Resveratrol ameliorates ortho- polychlorinated biphenyls' induced toxicity in ovary cells.

Polychlorinated biphenyls (PCBs) can induce chronic oxidative stress, inflammation, and cell death, leading to coronary heart disease, endothelial dysfunction, neurotoxicity, cancer, obesity, type 2 diabetes, reproductive dysfunction, etc. The aim of this study was to investigate possible protective effect of resveratrol (2.5-20 µM) in ovarian cells exposed to PCBs. An emphasis was on identifying mechanisms of resveratrol action upon distinct structure of the individual PCB congener-planar dioxin-like PCB 77 and non-planar di-ortho-substituted PCB 153. Multiple toxicity endpoint analysis was performed. Cell viability/proliferation was assessed by Trypan Blue exclusion method, Neutral Red, Kenacid Blue, and MTT bioassays. The level of oxidative stress was measured by fluorescent probes, and flow cytometry was applied to evaluate the mode of cell death. Resveratrol applied alone did not affect cell proliferation and viability in doses up to 20 µM, although significant antioxidative activity was observed. Toxic effects of ortho-PCB 153 (cytotoxicity, oxidative stress, and cell death) were mitigated by resveratrol. On the contrary pre-incubation with resveratrol did not result in cell viability protection when planar PCB 77 was applied. This indicates that resveratrol efficacy may be linked to specific structure of the individual congener, suggesting nutritional modulation of environmental insults caused by ortho-PCBs. We point out the importance of resveratrol dosage considering that synergistic cytotoxic effect with both PCB congeners is observed at concentrations ≥ 10 µM.

Novel ferrocene-containing triacyl derivative of resveratrol improves viability parameters in ovary cells.

Besides the use of resveratrol as a drug candidate, there are obstacles mainly due to its poor pharmacokinetic properties. Numerous studies are being conducted on the synthesis of resveratrol derivatives that exhibit enhanced biological activity. The aim of our research was to investigate activity of the newly synthesized ferrocene-containing triacyl derivative of resveratrol to achieve cell protection from endo/exogenous ROS and reduction in cell death by assessing multiple endpoints. Our research showed that both resveratrol and the resveratrol derivatives (1-100 µM) lower the levels of ROS in CHO-K1 cells. Resveratrol at doses <20 µM had little or no effect on cell proliferation, while at higher doses, a significant inhibitory effect on cell proliferation and viability has been noticed. The activity of the new derivative was significantly altered compared to resveratrol-cellular viability was not suppressed regardless of the concentration applied, and the extent of apoptosis was low. In summary, the new ferrocene-resveratrol derivative has the potential to protect cells from oxidative stress due to its low cytotoxicity and retained antioxidant properties, whereas caution should be exercised with resveratrol at higher doses, as it significantly impairs cell viability and induces cell death. By linking ROS to specific diseases (relevance in neurodegenerative, cardiovascular, and neoplastic diseases), we can assume that the new resveratrol derivative can prevent or alleviate the mentioned disorders. Furthermore, recognition of the resveratrol derivative as an anti-apoptotic chemical could be useful in the cultivation of various cell lines on a large scale in the industrial biotechnology.

Ortho-substituted PCB 153: effects in CHO-K1 cells.

Non-planar di-ortho-substituted PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl), one of the most abundant PCB congeners in the environment and in biological and human tissues, has been identified as potential endocrine disruptor affecting the reproductive and endocrine systems in rodents, wildlife, and humans. The aim of this study was to gain a deeper insight into its mode/mechanism of action in Chinese hamster ovary K1 cells (CHO-K1). PCB 153 (10-100 µmol/L) inhibited CHO-K1 cell proliferation, which was confirmed with four bioassays (Trypan Blue, Neutral Red, Kenacid Blue, and MTT), of which the MTT assay proved the most sensitive. PCB 153 also induced ROS formation in a dose-dependent manner. Apoptosis was seen after 6 h of exposure to PCB 153 doses ≥50 µmol/L, while prolonged exposure resulted in the activation of the necrotic pathway. PCB 153-induced disturbances in normal cell cycle progression were time-dependent, with the most significant effects occurring after 72 h.

Cell membrane-related toxic responses and disruption of intercellular communication in PCB mechanisms of toxicity: A review.

An understanding of polychlorinated biphenyl (PCB) congener-specific effects on cell membrane and intercellular communication is important within the studies of PCB absorption, organ-related PCB accumulation and exertion of toxic responses. Toxic potential of PCBs is linked to various deleterious effects on human health, including neurotoxicity, immunotoxicity, reproductive toxicity and genotoxicity and, recently in 2016 International Agency for Research on Cancer (IARC) has upgraded the classification of PCBs to Group 1 "Carcinogenic to humans." Proposed mechanisms of aforementioned PCBs adverse effects at cellular membrane level are: (i) downregulation of gap junction intercellular communication and/or connexins; (ii) compromised membrane integrity; and (iii) altered tight junction barrier function. This study, based on an extensive literature survey, shows the progress in scientific research of each of these three levels with the aim of pointing out the earliest toxic events of PCBs, which can result in serious cell/tissue/organ damage.

Reduced cytotoxicity in PCB-exposed Chinese Hamster Ovary (CHO) cells pretreated with vitamin E.

The aim of this study was to evaluate protective effects of vitamin E (50 -150 µM) in ovary cells upon cytotoxic effects induced by two structurally distinct PCB congeners - planar "dioxin-like" PCB 77 and non-planar di-ortho-substituted PCB 153 with an emphasis on identifying differences in the mechanism of vitamin E action depending on the structure of congeners. Application of three bioassays confirmed that PCBs decrease ovarian cell proliferation with slightly profound effects of PCB 77. PCB - induced ROS production and lipid peroxidation were significant for both congeners with also more noticeable effect for PCB 77. Vitamin E pre-incubation has improved viability of cells, reduced ROS formation and lipid peroxidation induced by PCBs' treatment. Preincubation with vitamin E was more effective when cells where treated with non-planar PCB 153. Altogether, vitamin E action was protective, congener specific and more effective when ovary cells were exposed to ortho-substituted PCB congener.

PCB 77 action in ovary cells--toxic effects, apoptosis induction and cell cycle analysis.

CONTEXT: PCB 77 (3,3',4,4'-tetrachlorobiphenyl), a non-ortho congener with planar configuration, has been identified as potential endocrine disrupter capable to increase the risk of reproductive and developmental failure. OBJECTIVE: In the present study, in vitro PCB 77 toxic potential, apoptosis induction and cell cycle alterations were investigated to reveal direct toxic effects on ovarian cells. METHODS: Chinese Hamster Ovary (CHO-K1) cell line was selected as a model system and decreased cell viability was confirmed by application of four bioassays. Cellular morphology and quantitative analysis of apoptotic, necrotic and viable cells were determined with fluorescent microscopy and cell cycle phase distributions by measuring DNA content using flow cytometry. RESULTS: We have indicated Trypan blue exclusion assay as the most sensitive for quantifying cytotoxicity of PCB 77 in terms of IC50 values, while the results obtained by other methods pointed to a possible localized effect on the lysosomes/endosomes (Neutral red), compromised intracellular metabolic processes (MTT) and possible interferation with the rate of protein synthesis (Kenacid blue). The loss of cell viability, as a consequence of treatment with 10-100 µM PCB 77, fundamentally was due to induction of apoptosis with observed common series of specific morphological changes characteristic to apoptotic phenomenon. The level of alterations of normal cell cycle progression was low without significant changes at analyzed time intervals. CONCLUSION: These results indicate toxic outcomes of PCB 77 at ovarian cellular level with regard to potential direct adverse effects to female reproductive system.

Lead content in multifloral honey from central Croatia over a three-year period.

Lead concentrations were analysed by atomic absorption spectrometry in multifloral honeys collected in central Croatia (Zagreb County) during a three-year period from 2009 to 2011. The mean levels of elements (µg/kg) in honey samples measured were: 90.8 in 2009, 189 in 2010 and 360 in 2011. Significant differences were observed, and Pb levels determined in 2009 were significantly lower than those in 2010 and 2011 (p < 0.05, both). In 2009 there was no concentration found above 300 µg/kg. However, in 2010 and 2011 levels exceeding 300 µg/kg were found in 28.6 % and 25 % of samples. Trace element levels of Pb determined in multifloral honey were generally higher than concentrations obtained from other geographical origins and neighbouring countries. These high concentrations of Pb may be related to the fact that the central region is becoming increasingly urban and the network of motorways is growing. Accordingly, the risk of positioning hives near zones of busy highways and railways is increasing. This underlines that particular attention should be paid to toxic Pb levels, due to their gradual increased during the study period.

Trace elements in tissues of wild carnivores and omnivores in Croatia.

The differences in metal exposure (As, Cd, Cu, Pb and Hg) in the muscle, liver and kidney tissues of brown bears (Ursus arctos), grey wolfs (Canis lupus), Eurasian lynxs (Lynx lynx), Eurasian badgers (Meles meles) and pine martens (Martes martes) from Croatia were observed. The highest mean Cd levels were found in kidney and liver of Eurasian badger (3.05 and 0.537 mg/kg). The highest Cu concentrations (mg/kg) measured in liver tissue were obtained in order: Eurasian badger (15.2) > brown bear (12.1) > pine marten (10.3) > Eurasian lynx (8.43) > grey wolf (6.44). Result presented that Eurasian badger accumulated the highest levels of elements: As, Cu and Pb in muscle; As, Cd, Cu and Pb in liver; Cd and Pb in kidney. Kidney of pine marten accumulated the highest concentrations of As, Cu and Hg. Omnivorous species observed present an important bioindicator for the accumulation of toxic elements indicating an enhanced vulnerability for response to ecological changes in forested terrain. Generally, element concentrations found in five species observed were lower in comparison to levels reported in previous studies and below levels related to toxicosis in mammals.

Wild boar tissue levels of cadmium, lead and mercury in seven regions of continental Croatia.

Concentrations of cadmium, mercury and lead were analysed by atomic absorption spectrometry in the kidney and muscle of free-living wild boar (n = 169) from hunting grounds in seven counties of continental Croatia. Mean levels of metals (mg/kg) in muscle and kidney of boars ranged as follows: Cd: 0.005-0.016 and 0.866-4.58, Pb: 0.033-0.15 and 0.036-0.441, Hg: 0.004-0.012 and 0.04-0.152. In all seven regions, concentrations exceeded the permitted values (muscle and kidney mg/kg: cadmium 0.05/1; lead 0.1/0.5; mercury 0.03/0.1) in 13.6% and 71.6% of samples (muscle and kidney, respectively) for cadmium; 13.6% and 8.9% for lead; 19.5% and 2.4% for mercury. There were significant differences among the regions. Vukovar-Srijem and Virovitica-Podravina Counties were highly contaminated with cadmium, Sisak-Moslavina and Virovitica-Podravina Counties with lead and Brod-Posavina County had highest mercury concentrations. These results suggest a detailed investigation of physiological and environmental factors contributing to accumulation of metals in boars.

Lindane-induced cytotoxicity and the role of vitamin E in Chinese Hamster Ovary (CHO-K1) cells.

Lindane, a toxic insecticide from the persistent organic pollutants (POP's) group, may act as an endocrine disrupter affecting crucial tissues of reproductive system. In this study a Chinese Hamster Ovary cell line (CHO-K1) was applied to assess the potential of lindane cytotoxicity at the cellular level. The methods of Trypan blue exclusion, MTT and Kenacid blue assays were used to assess cytotoxicity and confirmed a decrease in the number of viable CHO-K1 cells at 34.4-344 microM lindane during 24, 48 and 72 hours of exposure. The cell proliferation tests showed significant inhibition (p < 0.025-0.001 vs control) and a progressive increase in toxicity with increasing lindane concentrations. Corresponding IC(50) values were determined with each applied method. After 72 h of lindane exposure, IC(50) values were 184 microM according to the Trypan blue method and 272 and 256 microM with the Kenacid blue and MTT assays, respectively. Morphological changes induced by the cytotoxicity of lindane were followed by the fluorescence microscopy and only necrotic cells were detected. Vitamin E (25 and 50 microg/mL) was used for protection of ovarian cells against lidane-induced oxidative stress damage, and lipid peroxidation was postulated as a possible mechanism of lindane toxicity. The viability of cells pre-incubated with vitamin E was significantly enhanced (up to p < 0.025) compared to the results observed in cells exposed to lindane only, but vitamin E treatment could not prevent complete lindane-induced cytotoxicity. Results suggest that vitamin E may exert a slightly protective role in cell defense against lipophilic pro-oxidant xenobiotics such as lindane.

Lead and cadmium in red deer and wild boar from different hunting grounds in Croatia.

The concentration and relations of Cd and Pb as environmental risk factors were studied by atomic absorption spectrophotometry in the liver, kidney and muscle of free ranging wild boar (n=94) and red deer (n=45) from hunting grounds in four counties of north-east Croatia. In all four counties, the levels of Cd found in the kidney of red deer ranged from 2.28 to 5.91 mg/kg, and in wild boar from 3.47 to 21.10 mg/kg. The mean renal concentration of Cd was significantly higher in wild boar than in red deer from all four study areas. The mean hepatic (0.11 to 0.49 mg/kg, respectively) and muscle (0.01 to 0.04 mg/kg, respectively) Cd concentrations were similar in both species. The mean renal Cd concentration in wild boar and red deer exceeded 1 mg/kg in all four counties, ranging from 67.0% to 91.4% and from 45.5% to 69.2%, respectively. Also, the hepatic Cd/renal Cd ratio was lower than 1 in all animals. In all four counties, renal Pb concentration ranged from 0.058 to 3.77 mg/kg in red deer and from 0.056 to 11.60 mg/kg in wild boar. Hepatic Pb concentration was similar in both species (0.061 to 0.202 mg/kg in wild boar and 0.077 to 0.108 mg/kg in red deer). Because of the high Cd level in the organs of wild boar and red deer, further research is needed to identify the source of contamination in order to preserve the health of animals and humans.