42 Abstracts / Toxicology 290 (2011) 1–46 Shu, Y., Sheardown, S.A., Brown, C., Owen, R.P., Zhang, S.Z., Castro, R.A., Ianculescu, A.G., Yue, L., Lo, J.C., Burchard, E.G., Brett, C.M., Giacomini, K.M., 2007. Journal of Clinical Investigation 117, 1422–1431. doi:10.1016/j.tox.2011.09.074 P067 Reproductive, neuro- and immuno-toxicity in an extended one generation toxicity test with lead acetate David J. Esdaile 1,∗ , Raluca Kubaszky 1 , Peter Maslej 1 , Judit Hargitai 1 , Thomas Hanley 2 , Daniel Minnema 2 , Jayne Wright 3 , Lewis Richard 3 1 LAB Research International, Veszprem, Hungary 2 Syngenta Crop Protection LLC, Greensboro, NC, United States 3 Syngenta, Jealotts Hill, Bracknell, United Kingdom E-mail address: David.Esdaile@Labresearch.hu (D.J. Esdaile). This study was designed to demonstrate the utility of an extended one-generation reproduction study in rats as proposed by draft OECD guideline and the Agricultural Chemical Safety Assessment (ACSA) Technical Committee of the ILSI Health and Environmental Sciences Institute (HESI) and by Cooper et al. (2006). Lead Acetate was provided in drinking water to the F0 adult Wis- tar rats throughout pre-mating, gestation, and lactation, and to the F1 generation to adulthood. The adults (24 rats/sex/group) were given concentrations of 0 (sodium acetate control), 100, 800 and 1700 ppm Pb, beginning 2 to 4 weeks prior to mating. Adults were mated and evaluated for standard in-life parameters, reproduc- tive evaluations and histopathology. Resulting litters were culled to leave up to 10 pups per litter and then assigned to 3 subgroups for clinical pathology/thyroid function/neurotoxicity, immunotox- icity and reproductive toxicity assessments. Selected pups were evaluated for developmental milestones, growth, neurobehavior assessments (FOB, grip strength, landing foot splay, and motor activity by use of video recording, with automated image analysis), TDAR immunotoxicity (by IgM response to sheep-RBC challenge), thyroid hormones, standard pathology and neurohistopathology (with perfusion-fixation in situ and serial brain sectioning). Dose-related decreases in water consumption were observed in F0 adults (typically -21% in high dose) and F1 offspring (typ- ically -12 to -29% in high dose) at all concentrations. Decreases in sperm parameters (sperm counts, motility, and morphology) were observed in the F0 and F1 males. At the high dose, effects on early post-natal survival, lower pup body weights throughout lactation, decreases in anogenital distance, and delayed vaginal opening (2 day delay in 90% positive rate) and preputial separation were observed. Although not statistically significant from control, slightly lower serum IgM levels were noted for the 1700 ppm males. There was minimal evidence of neurobehavioral changes. Neuro- histopathology of the CNS revealed no evidence of any significant changes, however peripheral nerves (proximal sciatic, sural and tib- ial nerves) from both hind limbs of all animals revealed an increased incidence and severity of nerve fibre degeneration in the high-dose parents and 70-day pups, with no evidence of changes in 21-day weanling pups (Table 1). This outcome illustrates the utility of using an integrated study design to investigate the various aspects of developmental neuro- toxicity, immunotoxocity, and reprotoxicity. Table 1 Example results from affected parameters. Parameters n Mean or incidence SD Sperm count Control 24 253.93 30.29 1700 ppm 24 211.23** 30.27 Pup wt gain (g) to d21 Control 22 51.94 5.25 1700 ppm 22 49.09* 5.19 Vaginal opening on d31 Control 62 45 (73%) – 1700 ppm 62 19 (31%) – Reference Cooper, R.L., Lamb, J.C., Barlow, S.M., Bentley, K., Brady, A.M., Doerr, N., Eisenbrandt, D.L., Fenner-Crisp, P.A., Hines, R.N., Irvine, L.F.H., Kimmel, C.A., Koeter, H., Li, A.A., Makris, S.L., Sheets, L.P., Speijers, G.J.A., Whitby, K.E., 2006. A tiered approach to life stages testing for agricultural chemical safety assessment. Crit. Rev. Toxicol. 36, 69–98. doi:10.1016/j.tox.2011.09.075 P068 A possible role for BCRP in steroid hormone metabolism in endocrine organs Anita C.A. Dankers 1,∗ , Jorien W.H. Sanderink 1 , Jeanne C.L.M. Pertijs 1 , Fred C.G.J. Sweep 2 , Frans G.M. Russel 1 , Rosalinde Masereeuw 1 1 Department of Pharmacology and Toxicology, Nijmegen Centre for Molecular Life Sciences, The Netherlands 2 Department of Laboratory Medicine, Radboud University Nijmegen Medical Centre, The Netherlands E-mail address: A.Dankers@pharmtox.umcn.nl (A.C.A. Dankers). The efflux pump breast cancer resistance protein (BCRP) is known for its detoxification function by transporting drugs and their metabolites out of the cell. However, its physiological function is not yet elucidated. Preliminary in vivo data from our laboratory suggest a possible role of BCRP in regulating steroid hormone bal- ance. In this study, the expression and localization of Bcrp was investigated in endocrine organs of wild type FVB mice. Further- more, the interaction of various steroid hormones with human BCRP activity was studied. Quantitative PCR revealed Bcrp mRNA expression in the pitu- itary and adrenal glands, ovary, testis and adipose tissue. The localization of Bcrp in these organs was assessed by immuno- histochemistry. Perfusion-fixed, paraffin embedded organs were incubated with the primary antibody against mouse Bcrp (BXP- 9). Bcrp was present in the pituitary gland and in the cortex of the adrenal gland. Bcrp expression was also found in ovary, the interstitial cells of Leydig in the testis and in plasma membranes of adipocytes. The efflux pump was clearly absent in the pancreas. To study the interaction of several steroid hormones with BCRP, membrane vesicles of BCRP-overexpressing HEK293 cells were prepared. The effects of estradiol, testosterone, proges- terone and androstenedione on BCRP-mediated vesicular uptake of 3 H-estrone sulphate (E 1 S) were determined. E 1 S was trans- ported by BCRP with an apparent K m of 4.5 ± 0.4 M and a V max of 332.1 ± 11.5 pmol/mg min. The abovementioned steroids showed a clear concentration-dependent inhibition of this trans- port. Dixon plot analysis revealed inhibitory constant (K i ) values of 4.9 ± 0.2 M for estradiol, 36 ± 14 M for testos- terone, 14.7 ± 1.3 M for progesterone, and 216 ± 1.3 M for androstenedione. All compounds attenuated BCRP function in a