in non-target cells and suggest that IM can affect blood cells and induce oxidative stress.

Seasonal variations as predictive factors of the comet assay parameters: a retrospective study.

Since there are several predicting factors associated with the comet assay parameters, we have decided to assess the impact of seasonal variations on the comet assay results. A total of 162 volunteers were retrospectively studied, based on the date when blood donations were made. The groups (winter, spring, summer and autumn) were matched in terms of age, gender, smoking status, body mass index and medical diagnostic exposure in order to minimise the impact of other possible predictors. Means and medians of the comet assay parameters were higher when blood was sampled in the warmer period of the year, the values of parameters being the highest during summer. Correlation of meteorological data (air temperature, sun radiation and sun insolation) was observed when data were presented as the median per person. Using multivariate analysis, sampling season and exposure to medical radiation were proved to be the most influential predictors for the comet assay parameters. Taken together, seasonal variation is another variable that needs to be accounted for when conducting a cohort study. Further studies are needed in order to improve the statistical power of the results related to the impact of sun radiation, air temperature and sun insolation on the comet assay parameters.

Genotoxicity assessment of a selected cytostatic drug mixture in human lymphocytes: A study based on concentrations relevant for occupational exposure.

Cytostatic drugs are highly cytotoxic agents used in cancer treatment and although their benefit is unquestionable, they have been recognized as hazardous to healthcare professionals in occupational settings. In a working environment, simultaneous exposure to cytostatics may occur creating a higher risk than that of a single substance. Hence, the present study evaluated the combined cyto/genotoxicity of a mixture of selected cytostatics with different mechanisms of action (MoA; 5-fluorouracil, cyclophosphamide and paclitaxel) towards human lymphocytes in vitro at a concentration range relevant for occupational as well as environmental exposure. The results suggest that the selected cytostatic drug mixture is potentially cyto/genotoxic and that it can induce cell and genome damage even at low concentrations. This indicates not only that such mixture may pose a risk to cell and genome integrity, but also that single compound toxicity data are not sufficient for the prediction of toxicity in a complex working environment. The presence of drugs in different amounts and with different MoA suggests the need to study the relationship between the presence of genotoxic components in the mixture and the resulting effects, taking into account the MoA of each component by itself. Therefore, this study provides new data sets necessary for scientifically-based risk assessments of cytostatic drug mixtures in occupational as well as environmental settings.

Cytokinesis-block micronucleus cytome assay parameters in peripheral blood lymphocytes of the general population: Contribution of age, sex, seasonal variations and lifestyle factors.

The cytokinesis-block micronucleus cytome (CBMN Cyt) assay was used to evaluate the baseline frequency of cytogenetic damage in peripheral blood lymphocytes of the general population (average age, 38.28 ± 12.83 years) in relation to age, sex, body mass index, seasonal variations (season of sampling, period of sampling and different meteorological parameters) and lifestyle factors (smoking habit, alcohol consumption, exposure to medications and diagnostic radiation, physical activity, and family history of cancer). The background frequency of micronuclei (MNi) for the 200 subjects assayed was 5.06 ± 3.11 per 1000 binucleated cells, while the mean frequency of nucleoplasmic bridges (NPBs) was 1.21 ± 1.46 and of nuclear buds (NBUDs) 3.48 ± 2.14. The background frequency of apoptosis and necrosis was 1.58 ± 1.50 and 1.39 ± 1.56, respectively, while the mean nuclear division index (NDI) was 1.99 ± 0.14. The cut-off value, which corresponds to the 95th percentile of the distribution of 200 individual values, was 11 MNi, 4 NPBs and 7 NBUDs. The study also confirmed an association of the above mentioned parameters with age, sex and several lifestyle factors. Moreover, significant confounders based on our results are also sampling season, sampling period and different meteorological parameters that were dependent on the CBMN Cyt assay parameters. In line with the above mentioned, several factors should be taken into account when it comes to the monitoring of exposed populations using cytogenetic biomarkers. Moreover, the normal and cut-off values obtained in this study present background data for the general population, and can later serve as baseline values for further biomonitoring studies.

In vitro effects of simultaneous exposure to platinum and cadmium on the activity of antioxidant enzymes and DNA damage and potential protective effects of selenium and zinc.

Circulating platinum (Pt) is detectable in the blood of Pt-treated cancer patients for over a decade after the treatment. Prolonged exposure to Pt, in combination with adverse compounds from nutrition and lifestyle, such as cadmium (Cd), could increase the risk from second cancers. The aim of this study was to investigate the effects of simultaneous exposure to Cd- and Pt-compounds on oxidative and DNA damage and the possible protective effects of zinc (Zn) and selenium (Se). The aqueous solutions of PtCl4, CdCl2 × H2O, ZnCl2 and Na2SeO3 were added, alone or in combination, to whole blood and isolated erythrocytes to produce the final concentrations of 2000 µg/L of Pt, 8 µg/L of Cd, 100 µg/L of Se, and 1000 µg/L of Zn. The activity of copper, zinc-superoxide dismutase, glutathione peroxidase and glutathione in whole blood was determined after 1 h exposure in in vitro conditions. The induction of DNA strand-breaks in human peripheral blood leukocytes was determined with the alkaline comet assay after 24 h exposure. Exposure to Pt and/or Cd decreased the activities of antioxidant enzymes and elevated DNA damage compared to control. A statistically significant change in the activity of both enzymes and in the induction of DNA strand-breaks was observed in the cells treated with Pt + Cd combination, while the addition of Se and/or Zn resulted in partial recovery of these effects. The results indicate that combined exposure to Pt and Cd could disrupt antioxidant protection of the organism and increase DNA damage, whereas Se and Zn could partially ameliorate these harmful effects.

Chemotherapeutic potential of quercetin on human bladder cancer cells.

In an effort to improve local bladder cancer control, we investigated the cytotoxic and genotoxic effects of quercetin on human bladder cancer T24 cells. The cytotoxic effect of quercetin against T24 cells was examined by MTT test, clonogenic assay as well as DNA damaging effect by comet assay. In addition, the cytotoxic effect of quercetin on the primary culture of papillary urothelial carcinoma (PUC), histopathological stage T1 of low- or high-grade tumours, was investigated. Our analysis demonstrated a high correlation between reduced number of colony and cell viability and an increase in DNA damage of T24 cells incubated with quercetin at doses of 1 and 50 µM during short term incubation (2 h). At all exposure times (24, 48 and 72 h), the efficacy of quercetin, administered at a 10× higher dose compared to T24 cells, was statistically significant (P < 0.05) for the primary culture of PUC. In conclusion, our study suggests that quercetin could inhibit cell proliferation and colony formation of human bladder cancer cells by inducing DNA damage and that quercetin may be an effective chemopreventive and chemotherapeutic agent for papillary urothelial bladder cancer after transurethral resection.

Physico-chemical characterization and the in vitro genotoxicity of medical implants metal alloy (TiAlV and CoCrMo) and polyethylene particles in human lymphocytes.

BACKGROUND: The main objective of the present study was to investigate chemical composition and possible cyto/genotoxic potential of several medical implant materials commonly used in total hip joint replacement. METHODS: Medical implant metal alloy (Ti6Al4V and CoCrMo) and high density polyethylene particles were analyzed by energy dispersive X-ray spectrometry while toxicological characterization was done on human lymphocytes using multi-biomarker approach. RESULTS: Energy dispersive X-ray spectrometry showed that none of the elements identified deviate from the chemical composition defined by appropriate ISO standard. Toxicological characterization showed that the tested materials were non-cyto/genotoxic as determined by the comet and cytokinesis-block micronucleus (CBMN) assay. Particle morphology was found (by using scanning electron and optical microscope) as flat, sharp-edged, irregularly shaped fiber-like grains with the mean particle size less than 10µm; this corresponds to the so-called "submicron wear". The very large surface area per wear volume enables high reactivity with surrounding media and cellular elements. CONCLUSIONS: Although orthopedic implants proved to be non-cyto/genotoxic, in tested concentration (10µg/ml) there is a constant need for monitoring of patients that have implanted artificial hips or other joints, to minimize the risks of any unwanted health effects. GENERAL SIGNIFICANCE: The fractal and multifractal analyses, performed in order to evaluate the degree of particle shape effect, showed that the fractal and multifractal terms are related to the "remnant" level of the particles' toxicity especially with the cell viability (trypan blue method) and total number of nucleoplasmic bridges and nuclear buds as CBMN assay parameters.

Toxicity and antioxidant capacity of Frangula alnus Mill. bark and its active component emodin.

In the present study toxicity of Frangula alnus Mill. bark, widely used as laxative, was investigated. Human peripheral blood lymphocytes (HPBLs) were treated with F. alnus bark extract or emodin (emodin is bark component with laxative property), and cytotoxicity, genotoxicity and parameters of oxidative stress were assessed. Also, polyphenol content of bark extract and antioxidant activity of the extract and emodin measured by DPPH, ABTS and FRAP methods were examined. The bark extract (500 µg/ml) produced cell death and DNA damage, while level of ROS changed at 250 µg/ml. Emodin induced cell death and DNA damage at 150 µg/ml and 200 µg/ml, respectively, and the increase of ROS was observed at 25 µg/ml. These results suggest that both, bark extract and emodin, are cyto/genotoxic to HPBLs and that oxidative stress is involved in the mechanism of their toxicity. The results on antioxidant activity showed that, unlike emodin, bark extract possess moderate antioxidant capacity (44.6%, 46.8% and 2.25 mmol Fe(2+)/g measured by DPPH, ABTS and FRAP assay, respectively) that can be related to relatively high phenolic content (116.07 mg/g). However, due to toxicological properties use of F. alnus bark as well as emodin-containing preparations should be taken with caution.

Toxicological assessment and management options for boat pressure-washing wastewater.

Boats are washed periodically for maintenance in order to remove biofoulants from hulls, which results in the generation of wastewater. This study aimed at evaluating the cyto/genotoxic and mutagenic properties of wastewater produced by pressure washing of boats. The chemical characterisation of this wastewater showed that Cu, Zn, V, Cr, Fe, Pb, and select organic contaminants exceeded the maximum allowable values from 1.7 up to 96 times. The wastewater produced negative effects on human lymphocytes resulting in decreased cell viability after 4 and 24h of exposure. Chromosome aberration, micronucleus, and comet assay parameters were significantly higher after 24h of exposure. At the same time, the Salmonella typhimurium test showed negative for both TA98 and TA100 strains at all of the concentrations tested. After the treatment of wastewater using electrochemical methods/ozonation during real scale treatment plant, removal rates of colour, turbidity and heavy metals ranged from 99.4% to 99.9%, while the removal of total organic carbon (TOC) and chemical oxygen demand (COD) was above 85%. This was reflected in the removal of the wastewater's cyto/genotoxicity, which was comparable to negative controls in all of the conducted tests, suggesting that such plants could be implemented in marinas to minimise human impact on marine systems.

Combined antitumor effects of bee venom and cisplatin on human cervical and laryngeal carcinoma cells and their drug resistant sublines.

In the present study, we investigated the possible combined anticancer ability of bee venom (BV) and cisplatin towards two pairs of tumour cell lines: parental cervical carcinoma HeLa cells and their cisplatin-resistant HeLa CK subline,as well as laryngeal carcinoma HEp-2 cells and their cisplatin-resistant CK2 subline. Additionally, we identified several peptides of BV in the BV sample used in the course of the study and determined the exact concentration of MEL. BV applied alone in concentrations of 30 to 60 µg ml(­1) displayed dose-dependent cytotoxicity against all cell lines tested. Cisplatin-resistant cervical carcinoma cells were more sensitive to BV than their parental cell lines (IC(50) values were 52.50 µg ml(­1) for HeLa vs.47.64 µg ml(­1) for HeLa CK cells), whereas opposite results were obtained for cisplatin-resistant laryngeal carcinoma cells (IC(50) values were 51.98 µg ml(­1) for HEp-2 vs. > 60.00 µg ml(­1) for CK2 cells). Treatment with BV alone induced a necrotic type of cell death, as shown by characteristic morphological features, fast staining with ethidium-bromide and a lack of cleavage of apoptotic marker poly (ADP-ribose) polymerase (PARP) on Western blot. Combined treatment of BV and cisplatin induced an additive and/or weak synergistic effect towards tested cell lines, suggesting that BV could enhance the killing effect of selected cells when combined with cisplatin. Therefore, a greater anticancer effect could be triggered if BV was used in the course of chemotherapy. Our results suggest that combined treatment with BV could be useful from the point of minimizing the cisplatin concentration during chemotherapy, consequently reducing and/or postponing the development of cisplatin resistance.

γ-H2AX as a biomarker for DNA double-strand breaks in ecotoxicology.

The visualisation of DNA damage response proteins enables the indirect measurement of DNA damage. Soon after the occurrence of a DNA double-strand break (DSB), the formation of γ-H2AX histone variants is to be expected. This review is focused on the potential use of the γ-H2AX foci assay in assessing the genotoxicity of environmental contaminants including cytostatic pharmaceuticals, since standard methods may not be sensitive enough to detect the damaging effect of low environmental concentrations of such drugs. These compounds are constantly released into the environment, potentially representing a threat to water quality, aquatic organisms, and, ultimately, human health. Our review of the literature revealed that this method could be used in the biomonitoring and risk assessment of aquatic systems affected by wastewater from the production, usage, and disposal of cytostatic pharmaceuticals.

Exploring cytokinesis block micronucleus assay in Croatia: A journey through the past, present, and future in biomonitoring of the general population.

In this study, we used the cytokinesis-block micronucleus (CBMN) assay to evaluate the background frequency of cytogenetic damage in peripheral blood lymphocytes of the general population concerning different anthropometric data and lifestyle factors. The background frequency of CBMN assay parameters was analysed in 850 healthy, occupationally non-exposed male and female subjects (average age, 38±11 years) gathered from the general Croatian population from 2000 to 2023. The mean background values for micronuclei (MNi) in the whole population were 5.3±4.3 per 1000 binucleated cells, while the mean frequency of nucleoplasmic bridges (NPBs) was 0.7±1.3 and of nuclear buds (NBUDs) 3.1±3.2. The cut-off value, which corresponds to the 95th percentile of the distribution of 850 individual values, was 14 MNi, 3 NPBs, and 9 NBUDs. Results from our database also showed an association of the tested genomic instability parameters with age and sex but also with other lifestyle factors. These findings underscore the importance of considering several anthropometric and lifestyle factors when conducting biomonitoring studies. Overall, the normal and cut-off values attained here present normal values for the general population that can later serve as baseline values for further human biomonitoring studies either in Croatia or worldwide.

Cytogenetic status of healthy children assessed with the alkaline comet assay and the cytokinesis-block micronucleus cytome assay.

In the present study the alkaline comet assay and the cytokinesis-block micronucleus cytome (CBMN Cyt) assay were used to evaluate the baseline frequency of cytogenetic damage in peripheral blood lymphocytes (PBLs) of 50 healthy children from the general population in Croatia (age, 11.62±1.81 years). Mean values of tail length, tail intensity and tail moment, as comet assay parameters, were 12.92±0.10, 0.73±0.06 and 0.08±0.01, respectively. The mean frequency of micronuclei (MN) for all subjects was 2.32±0.28 per 1000 bi-nucleated cells, while the mean frequency of nucleoplasmic bridges (NPBs) was 1.72±0.24 and of nuclear buds (NBUDs) 1.44±0.19. The mean nuclear division index (NDI) was 1.70±0.05. When comet-assay parameters were considered, higher mean values for all three were found for the female population. According to the Mann-Whitney U test applied on the results of the comet assay, the only statistically significant difference between the male and female populations was found for tail length. Similar to the results obtained by the comet assay, girls showed higher mean values of all three measured parameters of the CBMN Cyt assay. This difference was statistically significant for total number of NPBs only. In the case of the NDI, a higher mean value was also obtained in girls, but this difference was not statistically significant. The results obtained present background data that could be considered as normal values for healthy children living in urban areas, and can later on serve as baseline values for further toxicological monitoring. Additionally, the usefulness of both techniques in measuring cytogenetic damage during bio-monitoring of children is confirmed.

Assessment of DNA damage and lipid peroxidation in diabetic mice: effects of propolis and epigallocatechin gallate (EGCG).

There is growing recognition that polyphenolic compounds present in many plants and natural products may have beneficial effects on human health. Propolis - a substance produced by honeybees - and catechins in tea, in particular (-)-epigallocatechin gallate (EGCG), are strong antioxidants that appear to have anti-obesity and anti-diabetic effects. The present study was designed to elucidate the anti-diabetic effect of the water-soluble derivative of propolis (WSDP), which contains phenolic acids as the main compounds, and EGCG in alloxan-induced (75mg/kg, iv) diabetes in mice. Intraperitoneal administration of EGCG or propolis at doses of 50mg/kg body weight (bw) to diabetic mice for a period of 7 days resulted in a significant increase in body weight and in haematological/immunological blood parameters, as well as in 100% survival of the mice. A significant decrease in lipid peroxidation in liver, kidney and brain tissue was also observed in diabetic mice treated with these two agents. Additionally, EGCG and propolis clearly reduced DNA damage in peripheral lymphocytes of diabetic mice. Our studies demonstrate the anti-oxidative and anti-inflammatory potential of WSDP and EGCG, which could exert beneficial effects against diabetes and the associated consequences of free-radical formation in kidney, liver, spleen and brain tissue. The results suggest that dietary supplementation with WSDP or EGCG could potentially contribute to nutritional strategies for the prevention and treatment of diabetes mellitus.

An alkaline comet assay study on the antimalarial drug atovaquone in human peripheral blood lymphocytes: a study based on clinically relevant concentrations.

Atovaquone, a hydroxynaphthoquinone, is an anti-parasite drug, selectively targeting the mitochondrial respiratory chain of malaria parasite. It is used for both the treatment and prevention of malaria, usually in a fixed combination with proguanil. Although atovaquone has not often been associated with severe adverse reactions in the recommended dosages and has a relatively favorable side effect profile, the present study was undertaken to evaluate its cytogenotoxic potential towards human peripheral blood lymphocytes. Two different concentrations of atovaquone found in plasma when used in fixed-dose combination with proguanile hydrochloride were used with and without S9 metabolic activation: 2950 ng ml(-1) used for prophylactic treatment and 11 800 ng ml(-1) used in treatment of malaria. The results showed that lymphocyte viability was not affected after the treatment, suggesting that atovaquone was not cytotoxic in the given concentrations. With the alkaline comet assay we demonstrated that in human peripheral blood lymphocytes no significant changes in comet parameters occurred after the treatment. There were no differences in tested parameters with the addition of S9 metabolic activation, indicating that atovaquone either has no metabolite or it is not toxic in the given concentrations. Since no effects were observed after the treatment, it is to be concluded that atovaquone is safe from the aspect of genototoxicity in the recommended dosages.

Presence of phthalate esters in intravenous solution evaluated using gas chromatography-mass spectrometry method.

Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer widely used in the production of poly-(vinyl) chloride (PVC) materials. It is a reproductive and developmental toxicant in animals and a suspected endocrine modulator in humans. DEHP is not covalently bound within the PVC molecule, which is why migration into a suitable medium can be expected. Since application of infusion solutions is one of the most common medical treatments, the objective of this study was to determine the migration of phthalates from softened PVC storage bags into infusion solution in different time periods within one year from date of production using a gas chromatography-mass spectrometry method. The measured values of DEHP ranged between 0.22 and 14.00 µg l(-1) , but the unexpected presence of other phthalate esters was also detected. It was concluded that values obtained in infusion solutions match the reference data and represent a minor risk for the patient. The presence of other phthalate esters leads to the conclusion that the pharmacopeic requirement for polymer cleanness was not fully met. Since phthalate esters are among the most extensively used industrial chemicals and are widely distributed in the environment, special precautions and further monitoring should be conducted to minimize any possible health risks.

Chemical composition and genotoxicity assessment of sanitary landfill leachate from Rovinj, Croatia.

Chemical analysis and an in vitro approach were performed to assess elemental composition and genotoxic effects of the samples of landfill leachate taken from Lokva Vidotto sanitary landfill the official landfill for Rovinj town, Croatia. Two samples of landfill leachate were collected and analyzed in order to evaluate macro, micro and trace elements by atomic absorption spectroscopy, energy dispersive X-ray spectrometry and colorimetry. Genotoxicity of sanitary landfill leachate was evaluated in human lymphocytes by the use of the micronucleus test and comet assay. Samples were characterized with relatively low concentrations of heavy metals while organic component level exceeded upper permissible limit up to 39 times. Observed genotoxic effects should be connected with high concentrations of ammonia nitrogen, which exceeded permissible limit up to 180 times. Leachate samples of both sanitary landfills increased the frequency of micronuclei, nucleoplasmic bridges and nuclear buds. Increase of DNA damage in human lymphocytes was also detected by virtue of measuring comet assay parameters. All parameters showed statistically significant difference compared to negative control. Increased micronucleus and comet assay parameters indicate that both samples of sanitary landfill leachate are genotoxic and could pose environmental and human health risk if discharged to an aquatic environment.

Chemical and toxicological characterization of the bricks produced from clay/sewage sludge mixture.

The present study aimed to characterize chemical properties of clay bricks containing 20 % of sewage sludge. After detection of potentially hazardous metals, we simulated precipitation exposure of such material to determine the amount of heavy metals that could leach out of the bricks. Metals, such as copper, zinc, nickel, cobalt, chromium, etc., were detected in leachate in low concentrations. Moreover, human peripheral blood lymphocytes were exposed to brick leachate for 24 h in order to evaluate its possible negative impact on human cells and genome in vitro. Cytotoxicity tests showed no effect on human peripheral blood lymphocytes viability after exposure to brick's leachate. On the contrary, the alkaline comet assay showed slight but significant increase in DNA damage with all three parameters tested. As we might predict, interactions of several heavy metals in low concentrations could be responsible for DNA damaging effect. In that manner, our findings suggest that leachates from sewage sludge-produced bricks may lead to adverse effects on the exposed human population, and that more stabile bricks should be developed to minimize leaching of heavy metals into the environment. Bricks with lower percentage of the sludge may be one of the solutions to reduce the toxic effect of the final product.

Application of dosimetry systems and cytogenetic status of the child population exposed to diagnostic X-rays by use of the cytokinesis-block micronucleus cytome assay.

Low-dose ionizing radiation used for medical purposes is one of the definite risk factors for cancer development, and children exposed to ionizing radiation are at a relatively greater cancer risk as they have more rapidly dividing cells than adults and have longer life expectancy. Since cytokinesis-block micronucleus cytome (CBMN Cyt) assay has become one of the standard endpoints for radiation biological dosimetry, we used that assay in the present work for the assessment of different types of chromosomal damage in children exposed to diagnostic X-ray procedures. Twenty children all with pulmonary diseases between the ages of 4 and 14 years (11.30 ± 2.74) were evaluated. Absorbed dose measurements were conducted for posterior-anterior projection on the forehead, thyroid gland, gonads, chest and back. Doses were measured using thermoluminescence and radiophotoluminescent dosimetry systems. It was shown that, after diagnostic X-rays, the mean total number of CBMN Cyt assay parameters (micronucleus, nucleoplasmic bridges and nuclear buds) was significantly higher than prior to diagnostic procedure and that interindividual differences existed for each monitored child. For the nuclear division index counted prior and after examination, no significant differences were noted among mean group values. These data suggest that even low-dose diagnostic X-ray exposure may induce damaging effect in the somatic DNA of exposed children, indicating that immense care should be given in both minimizing and optimizing radiation exposure to diminish the radiation burden, especially in the youngest population.

Cytogenotoxicity of sewage sludge leachate before and after calcium oxide-based solidification in human lymphocytes.

Present study aimed to establish the chemical composition of sewage sludge leachate before/after calcium oxide-based solidification using energy dispersive X-ray fluorescence (EDXRF). The other aim was to determine leachate effects on human lymphocyte and DNA integrity in vitro using a battery of bioassays (DNA diffusion assay, micronucleus test and comet assay) to determine effects of those complex mixtures of elements on cell and DNA integrity. EDXRF showed that nickel concentration in the leachate of untreated sludge was 18.5 times higher than the upper permissible limit for inert waste landfills. Other elements were kept below the permissible values. After sludge solidification, leachate concentrations of Cr, Mn, Fe, Ni, Cu, Zn, and Pb dropped 1.6, 2.7, 37, 5.9, 3.2, 7.8, and 2.6 times, respectively. Untreated sludge leachate was cytogenotoxic to lymphocytes, and may lead to adverse effects on the exposed human populations, but calcium oxide-based solidification reduced these effects in significant manner.