In vitro effect of organophosphate pesticides, Malathion and chlorpyriphos, on lipid peroxidation and antioxidant enzymes

Abstracts / Toxicology Letters 180S (2008) S32–S246

assess the molecular mechanism of arsenic toxicity, we investigated the effects of sodium arsenite on cell growth, apoptosis, telomeres length and telomerase activity in male and female human cord blood cells “in vitro”. Sodium arsenite at the concentration of 0.0001 ␮M increased telomerase activity in female, maintaining telomeres length and cellular growth in both genders. The concentration of 1 ␮M decreased either telomerase activity or telomeres length, inducing apoptosis and necrosis without gender difference. Our results suggest that telomerase activity and telomere length are associated with arsenic induced cell death, as well as with cell differentiation processes and rate of cell growth. At the same concentration of 0.0001 ␮M sodium arsenite exerted an upmodulation of two oncogenes RAS and MYC in both genders, even though either genes were more strongly modulated in female. This suggests that female is the most sensitive gender for telomerase stimulation at low concentrations and the modulation seems to be correlated with increased expression of RAS and MYC. On the contrary, higher concentrations of sodium arsenite are associated with increased loss of telomerase activity, telomeres shortening and apoptosis, without sex differences. doi:10.1016/j.toxlet.2008.06.417 V27 Intestinal absorption of arsenate sodium/phosphate cotransporter

through

type

IIb

Ricardo Villa-Bellosta ∗ , Ana Ferrer-Dufol, Victor Sorribas Toxicology Department Zaragoza University, Zaragoza, Spain Inorganic arsenate (AsV ) is a common contaminant of underground water. Following oral exposure, it is assumed that AsV is distributed and crosses cell membranes through inorganic phosphate (Pi) transporters. In this work we have analyzed the intestinal absorption of AsV by studying the AsV inhibition kinetics of Pi transport in rat intestinal Na/Pi-cotransporters expressed in Xenopus laevis oocytes and in the brush-border membrane vesicles (BBMV) of rat small intestine. We have found by western blot that type III Pi transporters, PiT-1, and PiT-2 are very abundant in the apical membrane of enterocytes. Conversely, NaPi-IIb is the only type II Pi transporter expressed in BBMV. PiT-1 and PiT-2 showed a low affinity for AsV (Ki ∼ 3.8 mM) compared to the affinity for Pi (Km 0.1–0.2 mM). In contrast, the high-affinity intestinal transporter, NaPi-IIb (Km ∼ 12 ␮M), was very efficiently inhibited with a Ki of 51 ␮M. Intestinal BBMV showed a moderate Pi affinity (Km 0.1 mM), which could be explained by the combined expression of NaPi-IIb, PiT-1, and PiT-2. Pi transport in BBMV was also efficiently inhibited by AsV , with a Ki of only three times the Pi Km . Given that Pi competition on AsV intestinal transport is very weak as a consequence of its low concentration in the intestinal lumen (depending mainly on the level of ingested water), and given the moderate affinity of AsV on the Pi intestinal transport system, our data suggest that NaPi-IIb could be the main entry route of AsV into the body after oral exposure. doi:10.1016/j.toxlet.2008.06.418 V28 Microcystin congener-specific in vitro neurotoxicity Daniel Feurstein ∗ , Andreas Fischer, Daniel R. Dietrich University of Konstanz, Konstanz, Germany Contamination of natural waters by cyanobacterial blooms is a worldwide problem, resulting in serious water pollution and

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health hazards for humans and livestock. The cyanobacterial microcystins (MCs) represent a group of >80 cyclic heptapeptide toxin congeners, known to induce hepato-, nephro-, and potentially neurotoxic effects via protein phosphatase (PP)-inhibition. Present evidence suggests that organic anion transporting polypeptides (rodent Oatps/human OATPs) are required for active uptake of MCs into hepatocytes and kidney epithelial cells. Based on the presence of Oatps/OATPs at the blood–brain-barrier (BBB) and blood–cerebrospinal fluid-barrier (BCFB) it was hypothesized that MCs can be transported across the BBB/BCFB and into neurons in an Oatp/OATP-dependent manner and will induce neurotoxic effects. To test this hypothesis, primary murine neurons (Cerebellar Granule Cells, mCGC) were analyzed for the presence of mCGC Oatps (mRNA level). Subsequently, the uptake, localization and neurotoxic effects of MC-LR, -LW, and -LF were investigated using MC-immunoblotting, confocal microscopy of immunostained neurons, PP-inhibition assay, TNF-␣ ELISA and caspase 3/7 activity assay. RT-PCR demonstrated the presence of six murine Oatps (Oatp1c1, 1a5, 3a1, 1a1, 1b2 and 6d1) in mCGC. MC-LR-specific immunodetection demonstrated a concentration-dependent accumulation of this congener and covalent binding to PP-1 and -2A. mCGC PP activity was reduced by 20% following 48 h exposure to ≥300 nM MC-LR, -LW and -LF concurrent with a congener and concentration-dependent up regulation of TNF-␣ expression and caspase 3/7 activity. In conclusion, the data suggest an MC congener-dependent uptake and neurotoxicity in primary mouse CGC. doi:10.1016/j.toxlet.2008.06.419 V29 In vitro effect of organophosphate pesticides, Malathion and chlorpyriphos, on lipid peroxidation and antioxidant enzymes Guillermina Font ∗ , Ana Juan-García, Monica Fernández, María Jose Ruiz Laboratori de Bromatologia i Toxicologia, Facultat de Farmacia, Universitat de Valencia, Valencia, Spain In vitro generation of reactive oxygen species (ROS) by organophosphates may produce oxidative stress. Effects of organophosphatesinduced lipid peroxidation (LPO) and the alteration of antioxidant defense system were studied. For this purpose, it was investigated the levels of malondialdehyde (MDA) and changes in the activity of antioxidant enzymes including superoxide dismutasa (SOD), catalase (CAT) and glutathione peroxidase (GPx) on CHO-K1 cells exposed to Malathion and chlorpyrifos. Addition of Malathion or chlorpyriphos provided an induction of LPO in CHO-K1 cells. Chlorpyriphos produced a greater increase in LPO than Malathion. Both of then increased GPx activity. The higher GPx activity occurred 24 h after exposure. Addition of organophosphates to cells resulted in a time-dependent increase of catalase activity. Catalase activity was higher with chlorpyriphos than Malathion exposure. SOD activity decreased after pesticides exposure. A smaller drop in the SOD activity, in relation to cell control, was observed at 24 h of organophosphate pesticide exposure. Experiments were repeated adding 100 ␮M of vitamin E to the medium, previously to determine MDA levels and GPx, catalase and SOD enzymatic activities, in order to determine if these enzymatic activities were inhibited even in conditions of LPO prevention. Vitamin E decreased the production of MDA and modified the enzymatic activity over control at all periods of incubation after organophosphate exposure. Decrease in MDA production confirms the antioxidant effect of vitamin E in CHO-K1 cells exposed to

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Abstracts / Toxicology Letters 180S (2008) S32–S246

pesticides, increasing protection with exposure time. Vitamin E provides adequate protection to mammal cells. Acknowledgements: This work was supported by the Science and Education Spanish Ministry (AGL2007–61493). doi:10.1016/j.toxlet.2008.06.420

cell suspension following with vortexing with glass beads prior the immobilization in the agarose to partially disrupt their rigid and highly insoluble cell wall, (2) ionic detergent N-laurylsarcosine for complete cell lysis was used. However, the problem of DNA unwinding still remains to be solved. The comet assay of this alga would undoubtedly prove to be a useful tool for testing the genotoxicity of chemicals and monitoring of environmental pollution.

V30 Genotoxicity of honeybee venom in human lymphocytes using Comet assay

Acknowledgements: The financial support by 1/0008/08, 2/70033/7, APVV-0321-07.

Goran Gajski ∗ , Vera Garaj-Vrhovac

doi:10.1016/j.toxlet.2008.06.422

Institute for Medical Research and Occupational Health, Mutagenesis Unit, Zagreb, Croatia Bee venom (BV) has a broad array of therapeutic applications in traditional medicine to treat variety of diseases. It is also known that BV possesses anti-inflammatory and anticancer effect and that it can inhibit proliferation and induces apoptosis in cancer cells but there is not enough information regarding genotoxicity of BV on normal human cells. To investigate the effect of whole BV, peripheral blood human lymphocytes from healthy donor were exposed in vitro to different concentration (5, 10, 25, 50 and 100 ␮g/ml) of whole BV at different exposure times (1, 6 and 24 h). Comet assay was used to evaluate the genotoxic effect of BV towards human cells. The results of the present study showed statistically significant increase in DNA damage caused in BV treated human lymphocytes compared to corresponding control cells for the tail length and tail moment. Our study has clearly shown genotoxic potential of whole BV on human peripheral blood lymphocytes in vitro determined by use of the alkaline comet assay in time and dose dependent manner. Based on the same results it is clear that whole BV induces DNA damage and has genotoxic potential on human lymphocytes. Results obtained by single cell gel electrophoresis indicate that comet assay is a useful tool for measuring DNA migration that is induced by natural venoms and the need for further genotoxic and cytogenetic researches using sensitive methods in detection of primary genome damage to establish the exact type and mechanism of DNA damage that is induced by BV. doi:10.1016/j.toxlet.2008.06.421 V31 The use of Chlamydomonas reinhardtii in Comet assay Eliska Galova ∗ , Andrea Sevcovicova, Katarina Hasplova, Katarina Klikova, Eva Miadokova Department of Genetics, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia Chlamydomonas reinhardtii is a unicellular green alga (Chlorophyta). Because of its quick generation time and the relative good adaptability C. reinhardtii is used as an important model organism. The single-cell gel electrophoresis (Comet assay) is widely used to test for various types of DNA damage and repair capacity of the cell. Breaks render nuclear DNA susceptible to movement by electrophoresis forming “comet tails” detectable by fluorescent dyes. In addition to bacterial, higher plant and animal model organisms, there are some fundamental purposes to apply the method to unicellular green alga C. reinhardtii. In order to overcome technical difficulties in Chlamydomonas assay (the lysis of the cells, DNA migration, and visualisation/quantification of DNA damage) we have standardized the protocol taking two main modifications: (1) Chlamydomonas gametic enzyme autolysine was applied to the

V32 Effects of plasticizers and phenols on estrogen and thyroid hormone functions Mandana Ghisari ∗ , Eva C. Bonefeld-Jorgensen University of Aarhus, Department of Public Health, Aarhus, Denmark Plasticizers and phenols are man-made chemicals mainly used to impart softness and flexibility to normally rigid plastic, and as additives in paint and adhesives. We have investigated the estrogenic and thyroid hormone-like activities in vitro of a range of widely used plasticizers and phenols including benzyl butyl phthalate, dibutyl phthalate, dioctyl phthalate, diisodecyl phthalate, diisononyl phthalate, bis (2-ethylhexyl) phthalate, bis (2-ethylhexyl) adipate, 4-tert-octylphenol, 4-chloro3-methylphenol, 2,4-dichlorophenol, 2-phenylphenol and resorcinol. Furthermore, the combined effect of selected compounds was investigated. The estrogenic activities of the compounds were assessed using luciferase reporter gene assay and the thyroid disrupting potential was determined by the effect on the TH-dependent rat pituitary GH3 cell proliferation. Our results showed that three of the compounds induced ER transactivity, whereas one antagonized the 17b-estradiol induced ER function. All the tested compounds, but one, significantly affected the GH3 cell proliferation. The mixture of six selected compounds significantly induced ER transactivity to a higher level than the compounds alone, whereas the effect of the mixture in the T-screen was not significantly different from the single compounds alone. Our in vitro data suggest that the tested plasticizers and phenols have endocrine-disrupting potential that can be mediated via interference with the estrogen and thyroid hormone system. It is of great concern since these two hormone systems are known to play important roles during fetus development with respect to brain as well as reproductive organs. Also the combined effects of the compounds should be taken into consideration for risk assessment of human health. doi:10.1016/j.toxlet.2008.06.423 V33 Investigation of biphasic effects of quercetin and genistein in vitro Burcu Eren ∗ , Hande Gurer-Orhan Ege University, Faculty of Pharmacy, Department of Toxicology, Izmir, Turkey Phytoestrogen is a general term given to plant-derived estrogenlike compounds. They have gained a reputation for being