Abstracts / Toxicology Letters 205S (2011) S180–S300 S233 nitrate caused independent or antagonistic effect on increasing of methemoglobin level in blood. Combination of sodium nitrate and imidacloprid was more dangerous and led to summation and – in same groups of animals – even to synergistic effect. Thus increas- ing the methemoglobin level in the blood plays an important role in the mechanism of toxicities of dimethoat and imidacloprid. doi:10.1016/j.toxlet.2011.05.795 P2172 Pesticides modulate the course of tularemia in European brown hares H. Bandouchova 1 , J. Pikula 1,* , M. Pohanka 2 , J. Osickova 1 , K. Ondracek 1 , J. Kral 1 , F. Vitula 1 , F. Treml 1 1 Department of Veterinary Ecology and Environmental Protection, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic, 2 Center of Advanced Studies, Faculty of Military Health Sciences, Hradec Kralove, Czech Republic The causative agent of tularemia Francisella tularensis is highly infectious and lagomorphs are important reservoirs and a source of human disease. The aim of the present study was to test the hypothesis that sublethal exposure to pesticides increases the susceptibility of hares to F. tularensis and modulates the course of the infection. Experimental hares were allocated to (a) paraoxon-treated, (b) F. tularensis-treated and (c) paraoxon-and- F. tularensis-treated groups of five specimens on a random basis and subcutaneously inoculated with a wild F. tularensis subsp. holarctica strain (a single dose of 9 × 10 8 CFU pro toto) and/or injected a sublethal dose of paraoxon (100 g/kg). Survival curves in the three groups were significantly different (logrank test, chi square = 12.01, df = 2, P = 0.0025). The paraoxon-and-F. tularensis- treated group showed a rapid onset of clinical sings and death occurred on day 2 and 3 post exposure. F. tularensis-inoculated hares survived from 3 to 10 days, while only one hare died on day 12 in the paraoxon-treated group. Group differences were also eval- uated using haematology, plasma biochemistry, antioxidant status parameters, and acetylcholinesterase activity. Results of this study can help us understand the pathogenesis of tularemia and the mor- tality in hares in agricultural habitats. Use of anticholinesterase agents in agriculture can pose a threat of infectious disease out- breaks and higher mortality in wildlife populations. Acknowledgements: The Ministry of Education, Youth and Sports (Czech Republic) and the Internal Grant Agency of the University of Veterinary and Pharmaceutical Sciences Brno supported projects MSM 6215712402 and 78/2010/FVHE, respectively. doi:10.1016/j.toxlet.2011.05.796 P2173 Glyphosate based herbicides toxicity, a new approach L. Ramírez Cebollero * , P. López-Aparicio, M. Natividad Recio, M.A. Pérez-Albarsanz Department of Biochemistry and Molecular Biology, Alcala University, Madrid, Spain Purpose: Glyphosate is the most commercialized broad- spectrum herbicide worldwide. People may be exposed to its residues by agricultural practices or along the food chain. Different harmful effects are described in several models, not only human. Our aim is to study glyphosate toxicity in the human prostate can- cer cell lines PC3 (androgen independent) and LNCap (androgen dependent). Methods: Anexin-V detection and propidium iodide cell cycle analysis by flow cytometry, LC3 and Procaspase-3 valuation by Western Blotting, cell proliferation assays performed by metab- olization of MTT salts and necrosis level determined by Lactate Dehydrogenase (LDH) release. Results: Cell proliferation assays show a decrease in cell num- ber up to a 60%, after three days of treatment with 50–100 M glyphosate. There is an increase of apoptotic cells referred to con- trol in both lines. LDH liberation assays, denote significant levels of necrosis in LNCap, but not in PC3. Primary studies of cell cycle, indi- cate there could be alterations at this level as well. These effects, mentioned in other models, are not enough to explain the dramatic decrease in cell viability observed. That is why we studied an indi- cator of another cell death pathway: autophagy. Results on LC3 protein expression are highly significant, reaching a 200% signal referred to the control, even before changes in apoptosis induc- tion or LDH liberation are seen. Commonly, all the effects are more intense in the androgen dependent cell line, what lead us to sus- pect that glyphosate has a differential effect between both models, resulting cytotoxic for LNCap and mainly cytostatic in PC3. doi:10.1016/j.toxlet.2011.05.797 P2174 Repeat dietary administration of an organic acid causes salivary gland alterations D. Saltmiras 1,* , A. Remick 2 , M. Haas 3 1 Monsanto, St Louis, USA, 2 WIL-Biotechnics, LLC, Hillsborough, USA, 3 Toxicology, WIL Research Laboratories, LLC, Ashland, USA Salivary gland weight increases and cytoplasmic alteration have been observed in rodents following repeat dietary dosing with glyphosate. Such data have been published and reviewed by regu- lators and authoritative experts for over a decade. Although not consider adverse effects by authorities, the toxicological signifi- cance of these findings has been questioned. One hypothesis is that increased salivary gland size and flow may be due to oral irrita- tion via ingestion of an organic acid. To characterize salivary gland effects of dietary organic acids, male Sprague-Dawley rats were dosed for eight weeks with: basal diet (control); 14,000 ppm citric acid in diet (low pH); 21,400 ppm trisodium citrate dihydrate in diet (high pH, stoichiometric equivalent citrate ion concentration); gav- age vehicle (control); and gavage citric acid (791–1316 mg/kg/day). Salivary glands (parotid and fused submaxillary/sublingual) were excised, weighed, fixed and examined microscopically. Statisti- cally significant increased parotid salivary gland weights were noted only for the low pH dietary citric acid group. Histopatho- logical evaluation revealed cytoplasmic alteration (characterized by basophilic tinctorial change and hypertrophy of acinar cells) in parotid salivary glands across all dose groups with varied sever- ity. Non-parametric statistical analysis of severity ratings (where minimal/mild/moderate = 1/2/3 respectively), noted a significant increase in parotid salivary gland cytoplasmic alteration severity in the low pH dietary citric acid group only. The results of this study support the hypotheses that salivary gland effects noted in repeat dose dietary studies with glyphosate are due to dietary adminis- tration of an organic acid and are considered adaptive responses, rather than adverse effects. doi:10.1016/j.toxlet.2011.05.798