Demasculinization and feminization of male gonads by atrazine: consistent effects across vertebrate classes.

Atrazine is the most commonly detected pesticide contaminant of ground water, surface water, and precipitation. Atrazine is also an endocrine disruptor that, among other effects, alters male reproductive tissues when animals are exposed during development. Here, we apply the nine so-called "Hill criteria" (Strength, Consistency, Specificity, Temporality, Biological Gradient, Plausibility, Coherence, Experiment, and Analogy) for establishing cause-effect relationships to examine the evidence for atrazine as an endocrine disruptor that demasculinizes and feminizes the gonads of male vertebrates. We present experimental evidence that the effects of atrazine on male development are consistent across all vertebrate classes examined and we present a state of the art summary of the mechanisms by which atrazine acts as an endocrine disruptor to produce these effects. Atrazine demasculinizes male gonads producing testicular lesions associated with reduced germ cell numbers in teleost fish, amphibians, reptiles, and mammals, and induces partial and/or complete feminization in fish, amphibians, and reptiles. These effects are strong (statistically significant), consistent across vertebrate classes, and specific. Reductions in androgen levels and the induction of estrogen synthesis - demonstrated in fish, amphibians, reptiles, and mammals - represent plausible and coherent mechanisms that explain these effects. Biological gradients are observed in several of the cited studies, although threshold doses and patterns vary among species. Given that the effects on the male gonads described in all of these experimental studies occurred only after atrazine exposure, temporality is also met here. Thus the case for atrazine as an endocrine disruptor that demasculinizes and feminizes male vertebrates meets all nine of the "Hill criteria".

Comparison of cytotoxicity induced by 17α-ethynylestradiol and diethylstilbestrol in fish CCO and mammalian CHO-K1 cell lines.

The effects of synthetic estrogens 17α-ethynylestradiol (EE2) and diethylstilbestrol (DES) were compared on cell proliferation and morphology in Channel Catfish Ovary (CCO) and Chinese Hamster Ovary (CHO-K1) cells. EE2 exposure (0.1 and 0.5 µg/mL) induced stimulatory effect on CCO and CHO-K1 cell proliferation, while higher concentrations (1-10 µg/mL) showed cytotoxic effects. Increase in DES concentrations mainly resulted in dose-depended increase in cytotoxicity in both cell lines. Morphological changes induced by EE2 and DES exposure (5 µg/mL) showed disrupted cell monolayer and increased number and size of lysosomes. Comparison of IC(50) values showed almost equal sensitivity towards cytotoxicity of tested compounds in CCO and CHO-K1 cells.

Influence of different ammonium, lactate and glutamine concentrations on CCO cell growth.

In this study the effects of ammonium and lactate on a culture of channel catfish ovary (CCO) cells were examined. We also made investigation on the influence of glutamine, since our previous research revealed that this amino acid stimulated CCO cell growth more than glucose in a concentration-dependent manner. The effect of ammonium in cell culture included the considerable decrease in cell growth rate with eventual growth arrest as well as the retardation of glucose consumption. At ammonium concentrations above 2.5 mM, the cells displayed specific morphological changes. The effect of lactate was different to that of ammonium since the cell growth rate was progressively decreasing with the increase of lactate concentration, whereas the glucose consumption rate remained almost unchanged. Besides that, it was found that lactate was steadily eliminated from the culture medium when its initial concentration was relatively high. The influence of glutamine on CCO cell propagation showed that nutrient requirements of this cell line were mainly dependent on glutamine rather than glucose. The increase in glutamine concentration led to the increase in cell growth rate and consequent ammonia accumulation while the glucose utilization and lactate production were reduced. Without glutamine in culture medium cell growth was arrested. However, the lack of glucose reversed the stimulating effect of glutamine by decreasing cell growth rate and affecting amino acid utilization.

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.

Effect of porcine brain growth factor on primary cell cultures and BHK-21 [C-13] cell line.

Growth factors from neural tissues have been described as potent mitogens for a wide variety of mesoderm- and ectoderm-derived cells in vitro. We used porcine brain extract for in vitro testing of proliferation properties on primary ovarian cells, uterine cells, and cardiomyocytes in culture as well as for BHK-21 [C-13] cell line. The addition of this extract accelerates proliferation in all examined cultures. It also lowers serum requirement and shortens the cultivation period for BHK-21 [C-13] cells. Fibroblast growth factors from brain of different species, but not porcine, are already characterized and their proliferative effect proved. Therefore, we purified, determined, and confirmed the presence of basic fibroblast growth factor in porcine brain extract by Western blot analysis and showed its biological activity on BHK-21 [C-13] cells.

Growth characteristics of channel catfish ovary cells-influence of glucose and glutamine.

The growth characteristics and influence of glucose and glutamine on the growth and maintenance of channel catfish ovary (CCO) cells were investigated. Besides glutamine, amino acids threonine, arginine, methionine and serine were found to be essential for CCO cell growth. In the glucose-free medium, glutamine is utilized as energy source and no cell growth limitation was observed. However, the lack of glutamine in culture medium did not stimulate CCO cells to efficient glucose consumption. When both glucose and glutamine were deficient, cell growth was also observed suggesting no rigorous nutritional requirements. Obtained results are useful for further understanding of culture processes using CCO cells.

Alternative models for toxicity testing of xenobiotics.

The alternatives to whole-animal testing include endpoint assays, cell and tissue cultures, use of tissue slices, toxicokinetic modelling, and structure-activity relationships and databases. The use of in vitro systems (subcellular fractions, cell lines, primary cell cultures, tissue slices, organ cultures, etc.) as research tools in toxicology is widespread. In the past few years, the apoptosis phenomena were followed by very precise intracellular changes where, through programmed cell death, a cell can be removed from a population. The in vitro systems are ideally suited for investigations of the molecular, cellular and physiological mechanisms of chemically induced toxicity, which cannot readily be studied in vivo for known target organ and target species toxicity studies and for answering specific questions about toxic effects. The main justification for developing in vitro toxicity tests is that they will make toxicology a more scientifically based practice. It is increasingly apparent that the development and incorporation of stepwise testing strategies, combining experimental data from a range of alternative methods (physicochemical techniques, quantitative structure-activity relationships--QSAR, metabolic and kinetic modelling and in vitro tests), provide the most advanced way to predict toxicity, reducing at the same time the number of laboratory animals used for testing.

BHK 21 C13 cells for Aujeszky's disease virus production using the multiple harvest process.

Production of Aujeszky's disease virus (ADV) from BHK 21 C13 suspension cells using a simple harvest and multiple harvest process mode was examined. We studied growth kinetics of BHK 21 C31 cells in 750 ml spinner flask containing 500 ml of culture medium. In the simple harvest process of ADV production, 425 ml of virus harvest was obtained with a virus titer of 10(6.4) TCID(50) ml(-1) which corresponds to 10,676 doses of vaccine. The multiple harvest process resulted in 850 ml of virus harvest with a virus titer of 10(6.5) TCID(50) ml(-1) corresponding to 26,877 AD vaccine doses. In conclusion, the multiple harvest process mode using BHK 21 C13 can be considered as a favorable process to produce ADV.