Phenotypic characterization of recessive gene knockout rat models of Parkinson's disease.

Recessively inherited loss-of-function mutations in the PTEN-induced putative kinase 1(Pink1), DJ-1 (Park7) and Parkin (Park2) genes are linked to familial cases of early-onset Parkinson's disease (PD). As part of its strategy to provide more tools for the research community, The Michael J. Fox Foundation for Parkinson's Research (MJFF) funded the generation of novel rat models with targeted disruption ofPink1, DJ-1 or Parkin genes and determined if the loss of these proteins would result in a progressive PD-like phenotype. Pathological, neurochemical and behavioral outcome measures were collected at 4, 6 and 8months of age in homozygous KO rats and compared to wild-type (WT) rats. Both Pink1 and DJ-1 KO rats showed progressive nigral neurodegeneration with about 50% dopaminergic cell loss observed at 8 months of age. ThePink1 KO and DJ-1 KO rats also showed a two to three fold increase in striatal dopamine and serotonin content at 8 months of age. Both Pink1 KO and DJ-1 KO rats exhibited significant motor deficits starting at 4months of age. However, Parkin KO rats displayed normal behaviors with no neurochemical or pathological changes. These results demonstrate that inactivation of the Pink1 or DJ-1 genes in the rat produces progressive neurodegeneration and early behavioral deficits, suggesting that these recessive genes may be essential for the survival of dopaminergic neurons in the substantia nigra (SN). These MJFF-generated novel rat models will assist the research community to elucidate the mechanisms by which these recessive genes produce PD pathology and potentially aid in therapeutic development.

Dietary administration of paraquat for 13 weeks does not result in a loss of dopaminergic neurons in the substantia nigra of C57BL/6J mice.

Several investigations have reported that mice administered paraquat dichloride (PQ·Cl2) by intraperitoneal injection exhibit a loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). In this study, male and female C57BL/6J mice were administered PQ·Cl2 in the diet at concentrations of 0 (control), 10, and 50ppm for a duration of 13weeks. A separate group of mice were administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) during week 12 as positive controls to produce a loss of dopaminergic neurons in the SNpc. The comparative effects of PQ and MPTP on the SNpc and/or striatum were assessed using neurochemical, neuropathological, and stereological endpoints. Morphological and stereological assessments were performed by investigators 'blinded' to the origin of the tissue. Neither dose of PQ·Cl2 (10 or 50 ppm in the diet) caused a loss of striatal dopamine or dopamine metabolite concentrations in the brains of mice. Pathological assessments of the SNpc and striatum showed no evidence of neuronal degeneration or astrocytic/microglial activation. Furthermore, the number of tyrosine hydroxylase-positive (TH(+)) neurons in the SNpc was not reduced in PQ-treated mice. In contrast, MPTP caused a decrease in striatal dopamine concentration, a reduction in TH(+) neurons in the SNpc, and significant pathological changes including astrocytic and microglial activation in the striatum and SNpc. The MPTP-induced effects were greater in males than in females. It is concluded that 13weeks of continuous dietary exposure of C57BL/6J mice to 50ppm PQ·Cl2 (equivalent to 10.2 and 15.6mg PQ ion/kg body weight/day for males and females, respectively) does not result in the loss of, or damage to, dopaminergic neurons in the SNpc.

Characterization of the T-dependent antibody response (TDAR) to keyhole limpet hemocyanin (KLH) in the Göttingen minipig.

Recently, there has been a renewed interest in the use of the minipig as an alternative to dogs and non-human primates for conducting toxicological assessments in non-rodent species. Since the T-dependent antibody response (TDAR) is one of the most widely-accepted assays used in the assessment of immunocompetence, the present study was undertaken to characterize the primary and secondary TDAR to keyhole limpet hemocyanin (KLH) in the Göttingen Minipig(®). Following primary immunization with either 2 or 10 mg KLH, anti-swine IgM and IgG ELISAs were optimized and individual animal responses were evaluated over time. Immunization with 10 mg KLH on Day 0 promoted primary IgM responses that peaked 6-9 days after antigen administration, while primary IgG levels peaked on Day 13 or 14. Secondary IgG antibody levels (following secondary injection with 2 mg KLH on Day 14) plateaued on Days 20-22. Anti-KLH antibody levels were decreased in minipigs treated with cyclophosphamide (CPS), a known immunosuppressant, at doses ranging from 12.5-50 mg/kg/day, while antibody levels in animals treated with 2.5 mg CPS/kg/day were similar to levels in saline-treated swine. These results demonstrate that the Göttingen Minipig(®) can be a useful alternative non-rodent species to the dog and the non-human primate for evaluating the TDAR to KLH in regulatory assessments of immunotoxicity.

Loss of leucine-rich repeat kinase 2 (LRRK2) in rats leads to progressive abnormal phenotypes in peripheral organs.

The objective of this study was to evaluate the pathology time course of the LRRK2 knockout rat model of Parkinson's disease at 1-, 2-, 4-, 8-, 12-, and 16-months of age. The evaluation consisted of histopathology and ultrastructure examination of selected organs, including the kidneys, lungs, spleen, heart, and liver, as well as hematology, serum, and urine analysis. The LRRK2 knockout rat, starting at 2-months of age, displayed abnormal kidney staining patterns and/or morphologic changes that were associated with higher serum phosphorous, creatinine, cholesterol, and sorbitol dehydrogenase, and lower serum sodium and chloride compared to the LRRK2 wild-type rat. Urinalysis indicated pronounced changes in LRRK2 knockout rats in urine specific gravity, total volume, urine potassium, creatinine, sodium, and chloride that started as early as 1- to 2-months of age. Electron microscopy of 16-month old LRRK2 knockout rats displayed an abnormal kidney, lung, and liver phenotype. In contrast, there were equivocal or no differences in the heart and spleen of LRRK2 wild-type and knockout rats. These findings partially replicate data from a recent study in 4-month old LRRK2 knockout rats and expand the analysis to demonstrate that the renal and possibly lung and liver abnormalities progress with age. The characterization of LRRK2 knockout rats may prove to be extremely valuable in understanding potential safety liabilities of LRRK2 kinase inhibitor therapeutics for treating Parkinson's disease.

An oral developmental neurotoxicity study of decabromodiphenyl ether (DecaBDE) in rats.

BACKGROUND: Decabromodiphenyl ether (DecaBDE; CASRN 1163-19-5) is a flame retardant used in a variety of manufactured products. A single oral dose of 20.1 mg/kg administered to mice on postnatal day 3 has been reported to alter motor activity at 2, 4, and 6 months of age. METHODS: To further evaluate these results, a developmental neurotoxicity study was conducted in the most commonly used species for studies of this type, the rat, according to international validated testing guidelines and Good Laboratory Practice Standards. DecaBDE was administered orally via gavage in corn oil to dams from gestation day 6 to weaning at doses of 0, 1, 10, 100, or 1,000 mg/kg/day. Standard measures of growth, development, and neurological endpoints were evaluated in the offspring. Motor activity was assessed at 2 months of age. Additional motor activity assessments were conducted at 4 and 6 months of age. Neuropathology and morphometry evaluations of the offspring were performed at weaning and adulthood. RESULTS: No treatment-related neurobehavioral changes were observed in detailed clinical observations, startle response, or learning and memory tests. No test substance-related changes were noted in motor activity assessments performed at 2, 4, or 6 months of age. Finally, no treatment-related neuropathological or morphometric alterations were found. CONCLUSIONS: Under the conditions of this study, the no-observed-adverse-effect level for developmental neurotoxicity of DecaBDE was 1,000 mg/kg/day, the highest dose tested.

Effects of dose, administration route, and/or vehicle on decabromodiphenyl ether concentrations in plasma of maternal, fetal, and neonatal rats and in milk of maternal rats.

The effects of route and vehicle on blood and milk levels of decabromodiphenyl ether (DecaBDE; CASRN 1163-19-5) were investigated in the rat to assist in the design and conduct of a developmental neurotoxicity study. Blood plasma and/or milk concentrations were determined in dams, fetuses, and/or nursing pups after repeated DecaBDE administration by gavage throughout gestation or gestation and lactation using corn oil (CO) or soyaphospholipon/Lutrol F 127-water (SPL) as the vehicle. The impact of vehicle on plasma levels was also investigated in pups derived from naive dams after a single postnatal dose. This study reports for the first time fetal and neonatal plasma concentrations concurrent with those of maternal plasma and/or milk. Higher concentrations of DecaBDE were achieved in plasma and in milk with CO than with SPL. Furthermore, pups derived from dams treated with only SPL were lower in body weight, compared with those from dams treated with either CO, CO and DecaBDE, or SPL and DecaBDE. The study further shows that exposure to DecaBDE is relatively consistent across the dose range of 100 to 1000 mg/(kg · day) when administered in CO.

Developmental neurotoxicity study of dietary bisphenol A in Sprague-Dawley rats.

This study was conducted to determine the potential of bisphenol A (BPA) to induce functional and/or morphological effects to the nervous system of F(1) offspring from dietary exposure during gestation and lactation according to the Organization for Economic Cooperation and Development and U.S. Environmental Protection Agency guidelines for the study of developmental neurotoxicity. BPA was offered to female Sprague-Dawley Crl:CD (SD) rats (24 per dose group) and their litters at dietary concentrations of 0 (control), 0.15, 1.5, 75, 750, and 2250 ppm daily from gestation day 0 through lactation day 21. F(1) offspring were evaluated using the following tests: detailed clinical observations (postnatal days [PNDs] 4, 11, 21, 35, 45, and 60), auditory startle (PNDs 20 and 60), motor activity (PNDs 13, 17, 21, and 61), learning and memory using the Biel water maze (PNDs 22 and 62), and brain and nervous system neuropathology and brain morphometry (PNDs 21 and 72). For F(1) offspring, there were no treatment-related neurobehavioral effects, nor was there evidence of neuropathology or effects on brain morphometry. Based on maternal and offspring body weight reductions, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was 75 ppm (5.85 and 13.1 mg/kg/day during gestation and lactation, respectively), with no treatment-related effects at lower doses or nonmonotonic dose responses observed for any parameter. There was no evidence that BPA is a developmental neurotoxicant in rats, and the NOAEL for developmental neurotoxicity was 2250 ppm, the highest dose tested (164 and 410 mg/kg/day during gestation and lactation, respectively).

Safety assessment of heat-sterilized green tea catechin preparation: a 6-month repeat-dose study in rats.

Evidence suggests that the purported health benefits associated with green tea consumption are related to tea catechins. In the present study, potential adverse effects of a standardized heat-sterilized green tea catechin (GTC-H) preparation was investigated following gavage administration to rats at doses of 0, 120, 400, 1200 mg/kg/day for 6 months. A decaffeinated high-dose group (1200 mg/kg/day) (GTC-HDC), was included for comparison. A possibly test article-related clinical finding of intermittent increased activity was noted in the 400 and 1200 mg/kg/day GTC-H groups, but was not considered to be adverse. Lower body weight gains without any decrease in food consumption were noted in the high-dose (1200 mg/kg/day)-treated GTC-H and GTC-HDC females. In the high-dose male GTC-H group, a lower total motor activity count for the 60-min session was noted prior to dosing at the study week 25 evaluations compared to the control group. Similar changes were not observed in the GTC-HDC group. Based on the results of this study, the no-observed-adverse-effect level (NOAEL) for GTC-H was 1200 mg/kg/day for males, the highest dose tested, and 400mg/kg/day for females based on reduced body weight gains. The NOAEL for GTC-HDC was 1200 mg/kg/day for males and could not be determined in females.

28-Day oral (gavage) toxicity studies of green tea catechins prepared for beverages in rats.

The beneficial health effects associated with drinking green tea are widely considered to be due primarily to tea catechins. Heat treatment of marketed green tea beverages for sterilization causes epimerization and/or polymerization of tea catechins. Safety studies on heat-treated tea catechins are limited. The objective of the present study was to evaluate potential adverse effects, if any, of two standardized green tea catechin (GTC) preparations: one that underwent heat sterilization (GTC-H) and one that was not heat-sterilized (GTC-UH). A decaffeinated preparation of the GTC-H (GTC-HDC) was also evaluated to ascertain if any effects were due to caffeine. The GTC preparations were administered to rats once daily at levels up to 2000 mg/kg/day for 28 days. There were no deaths attributable to the GTC preparations. The clinical condition of the animals, functional observational battery, motor activity, clinical pathology evaluations, organ weights, and gross necropsy findings were unaffected by any of the GTC preparations. GTC-HDC or GTC-UH dosing had no effects on body weights or microscopic findings, whereas lower body weights and food consumption were observed in the 1000 and 2000 mg/kg/day GTC-H group males. The no observed-adverse-effect level (NOAEL) for localized gastric effects for GTC-H was 1000 mg/kg/day. No other target organs were identified. Thus, the NOAEL for systemic toxicity following oral administration was 2000 mg/kg/day for GTC-H, GTC HDC, and GTC-UH under the conditions of this study.

Developmental neurotoxicity study of styrene by inhalation in Crl-CD rats.

This study was conducted to assess potential adverse functional and/or morphological effects of styrene on the neurological system in the F2 offspring following F0 and F1 generation whole-body inhalation exposures. Four groups of male and female Crl:CD (SD)IGS BR rats (25/sex/group) were exposed to 0, 50, 150, and 500 ppm styrene for 6 hr daily for at least 70 consecutive days prior to mating for the F0 and F1 generations. Inhalation exposure continued for the F0 and F1 females throughout mating and through gestation day 20. On lactation days 1 through 4, the F0 and F1 females received styrene in virgin olive oil via oral gavage at dose levels of 66, 117, and 300 mg/kg/day (divided into three equal doses, approximately 2 hr apart). Inhalation exposure of the F0 and F1 females was re-initiated on lactation day 5 and continued through weaning of the F1 or F2 pups on postnatal day (PND) 21. Developmental landmarks were assessed in F1 and F2 offspring. The neurological development of randomly selected pups from the F2 generation was assessed by functional observational battery, locomotor activity, acoustic startle response, learning and memory evaluations, brain weights and dimension measurements, and brain morphometric and histologic evaluation. Styrene exposure did not affect survival or the clinical condition of the animals. As expected from previous studies, slight body weight and histopathologic effects on the nasal olfactory epithelium were found in F0 and F1 rats exposed to 500 ppm and, to a lesser extent, 150 ppm. There were no indications of adverse effects on reproductive performance in either the F0 or F1 generation. There were exposure-related reductions in mean body weights of the F1 and F2 offspring from the mid and high-exposure groups and an overall pattern of slightly delayed development evident in the F2 offspring only from the 500-ppm group. This developmental delay included reduced body weight (which continued through day 70) and slightly delayed acquisition of some physical landmarks of development. Styrene exposure of the F0 and F1 animals had no effect on survival, the clinical condition or necropsy findings of the F2 animals. Functional observational battery evaluations conducted for all F1 dams during the gestation and lactation periods and for the F2 offspring were unaffected by styrene exposure. Swimming ability as determined by straight channel escape times measured on PND 24 were increased, and reduced grip strength values were evident for both sexes on PND 45 and 60 in the 500-ppm group compared to controls. There were no other parental exposure-related findings in the F2 pre-weaning and post-weaning functional observational battery assessments, the PND 20 and PND 60 auditory startle habituation parameters, in endpoints of learning and memory performance (escape times and errors) in the Biel water maze task at either testing age, or in activity levels measured on PND 61 in the 500-ppm group. Taken together, the exposure-related developmental and neuromotor changes identified in F2 pups from dams exposed to 500 ppm occurred in endpoints known to be both age- and weight-sensitive parameters, and were observed in the absence of any other remarkable indicators of neurobehavioral toxicity. Based on the results of this study, an exposure level of 50 ppm was considered to be the NOAEL for growth of F2 offspring; an exposure level of 500 ppm was considered to be the NOAEL for F2 developmental neurotoxicity.

1,3-Dinitrobenzene inhibits mitochondrial complex II in rat and mouse brainstem and cortical astrocytes.

1,3-Dinitrobenzene (DNB) produces edematous, glio-vascular lesions that are initially confined to brainstem nuclei with high energy requirements in rats and mice. Perturbation of energy producing processes in the cell is known to induce formation of the mitochondrial permeability transition pore (mtPTP) complex. Selective vulnerability of brainstem astrocytes to DNB is mediated by a 10-fold lower threshold for opening of the cyclosporin A-inhibitable mitochondrial permeability transition (MPT) pore than their cortical counterparts. Other nitrocompounds, such as 3-nitropropionic acid, selectively interfere with regional energy metabolism, including mitochondrial succinate dehydrogenase activity. However, the link between DNB-induced onset of the MPT and disruption of energy producing processes in the astrocyte remains unclear. The effects of DNB on succinate dehydrogenase activity were evaluated in cultured neonatal rat and mouse brainstem and cortical astrocytes. Both histochemical and spectrophotometric assays confirmed significant temporal inhibition of SDH activity in brainstem and cortical astrocytes 0.5, 2 and 5h following exposure to 100 microM DNB in vitro. Although DNB-induced inhibition of SDH was significantly decreased by CsA pretreatment in brainstem astrocytes after 0.5 and 2h and with a second pore inhibitor, bongkrekic acid (BKA) after 5h, both inhibitors failed to reduce inhibition of SDH activity in cortical astrocytes. These data suggest that DNB-induced inhibition of SDH may be independent of differential regional activation of the mtPTP complex in astrocytes and that an unidentified cyclosporin A-inhibitable factor mediates DNB-induced loss of SDH function.