Subchronic, Low-Level Intraperitoneal Injections of Manganese (IV) Oxide and Manganese (II) Chloride Affect Rat Brain Neurochemistry.

Manganese (Mn) is neurotoxic and can induce manganism, a Parkinson-like disease categorized as being a serious central nervous system irreversible neurodegenerative disease. An increased risk of developing symptoms of Parkinson disease has been linked to work-related exposure, for example, for workers in agriculture, horticulture, and people living near areas with frequent use of Mn-containing pesticides. In this study, the focus was placed on neurochemical effects of Mn. Rats were dosed intraperitoneally with 0.9% NaCl (control), 1.22 mg Mn (as MnO2)/kg bodyweight (bw)/day, or 2.5 mg Mn (as MnCl2)/kg bw/day for 7 d/wk for 8 or 12 weeks. This dosing regimen adds relevant new knowledge about Mn neurotoxicity as a consequence of low-dose subchronic Mn dosing. Manganese concentrations increased in the striatum, the rest of the brain, and in plasma, and regional brain neurotransmitter concentrations, including noradrenaline, dopamine (DA), 5-hydroxytrytamine, glutamate, taurine, and γ-amino butyric acid, and the activity of acetylcholinesterase changed. Importantly, a target parameter for Parkinson disease and manganism, the striatal DA concentration, was reduced after 12 weeks of dosing with MnCl2. Plasma prolactin concentration was not significantly affected due to a potentially reduced dopaminergic inhibition of the prolactin release from the anterior hypophysis. No effects on the striatal α-synuclein and synaptophysin protein levels were detected.

Probabilistic cumulative risk assessment of anti-androgenic pesticides in food.

In this paper, we present a cumulative risk assessment of three anti-androgenic pesticides (vinclozolin, procymidone and prochloraz) using the relative potency factor (RPF) approach and an integrated probabilistic risk assessment (IPRA) model. RPFs for each substance were estimated for three reproductive endpoints (ano-genital distance, and weights of the seminal vesicles and the musculus levator ani/bulbocavernosus) in male rat foetuses exposed in utero. The cumulative dietary intake was estimated based on consumption data and residue data from the Netherlands. The IPRA model combines variability in both exposure and sensitivity between individuals into a distribution of individual margins of exposures (IMoEs) and IMoEs of 1 or less indicate a possible concern. The assessment did not result in IMoEs < or = 1. The endpoint 'weight of seminal vesicles' resulted in the lowest IMoEs (0.1th percentile: 198) and the fraction of individuals with IMoEs<1000 was 1.43%. For the two other endpoints, the fractions were slightly lower. Thus, cumulative dietary exposure of Dutch women to vinclozolin, procymidone and prochloraz is not likely to be of concern for the reproductive development of their male foetuses. However, other anti-androgenic substances and exposure routes should also be included in the cumulative assessment to make it more comprehensive.

Lung inflammation and genotoxicity following pulmonary exposure to nanoparticles in ApoE-/- mice.

BACKGROUND: The toxic and inflammatory potential of 5 different types of nanoparticles were studied in a sensitive model for pulmonary effects in apolipoprotein E knockout mice (ApoE-/-). We studied the effects instillation or inhalation Printex 90 of carbon black (CB) and compared CB instillation in ApoE-/- and C57 mice. Three and 24 h after pulmonary exposure, inflammation was assessed by mRNA levels of cytokines in lung tissue, cell composition, genotoxicity, protein and lactate dehydrogenase activity in broncho-alveolar lavage (BAL) fluid. RESULTS: Firstly, we found that intratracheal instillation of CB caused far more pulmonary toxicity in ApoE-/- mice than in C57 mice. Secondly, we showed that instillation of CB was more toxic than inhalation of a presumed similar dose with respect to inflammation in the lungs of ApoE-/- mice. Thirdly, we compared effects of instillation in ApoE-/- mice of three carbonaceous particles; CB, fullerenes C60 (C60) and single walled carbon nanotubes (SWCNT) as well as gold particles and quantum dots (QDs). Characterization of the instillation media revealed that all particles were delivered as agglomerates and aggregates. Significant increases in Il-6, Mip-2 and Mcp-1 mRNA were detected in lung tissue, 3 h and 24 h following instillation of SWCNT, CB and QDs. DNA damage in BAL cells, the fraction of neutrophils in BAL cells and protein in BAL fluid increased statistically significantly. Gold and C60 particles caused much weaker inflammatory responses. CONCLUSION: Our data suggest that ApoE-/- model is sensitive for evaluating particle induced inflammation. Overall QDs had greatest effects followed by CB and SWCNT with C60 and gold being least inflammatory and DNA-damaging. However the gold was used at a much lower mass dose than the other particles. The strong effects of QDs were likely due to Cd release. The surface area of the instilled dose correlated well the inflammatory response for low toxicity particles.

Neurological Effects of White Spirit: Contribution of Animal Studies during a 30-Year Period*.

Numerous studies have suggested that long-term occupational exposure to white spirit may cause chronic toxic encephalopathy (WHO 1996). This review summarizes the chronic nervous system effects of white spirit in animal studies during a 30-year period. First, routine histopathology was consistently unable to reveal adverse peripheral or central nervous system effects after inhalation of white spirit. Second, neurobehavioural studies in animals showed no adverse effect after inhalation of white spirit with a high content of aromatics in contrast to what was found with products with a low content. Third, white spirit with a high content of aromatics induced adverse neurochemical changes at inhalation of 400 ppm and possibly already at 100 ppm. In the studied parameters, white spirit with a low content of aromatics showed no clear adverse neurochemical effects at inhalation of 400 ppm, but the neurophysiological tests showed adverse effects at this level. Fourth, neurophysiological methods may be more sensitive than histopathological, neurobehavioural and neurochemical methods. Overall, white spirit with a high and a low content of aromatics showed no overt difference in long-term effects in animals, taking all studied end-points into account. The differences in sensitivity of the test methods should be taken into consideration if new toxicological studies are conducted on this type of solvents.

Neurotoxic effect of maneb in rats as studied by neurochemical and immunohistochemical parameters.

Epidemiological investigations document that workers in agriculture, horticulture and people living near areas with frequent use of pesticides have increased risk of developing symptoms of Parkinson's disease. This study investigated the neurotoxic effect of the fungicide maneb by morphological, immunohistochemical and neurochemical methods applying young Sprague-Dawley male rat as the model. Intraperitoneal dosing (7.5, 15 or 30mg maneb/kg bodyweight/week for 12 weeks) demonstrated dose-related increased manganese concentration in corpus striatum. The striatal concentration of 5-hydroxytryptamine (5-HT) increased in a dose-related manner, as did the 5-HT concentrations in the rest of the brain indicating early sign of neurotoxicity. Striatal acetylcholinesterase activity was not affected. The concentrations of noradrenaline, dopamine, neurotransmitter amino acids and the levels of the proteins α-synuclein and synaptophysin in corpus striatum and the rest of the brain were not changed. No histological parameter was affected when studied in corpus striatum and substantia nigra.

Antiandrogenic effects in male rats perinatally exposed to a mixture of di(2-ethylhexyl) phthalate and di(2-ethylhexyl) adipate.

Di(2-ethylhexyl) phthalate (DEHP) is a well-known testicular toxicant inducing adverse effects in androgen responsive tissues. Therefore, di(2-ethylhexyl) adipate (DEHA) is currently being evaluated as a potential substitute for DEHP. Similarities in structure and metabolism of DEHP and DEHA have led to the hypothesis that DEHA can modulate the effects of DEHP. Wistar rats were gavaged with either vehicle, DEHP (300 or 750mg/kg bw/day) or DEHP (750mg/kg bw/day) in combination with DEHA (400mg/kg bw/day) from gestation day (GD) 7 to postnatal day (PND) 17. Decreased anogenital distance (AGD) and retention of nipples in male offspring were found in all three exposed groups. Dosed males exhibited decreased weights of ventral prostate and m. levator ani/bulbocavernosus. Histopathological investigations revealed alterations in testis morphology in both juvenile and adult animals. The litter size was decreased and postnatal mortality was increased in the combination group only, which is likely a combined effect of DEHP and DEHA. However, no combination effect was seen with respect to antiandrogenic effects, as males receiving DEHP in combination with DEHA did not exhibit more pronounced effects in the reproductive system than males receiving DEHP alone.

Early testicular effects in rats perinatally exposed to DEHP in combination with DEHA--apoptosis assessment and immunohistochemical studies.

This study aimed to characterize the effects of di(2-ethylhexyl) phthalate (DEHP) on the fetal rat testes and relate them to the effects seen in adults. Histopathological effects in fetal testes were examined with immunohistochemistry for anti-Mullerian hormone (AMH), 3beta-hydroxysteroid dehydrogenase, smooth muscle actin (SMA), proliferating cell nuclear antigen (PCNA), histone H3 and vimentin. Additionally, testicular apoptosis levels were assessed in fetal, prepubertal and adult rats. As the plasticizer di(2-ethylhexyl) adipate (DEHA) has similarities with DEHP in chemical structure and metabolism, we investigated if the testicular effects of DEHP were modulated by co-administration with DEHA. Wistar rats were gavaged during gestation and lactation with vehicle, DEHP (300 or 750 mg/kg/day), or DEHP (750 mg/kg/day) in combination with DEHA (400mg/kg/day), and male offspring were examined at gestation day (GD) 21, postnatal day (PND) 22, 26 and 190. In fetal testes, Leydig cells were found in large clusters containing AMH positive Sertoli cells. At GD 21, seminiferous chords appeared enlarged with an apparently increased number of gonocytes. However, proliferation of gonocytes did not appear increased. A few animals had a high number of TUNEL positive apoptotic cells in degenerating seminiferous tubules at PND 22 and 190, whereas most exposed animals had low levels of germ cell apoptosis at GD 21, PND 22 or PND 26, as evaluated by DNA laddering, TUNEL staining, Caspase-3 immunohistochemistry and Caspase-3 activity measurement. No differences between DEHP and DEHP+DEHA exposed groups were observed.

Effects of combined prenatal stress and toluene exposure on apoptotic neurodegeneration in cerebellum and hippocampus of rats.

Pregnant Wistar rats were exposed to 1500 ppm toluene 6 hr/day from gestational day 7-20 or to chronical mild stress from gestational day 9-20 as single exposure or in combination. Behavioural, immunohistopathological, molecular biological, and neurochemical methods were applied to investigate the offspring for developmental neurotoxicity and level of apoptosis in the brain. The number of apoptotic cells in cerebellum postnatal day 22, 24, and 27 and in hippocampus (postnatal day 22, 24, and 27) were counted after visualization by the TUNEL staining or measured by DNA-laddering technique. Caspase-3 activity was determined in cerebellum (postnatal day 6, 22, 24, and 27) and in hippocampus (postnatal day 6 and 22). TUNEL staining and DNA-laddering technique showed a marked decrease in number of apoptotic cells from postnatal day 22 to 27 in both cerebellum and hippocampus. Apparently, a peak in the number of TUNEL positive cells was identified in cerebellum at postnatal day 22. There was no statistically significant influence of exposure except that DNA-laddering in cerebellum at postnatal day 27 was increased by toluene exposure. Caspase-3 activity decreased in cerebellum and hippocampus with age. At postnatal day 6 stress and toluene, when singly exposed, increased activity in cerebellum whereas co-exposure to stress and toluene did not. Stress increased caspase-3 activity in hippocampus postnatal day 22. There was overall consistency between the results obtained by the three supplementary methods regarding the influence of exposure and age on apoptotic activity in cerebellum and hippocampus. New methods to quantitate the relative level of apoptosis measured as DNA-laddering and the caspase-3 activity in tissue are presented.

Steroidogenesis in fetal male rats is reduced by DEHP and DINP, but endocrine effects of DEHP are not modulated by DEHA in fetal, prepubertal and adult male rats.

The plasticizer di(2-ethylhexyl)phthalate (DEHP) exhibits antiandrogenic effects in perinatally exposed male rats. Di(2-ethylhexyl) adipate (DEHA) and diisononyl phthalate (DINP) are currently being evaluated as potential substitutes for DEHP, but similarities in structure and metabolism of DEHP with DEHA and DINP have led to the hypothesis that similarities in action may also exist. Pregnant Wistar rats were gavaged during gestation and lactation with vehicle, DEHP (300 or 750 mg/kg bodyweight per day), DINP (750 mg/kg bodyweight per day), DEHP (750 mg/kg bodyweight per day) in combination with DEHA (400 mg/kg bodyweight per day), or DEHP (300 mg/kg bodyweight per day) in combination with DINP (750 mg/kg bodyweight per day). DINP and DEHP were both shown to reduce testicular testosterone production ex vivo and testosterone levels in testes and plasma of male fetuses at gestation day 21, indicating a similar mechanism of action for DINP and DEHP. Additionally, plasma LH levels in male fetuses were elevated. Neonatal anogenital distance was reduced and the number of nipples at postnatal day 13 increased in DEHP-exposed male offspring. Serum inhibin B levels were significantly reduced in DEHP-exposed prepubertal male offspring, and in a few adult males. No modulating effects of DEHA on the endocrine effects of DEHP were detected, but a tendency towards an accumulating effect of DEHP and DINP in combination on suppression of testosterone synthesis was seen.

Di(2-ethylhexyl) adipate (DEHA) induced developmental toxicity but not antiandrogenic effects in pre- and postnatally exposed Wistar rats.

Di(2-ethylhexyl) adipate (DEHA) has replaced the phthalates in thin plasticized polyvinyl chloride films used for food packaging, mainly because some phthalates induce testis toxicity and antiandrogenic effects. A dose-range finding study followed by a dose-response/effect study in Wistar rats investigated whether pre- and postnatal DEHA doses of 0, 800, or 1200mg/kg/day body weight and doses of 0, 200, 400, or 800mg/kg/day (main study) elicited developmental toxicity including antiandrogenic effects. In the main study, DEHA induced a prolonged gestation period (800mg/kg/day) and a dose-related increase in postnatal death (400 and 800mg/kg/day). DEHA also induced a permanent decrease in offspring body weight (800mg/kg/day). No antiandrogenic endpoints were affected. We conclude that DEHA induced developmental toxicity and the NOAEL is 200mg/kg. DEHA did not induce antiandrogenic effects similar to those of di(2-ethylhexyl) phthalate even though the chemical structures have similarities and the two chemicals have a common metabolite.

In utero exposure to diethylstilboestrol or 4-n-nonylphenol in rats: number of sertoli cells, diameter and length of seminiferous tubules estimated by stereological methods.

The effects on testis weight and histopathology were studied in 11-day-old male Wistar rats after prenatal exposure to peanut oil (control), diethylstilboestrol 30 microg/kg b.wt./day, or 4-n-nonylphenol 75 mg/kg b.wt./day from gestational day 11 to 18. Additionally, the diameter and length of seminiferous tubules, and the number of Sertoli cells were investigated with stereological methods. Such unbiased methods have not previously been applied on testis diameter and length or on Sertoli cell number of 11-day-old rats. In the control group, the mean length of the seminiferous tubule was 3.0 m+/-0.6, the mean diameter of the seminiferous tubule was 83 microm+/-6, and the mean number of Sertoli cells was 26.1x10(6)+/-4.6. No differences in testis weight, histopathology, or length or diameter of the seminiferous tubules were observed in the diethylstilboestrol and nonylphenol exposed groups when compared to the control group. In the diethylstilboestrol-treated group, a statistically significant decrease in the number of Sertoli cells was observed (P<0.01) when compared to the control group, whereas nonylphenol had no effect. The result suggests that diethylstilboestrol decreases Sertoli cell proliferation in the foetal testis and furthermore indicate that oestrogens may pose a risk to the reproductive capacity in sensitive species, including man.

Development and vulnerability of rat brain and testes reflected by parameters for apoptosis and ornithine decarboxylase activity.

BACKGROUND AND PURPOSE: Awareness of effects of chemicals on brain and sex organs during organogenesis is increasing. Balance between apoptosis and ornithine decarboxylase (ODC) activity has an essential role for final structure and function of these organs. It is important to localize stages in development where these processes may be particularly vulnerable to chemicals. We describe reference data on apoptosis and ODC activity in brain and testes. METHODS: Brain and testes specimens were obtained during gestational days (G) 15 to 21 and on postnatal days (P) 1 to 60, and ODC activity and parameters of apoptosis (DNA laddering and Terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling-staining) were investigated. RESULTS: Brain ODC activity reaches maximum at G19 and thereafter rapidly decreases until P7. Apoptotic DNA laddering occurs in the brain from G17 to P7. Significant apoptotic ladders were not detected between P9 and 60. In the testes, apoptotic laddering was weak from G21 to P15, but increased significantly from P15 to 60. Histologic examination and DNA laddering analyses revealed low-level germ cell apoptosis from G15 to P11. At onset of spermatogenesis at P15, the number of apoptotic germ cells increased markedly. CONCLUSIONS: Brain ODC activity and apoptosis from G15 to P7 and at the onset of testes apoptosis at P15 are relevant markers for chemically induced developmental toxicity in these organs.