Effects of the chloro-s-triazine herbicide terbuthylazine on DNA integrity in human and mouse cells.

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 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.

Cytotoxic, genotoxic and biochemical markers of insecticide toxicity evaluated in human peripheral blood lymphocytes and an HepG2 cell line.

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.

Does the Serum Metallothionein Level Reflect the Stage of Testicular Germ Cell Tumor?

Increased levels of metallothionein (MT) have recently been found in the blood serum of men with newly diagnosed testicular germ cell tumors (TGCT). In light of previously published results, the aim of this study was to investigate the difference in serum MT levels among patients with different stages of TGCT and compare MT with commonly used markers (α-fetoprotein, ß-human chorionic gonadotropin and lactate dehydrogenase). The concentration of total MT was determined in the serum of 25 men with TGCT (seminoma or non-seminoma) by differential pulse voltammetry. Serum samples were obtained prior to chemotherapy, after two cycles of chemotherapy and 1 year after chemotherapy. A statistically significant difference in MT levels in patients with different stages of TGCT was observed in the serum of patients with non-seminoma obtained before chemotherapy. Although not significant, an increase in serum MT levels commensurate with the disease stage increase was also observed in patients with seminomatous TGCT. The results indicate that, in combination with the existing markers, MT could be useful for the identification of the histological type of tumor and stage of the disease before biopsy diagnosis.

Assessment of oxidative stress responses and the cytotoxic and genotoxic potential of the herbicide tembotrione in HepG2 cells.

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.

Serum metallothionein in patients with testicular cancer.

PURPOSE: Metallothioneins (MTs) have been disclosed as a useful diagnostic factor for tumour progression and drug resistance in a variety of malignancies. Increased levels of MT in blood serum have been found in patients with several types of cancer, but there is no available information on serum MT levels in patients with testicular germ cell tumour (TGCT). The aim of the study was to determine MT levels in serum of patients with TGCT and to evaluate the portion of platinum (Pt) that binds to MT after cisplatin administration since MTs could be involved in drug resistance. METHODS: Concentration of total MT was determined in serum of 25 men with newly diagnosed TGCT by differential pulse voltammetry. The fractionation of serum was carried out by size exclusion high-performance liquid chromatography (SE-HPLC), while concentration of Pt in collected fractions was determined by inductively coupled plasma mass spectrometry. RESULTS: Concentration of serum MT was significantly higher in TGCT patients than in healthy volunteers. The results of SE-HPLC analysis showed that only a small amount of Pt was bound to proteins in the area of MT elution. CONCLUSIONS: Significant increase in MT levels in individuals with TGCT indicates certain health problem and, in combination with other commonly used diagnostic tools, could improve early diagnosis.

Relevance of serum copper elevation induced by oral contraceptives: a meta-analysis.

BACKGROUND AND STUDY DESIGN: It is well documented that copper (Cu) blood levels are elevated in combination oral contraceptive (COC) users. The aim of this study was to establish the range of Cu increase in OC users compared to nonusers through a systematic literature overview and quantitative data analysis. RESULTS: Twenty-six articles were included in the meta-analysis. The increase in Cu level exponentially decreased in COC users over time, with a rapid decline through the 1960s and 1970s. After controlling for the publication year, use of COC increases the mean serum/plasma Cu level by 0.57 mg/L (95% confidence interval 0.49-0.66 mg/L). CONCLUSION: COCs commonly raise serum Cu to levels between 1.5 and 2 mg/L, which are above reference levels. Although these levels are not considered toxic, there are suggestions that such Cu increase could be implicated in oxidative pathophysiological processes in the body. Further research on safety of COCs use, including oxidative-stress-related effects, is warranted.

Reproductive toxicity of metals in men.

A combination of genetic, environmental and lifestyle factors contributes to adverse effects on the reproductive health in men. Metals are pervasive in food, water, air, tobacco smoke, and alcoholic beverages. Experimental studies suggest that many metals have adverse effects on the male reproductive function. However, information about reproductive effects of human exposure to metals is scarce and/or inconsistent. This review summarises the information from epidemiological studies of the effects of metal exposure on reproductive function in men. Factors capable of affecting these relationships were identified and discussed. A particular attention is given to the studies considering influence of concomitant exposure to various metals. These studies have generally confirmed that even moderate- to low-level exposure to lead affects certain reproductive parameters, and that exposure to cadmium affects the prostate function and serum testosterone levels. Adverse effects of mercury, manganese, chromium and arsenic on semen quality and altered serum hormone are less well documented. There is no clear evidence that boron exposure may impair reproductive health in men. Only a few studies have investigated reproductive effects of concomitant exposure to several metals and controlled for potential confounders. Future studies should consider the contribution of combined exposure to various metals and/or other factors that may influence individual susceptibility to reproductive health impairment in men.

Estimation of copper intake in moderate wine consumers in croatia.

To estimate Cu exposure level from wine consumption and to assess possible health risk for moderate wine consumers, wine samples were collected from different wine-growing areas of Croatia. Median concentrations were 180 µg L-1, range (76 to 292) µg L-1, in commercial wines and 258 µg L-1, range (115 to 7600) µg L-1, in homemade wines (P>0.05). Maximum permitted level of 1000 µg L-1 was exceeded in three homemade wines. However, daily intake of Cu from wine (in the range from 0.02 mg d-1 to 1.52 mg d-1) estimated from Cu concentration in all wine samples is lower than the tolerable upper intake level of 5 mg d-1 proposed by the EU Scientific Committee on Food and does not present a risk to moderate wine consumers.

Metals in wine--impact on wine quality and health outcomes.

Metals in wine can originate from both natural and anthropogenic sources, and its concentration can be a significant parameter affecting consumption and conservation of wine. Since metallic ions have important role in oxide-reductive reactions resulting in wine browning, turbidity, cloudiness, and astringency, wine quality depends greatly on its metal composition. Moreover, metals in wine may affect human health. Consumption of wine may contribute to the daily dietary intake of essential metals (i.e., copper, iron, and zinc) but can also have potentially toxic effects if metal concentrations are not kept under allowable limits. Therefore, a strict analytical control of metal concentration is required during the whole process of wine production. This article presents a critical review of the existing literature regarding the measured metal concentration in wine, methods applied for their determination, and possible sources, as well as their impact on wine quality and human health. The main focus is set on aluminum, arsenic, cadmium, chromium, copper, iron, manganese, nickel, lead, and zinc, as these elements most often affect wine quality and human health.

Determination of lead in croatian wines by graphite furnace atomic absorption spectrometry.

A method has been developed for direct determination of lead in wine by graphite furnace atomic absorption spectrometry (GFAAS) with Zeeman-effect background correction. The thermal behaviour of Pb during pyrolysis and atomisation stages was investigated without matrix modifier and in the presence of Pd(NO(3))(2), Pd(NO(3))(2) + Mg(NO(3))(2) × 6H(2)O, and NH(4)H(2)PO(4) + Mg(NO(3))(2) × 6H(2)O as matrix modifiers. A simple 1:1 dilution of wine samples with Pd(NO3)2 as a matrix modifier proved optimal for accurate determination of Pb in wine. Mean recoveries were 106 % for red and 114 % for white wine, and the detection limit was 3 µg L(-1). Within-run precision of measurements for red and white wine was 2.1 % and 1.8 %, respectively. The proposed method was applied for analysis of 23 Croatian wines. Median Pb concentrations were 33 µg L(-1), range (16 to 49) µg L(-1) in commercially available wines and 46 µg L(-1), range (14 to 559) µg L(-1) in home-made wines. There were no statistically significant differences (P<0.05) in Pb concentration between commercial and home-made wines or between red and white wines.