Comparative male reproductive toxicity of selected phthalates

Abstracts / Toxicology Letters 205S (2011) S180–S300

ters and histopathological analysis. Results: The testis and plasma of CP-exposed rats showed significant increase in the level of nitric oxide (NO) and lipid peroxidation, along with a significant decrease in superoxide dismutase, catalase and glutathione peroxidase activities. Also, treatment of male rats with CP caused a significant decrease in the sperm count and motility, while dead and abnormal sperms increased as compared to control. The histopathological study revealed that CP induced marked reductions in sperm’s content of epididymis, obstruction in seminiferous tubules, hypospermia and dilated congested blood vessels in testicular sections. Co-administration of ALC minimized all toxic effects of CP. Conclusion: The administration of CP causes elevation of oxidative stress leading to a marked testicular dysfunction and ALC ameliorates these effects by antioxidant mechanisms suggesting a protective role against male infertility. doi:10.1016/j.toxlet.2011.05.841

P2218 Pioglitazone ameliorates letrozole-induced hyperandrogenism and oxidative stress-mediated polycystic ovary in female rat M.A. Rezvanfar 1 , A. Ahmadi 2 , M. Baeeri 1 , A. Kebriaeezadeh 1 , M. Abdollahi 1,∗ 1

Toxicology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Centre, Tehran University of Medical Sciences, Tehran, Iran, 2 Histology and Embryology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran Purpose: Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism and chronic anovulation and studies have shown that thiazolidinediones (TZDs) improve metabolic disturbances in PCOS patients. The aim of the present work was to investigate the relationship between oxidative stress and the underlying mechanisms related to letrozole-induced polycystic ovary and the possible protection of these complications by thiazolidinedione derivative pioglitazone. Methods: Treatment with letrozole (1 mg/kg body weight) for 21 consecutive days, recreates a rat model that resembles many aspects of the human polycystic ovary syndrome (PCOS). Pioglitazone was administrated orally at dose of 20 mg/kg/day for 21 days. Serum hormone levels, histological changes in ovaries and biomarkers of oxidative stress were examined. Results: Severe pathologic changes such as marginal large fluid-filled follicular cysts and loss of corpus luteum are the characteristics of letrozole-induced PCO in rat ovary. Also we found that hyperandrogenism produced by letrozole administration, induced a pro-inflammatory and a pro-oxidant condition represented by increased levels of ovarian prostaglandin E production and blood and ovarian nitric oxide activity and a significant decrease in superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities together with an increase in the level of serum TNF-␣. Serum estradiol and progesterone levels were reduced and testosterone levels were markedly increased after letrozole administration. All measured parameters were improved by pioglitazone treatment and reached close to normal levels. Conclusion: The present study for the first time determined the role of oxidative/nitrosative stresses in the pathogenesis of letrozole induced-PCO and the protective effects of pioglitazone. doi:10.1016/j.toxlet.2011.05.842

S247

P2219 Inhibition of carbendazim-induced steroidogenic dysfunction and apoptosis in rat testis by kolaviron, a natural biflavonoid from Garcinia kola seed I.A. Adedara 1,∗ , S. Vaithinathan 2 , R. Jubendradass 2 , P.P. Mathur 2 , E.O. Farombi 1 1

Department of Biochemistry, University of Ibadan, Ibadan, Nigeria, Biochemistry and Molecular Biology, Pondicherry University, Pondicherry, India 2

We evaluated the protective role of kolaviron (a natural biflavonoid from the seed of Garcinia kola) and vitamin E on carbendazim-induced reproductive dysfunction in male rats. Thirty adult male rats were divided into five groups. Control rats received corn oil only. A group of rats received a single oral dose of carbendazim (200 mg/kg bw) only. Other groups of rats were pretreated with kolaviron (100 and 200 mg/kg bw) or vitamin E (50 mg/kg bw) for 7 days before challenging with a single oral dose of carbendazim. All the animals were sacrificed 24 h after the last treatment. The results showed that a single oral dose of carbendazim significantly decreased the activities of superoxide dismutase and catalase but markedly increased sialic acid concentration and lipid peroxidation in rat testes. Western blot analysis revealed that carbendazim treatment decreased the expression of steroid acute regulatory protein and androgen binding protein with concomitant decrease in activities of 3␤-hydroxysteroid dehydrogenase and 17␤-hydroxysteroid dehydrogenase. The elevated cytosolic level of cytochrome c with increased caspase 3 and Fas levels in testicular sections from immunofluorescence staining was consistent with the increased TUNEL-positive nuclei in carbendazim-treated rats. However, kolaviron and vitamin E restored the testicular antioxidant status, sialic acid concentration and steroidogenesis in carbendazim-treated rats. Also, testicular caspase 3 and Fas expression and the apoptotic nuclei were significantly decreased to near control level by kolaviron and vitamin E. Collectively, the result suggests that kolaviron may prove useful in combating carbendazim-induced reproductive toxicity possibly by decreasing germ cell apoptosis. doi:10.1016/j.toxlet.2011.05.843

P2220 Comparative male reproductive toxicity of selected phthalates U. Bernauer ∗ , E. Rosenthal, B. Heinrich-Hirsch, A. Schulte Safety of Chemicals, Federal Institute for Risk Assessment (BfR), Berlin, Germany Purpose: The reproductive toxicity of several phthalates leads to regulatory measures for these plasticisers. However, only few phthalates have been evaluated for their toxicological effects. Therefore, regulations exist only for a limited number of phthalates. It has been claimed that phthalates, which are structurally similar to already classified and regulated phthalates, possess comparable reprotoxic properties (especially concerning the male reproductive system). In order to compare the toxic potency of phthalates, it was the aim of this study to identify appropriate sensitive parameters for reproductive effects of phthalates. Methods: The male reproductive toxicity of dialkyl phthalates of side chains in the range of 3–8 carbon atoms was reviewed based on about 200 publications in the open literature. The reproductive toxic potential was analysed for di-n-propyl-, di-iso-butyl-, di-n-butyl-, di-n-pentyl-, di-cyclo-

S248

Abstracts / Toxicology Letters 205S (2011) S180–S300

hexyl-, di-n-hexyl-, di-iso-heptyl-, di-2-ethylhexyl-, di-n-heptyl-, di-n-octyl-, and di-iso-nonyl-phthalate. Adverse effects on various endpoints of male fertility and offspring development were compared. Results: Selected phthalates with side chain lengths of 4–6 C-atoms adversely affect male fertility with comparable potential and potency. Overall, impairment of male development was more pronounced compared to the impairment of male fertility. Concerning fertility, adverse effects on sperm parameters and Sertoli cells were the most sensitive endpoints. Concerning development, reduction of anogenital distance and changes in testosterone levels in serum or testes were the most sensitive endpoints. Concerning development, doses inducing adverse effects were lower for phthalates with 4–6 C-atoms in the side chain when compared to phthalates with longer or shorter side chains. doi:10.1016/j.toxlet.2011.05.844

P2221 Evaluation of the embryotoxicity of misoprostol using the whole embryo culture assay M.F. Cavieres ∗ , C.A. Campos Faculty of Pharmacy, Universidad de Valparaíso, Valparaíso, Chile Purpose: Misoprostol, a synthetic analog of prostaglandin E1, is indicated for pregnancy termination. Misoprostol-inducedabortions may not be successful leading to in utero exposure to the drug. A strong epidemiological relationship has been found between in utero exposure to misoprostol and the induction of developmental defects. However, reports of experimental studies are practically non-existent, probably indicating lack of experimental models suitable for the study of the teratogenicity of the drug. Here we used the whole embryo culture assay (WEC) to examine the effect of misoprostol on development. Methods: Embryos were collected from Sprague Dawley rats on gestation day 9.5 and cultured according to the ECVAM method. In brief, embryos with attached yolk sacs and ectoplacental cones were cultured for 48 h at 37 ◦ C in rat serum supplemented with streptomycin and penicillin and a gas mixture of CO2 , O2 and N2 . Misoprostol concentrations were 200, 2000 and 20,000 pg/ml. At the end of culture, embryonic growth was assessed by measurement of crown–rump length, head length, and yolk sac diameter. Development was evaluated by counting the number of somites and arches. Rotation, forelimb bud, optic and otic vesicles, and other embryo structures were evaluated for stage of development. Heartbeat and presence of functional blood vessels in the visceral yolk sac were also determined. Results: Misoprostol resulted in concentration-dependent induction of morphologic abnormalities and in the decrease of embryo growth parameters. The data confirms that WEC is an experimental alternative suitable for the study of mechanisms that mediate misoprostol teratogenicity. doi:10.1016/j.toxlet.2011.05.845

P2222 Protective effect of Spirulina (Arthrospira) maxima against cadmium-induced genotoxicity and teratogenicity in mice G.A. Chamorro-Cevallos 1,∗ , N. Argüelles-Velázquez 1 , I. Alvarez González 2 , E. Madigal-Bujaidar 2 1

Pharmacy, Escuela Nacional de Ciencias Biològicas, IPN, Mèxico D.F., Mexico, 2 Morphology, Escuela Nacional de Ciencias Biològicas, IPN, Mèxico D.F., Mexico The role of Spirulina (Arthrospira) maxima (SP) in preventing cadmium (Cd) genotoxicity and teratogenicity in ICR mice was studied. Cd was administered intraperitoneally to pregnant female mice at 1.5 mg/kg on gestation day (GD) 7 and SP was given by gastric intubation at 0, 200, 400 or 800 mg/kg from GD-0 through GD-17 (until sacrifice). For the genotoxicity study, blood samples were collected through a neck incision in each fetus and a micronucleous assay was used. The frequency of micronucleated polychromatic erythrocytes (MNPE) was counted among 1000 polychromatic erythrocytes (PE). Cytotoxicity was also determined as the PE/NE (normochromatic erythrocyte) index. On the other hand, for the teratogenic study, the fetuses were weighed and inspected for external malformations. Total implantations sites and reabsorptions were noted in the uterus. One third of the offspring was designed for skeletal analysis. The remaining fetuses were fixed in Bouin’s solution and prepared for Wilson serial slices. The group treated with Cd showed a significant increase in the quantity of MNPE. The SP treatment at the three doses diminished genotoxic damage in a dose-dependent manner. The cytotoxic assay revealed no alterations in the evaluated index. SP significantly decreased the frequency of fetuses with exencephaly, micrognathia, micropthalmia and skeletal abnormalities at these doses. The results of the present study clearly demonstrate the therapeutic potential of SP in Cd-induced mutagenicity and teratogenicity. It is possible that the mechanism of action of SP could be related to its antioxidant capacity, which has been clearly demonstrated in previous experiments. doi:10.1016/j.toxlet.2011.05.846 P2223 Impact of food products from crude oil polluted area of Nigeria on the reproductive organs of male rats A.P. Ebokaiwe ∗ , E.O. Farombi Biochemistry, University of Ibadan, Ibadan, Nigeria This study was designed to investigate the effect of the secondary exposure of Bonny Light crude oil (BLCO) via food chain on the reproductive system of male rats. Four weeks old male rats were fed with food products from crude oil contaminated area (Imiringi, Bayelsa State) and non contaminated area (Ibadan, Oyo State) for 90 days. Normal commercial rat feed served as control. The food products include 20% fish meal, 33% corn starch, 33% whole grain (corn), 07% fluted pumpkin, 06% vegetable oil, 1% CaCl2 , 0.4% NaCl, 0.6% mixture of minerals and vitamins. Weights and histology of reproductive organs, serum Leutenizing Hormone (LH), Follicle Stimulating Hormone (FSH) and testosterone levels, sperm analysis and morphology, antioxidant system status, and levels of bio-metals were evaluated. The result showed significant increase in weights of seminal vesicle and prostate gland with no effect in testes and epididymal weight. BLCO contaminated food products increased FSH level, whereas LH and testosterone decreased significantly. Although there was a significant increase in bio-metals with