Effects of exposure to 1-bromopropane on astrocytes and oligodendrocytes in rat brain.

OBJECTIVES: Human cases of 1-bromopropane (1-BP) toxicity showed ataxic gait and cognitive dysfunction, whereas rat studies showed pyknotic shrinkage in cerebellar Purkinje cells and electrophysiological changes in the hippocampus. The present study investigated the effects of 1-BP on astrocytes and oligodendrocytes in the rat cerebellum and hippocampus to find sensitive markers of central nervous system toxicity. METHODS: Forty-eight F344 rats were divided into four equal groups and exposed to 1-BP at 0, 400, 800 and 1,000 ppm for 8 h/day; 7 days/week, for 4 weeks. Nine and three rats per group were used for biochemical and histopathological studies, respectively. RESULTS: Kluver-Barrera staining showed pyknotic shrinkage in the cytoplasm of Purkinje cells and nuclei of granular cells in the cerebellum at 1,000 ppm. Immunohistochemical analysis showed increased length of glial fibrillary acidic protein (GFAP)-positive processes of astrocytes in the cerebellum, hippocampus and dentate gyrus at 800 and 1,000 ppm. The myelin basic protein (MBP) level was lower at 1,000 ppm. The numbers of astrocytes and granular cells per tissue volume increased at 400 ppm or higher. CONCLUSION: The present study showed that elongation of processes of astrocytes accompanies degeneration of granular cells and Purkinje cells in the cerebellum of the rats exposed to 1-BP. The decrease in MBP and number of oligodendrocytes suggest adverse effects on myelination. The increase in astrocyte population per tissue volume in the cerebellum might be a sensitive marker of 1-BP neurotoxicity, but the underlying mechanism for this change remains elusive.

Exposure to 1-bromopropane induces microglial changes and oxidative stress in the rat cerebellum.

1-Bromopropane (1-BP), an alternative to ozone-depleting solvents, is reported to exhibit neurotoxicity and reproductive toxicity in animals and humans. However, the underlying mechanism of the toxicity remains elusive. This study was designed to identify the microglial changes and oxidative stress in the central nervous system (CNS) after 1-BP exposure. Four groups of Wistar-ST rats (n=12 each) were exposed to 0, 400, 800 and 1000ppm of 1-BP, 8h/day for 28 consecutive days. The cerebellum was dissected out in 9 rats of each group and subjected to biochemical analysis, while the brains of the remaining 3 rats were examined immunohistochemically. Exposure to 1-BP increased the levels of oxidative stress markers [thiobarbituric acid reactive substances (TBARS), protein carbonyl and reactive oxygen species (ROS)] in a dose-dependent manner. Likewise, there was also 1-BP dose-dependent increase in nitric oxide (NO) and dose-dependent decrease in protein concentrations in the cerebellum. Immunohistochemical studies showed 1-BP-induced increase in cd11b/c-positive microglia area in the white matter of the cerebellar hemispheres. The results showed that exposure to 1-BP induced morphological change in the microglia and oxidative stress, suggesting that these effects are part of the underlying neurotoxic mechanism of 1-BP in the CNS.

Increased susceptibility of Nrf2-null mice to 1-bromopropane-induced hepatotoxicity.

1-Bromopropane (1-BP) was introduced as an alternative to ozone-depleting solvents. However, it was found to exhibit neurotoxicity, reproductive toxicity, and hepatotoxicity in rodents and neurotoxicity in human. However, the mechanisms underlying the toxicities of 1-BP remain elusive. The present study investigated the role of oxidative stress in 1-BP-induced hepatotoxicity using nuclear factor erythroid 2-related factor 2 (Nrf2)-null mice. Groups of 24 male Nrf2-null mice and 24 male wild-type (WT) C57BL/6J mice were each divided into three groups of eight and exposed to 1-BP at 0, 100, or 300 ppm for 8 h/day for 28 days by inhalation. Liver histopathology showed significantly larger area of necrosis in Nrf2-null mice relative to WT mice at the same exposure level. Nrf2-null mice also had greater malondialdehyde (MDA) levels, higher ratio of oxidized glutathione/reduced form of glutathione, and lower total glutathione content. The constitutive level and the increase in ratio per exposure level of glutathione S-transferase (GST) activity were lower in the liver of Nrf2-null mice than WT mice. Exposure to 1-BP at 300 ppm increased the messenger RNA levels of heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GcLm), glutamate-cysteine synthetase (GcLc), glutathione reductase, and NAD(P)H: quinone oxidoreductase 1 (NQO1) in WT mice but not in Nrf2-null mice except for GST Yc2. Nrf2-null mice were more susceptible to 1-BP-induced hepatotoxicity. That oxidative stress plays a role in 1-BP hepatotoxicity is deduced from the low expression levels and activities of antioxidant enzymes and high MDA levels in Nrf2-null mice.

Comparative study on susceptibility to 1-bromopropane in three mice strains.

Previous studies indicate that 1-bromopropane (1BP) has neurotoxicity and reproductive toxicity both in humans and animals. The present study investigated strain differences in susceptibility to 1BP and identified possible biological factors that determine such susceptibility. Twenty-four male mice of each of the three strains (C57BL/6J, DBA/2J, and BALB/cA) were divided into four groups of six each and exposed to 1BP at 0, 50, 110, and 250 ppm for 8 h/day for 28 days by inhalation. At the end of exposure period, the relative susceptibilities of each strain to 1BP-mediated hepatotoxicity and male reproductive toxicity were evaluated. The contributing factors to strain-dependent susceptibility were assessed by determination of hepatic CYP2E1 levels, glutathione-S-transferase (GST) activity, glutathione (GSH) status, and NAD(P)H:quinone oxidoreductase and heme oxygenase-1 mRNA levels. Liver histopathology showed significantly larger area of liver necrosis and more degenerative lobules in BALB/cA in the order of BALB/cA > C57BL/6J > DBA/2J. BALB/cA showed higher CYP2E1 protein level and lower total GSH content and GST activity in the liver than DBA/2J. These results indicate that BALB/cA mice are the most susceptible to hepatotoxicity of 1BP among the three strains tested, and that CYP2E1, GSH level/GST activity may contribute to the susceptibility to 1BP hepatotoxicity. Exposure to > or = 50 ppm of 1BP also decreased sperm count and sperm motility and increased sperms with abnormal heads in all three strains mice in a dose-dependent manner. Comparison with previous studies in rats indicates that mice are far more susceptible than rats to 1BP regarding hepatotoxicity and reproductive toxicity.

Changes in neurotransmitter receptor expression levels in rat brain after 4-week exposure to 1-bromopropane.

1-Bromopropane (1-BP), an alternative to ozone-depleting solvents, exhibits neurotoxicity and reproductive toxicity in animals and humans. The present study investigated the effects of exposure to 1-BP on expression of neurotransmitter receptor genes in the rat brain to explore possible biomarkers for central neurotoxicity and find brain regions sensitive for microarray analysis. Thirty-six F344 rats were divided at random into four equal groups of nine and exposed to 1-BP at 0, 400, 800 and 1000 ppm for 8 h/day; 7 days/week for 4 weeks. Total RNA from different brain regions was extracted and real-time PCR was conducted to quantify the mRNA levels of serotonin, dopamine and GABA receptors. Western blot analysis for specific regions of interest was also carried out to determine the protein levels. The mRNAs of 5HTr2a, D2R and GABAa1 were down regulated in a 1-BP dose-dependent manner in the hippocampus. The mRNA levels of 5HTr1a, 5HTr2a, D1R and GABAa1 were significantly decreased in the cortex of rats exposed to 800 ppm, but not to 1000 ppm. The mRNAs of 5HTr1a and 5HTr3a in the pons-medulla were decreased in rats exposed to 400 ppm or higher concentrations. The mRNA expression of D2R in the hippocampus and 5HTr1a and 5HTr3a in the pons-medulla oblongata were the most sensitive indicators of 1-BP neurotoxicity. The results suggest that mRNA expression analysis is useful in identifying brain regions susceptible to 1-BP, as well as providing potential biomarkers for central nervous system toxicity.

Reversibility of the adverse effects of 1-bromopropane exposure in rats.

Previous experiments indicated that 1-bromopropane (1-BP), an alternative to chloroflurocarbons, is neurotoxic and inhibits spermiation in the testis. Here we investigated the reversibility of the toxic effects of 1-BP in rats. Male Wistar rats were divided into three equal groups of 24 each and exposed by inhalation to 0, 400 or 1000 ppm of 1-BP for 6 weeks (8 hrs/day, 7 days/week). Eight rats from each group were sacrificed at the end of 6 weeks exposure, and at 4 and 14 weeks after the end of exposure, to assess the recovery processes. We studied sperm count, motility, morphology and testicular histopathology, as well as blood pressure, skin temperature and hindlimb muscle strength. At the end of 6 weeks of exposure to 1000 ppm (0 week recovery), testicular weight, epididymal weight, sperm count and motility were low, morphologically abnormal sperm were increased and spermatogenic cells showed diffuse degeneration. These changes did not show full recovery at 14 weeks recovery, with the exception of the prostate and seminal vesicular weights, which recovered back to control values. At 400 ppm, increased retained spermatids at 0 week recovery returned to normal at 4 weeks recovery. Exposure to 1000 ppm produced sustained reduction of hindlimb muscle strength at 14 weeks recovery, whereas normalization of the skin temperature and blood pressure was noted after transient changes. Our study showed that the effect of 1-BP on spermatogenesis is dose-dependent; low exposure inhibited spermiation and hormone-dependent organ weight reduction and these changes were transient, while a higher dose of 1000 ppm 1-BP caused persistent depletion of spermatogenic cells.

Effects of exposure of rat dams to 1-bromopropane during pregnancy and lactation on growth and sexual maturation of their offspring.

1-Bromopropane (1-BP) exhibits neuroreproductive toxicities in adult rats and humans. Here, we determined the effects of exposure of rat dams to 1-BP during pregnancy and lactation on the growth and sexual maturation of their offspring. In Experiment 1, 40 rats were exposed to 0, 100, 400 and 800ppm 1-BP during pregnancy and lactation for 8h/day. Ten rats that were not placed in chambers throughout the experiment served to observe the effect of separation of dams from offspring. In Experiment 2, three groups of 10 pregnant rats each were exposed to fresh air in three chambers and 10 other rats were exposed to 800ppm 1-BP during pregnancy and lactation for 8h/day. After delivery, offspring of the exposed and non-exposed dams were swapped so that they were nursed by the opposite dams. In Experiment 1, the survival rate and body weight of offspring were lower than the non-exposed in 1-BP dose-dependent manner. In Experiment 2, the survival rate and body weight of offspring (Group A) nursed by exposed dams and those (Group B) of exposed dams were significantly lower than non-exposed groups. The body weight of Group A was lower than that of Group B, although the two groups showed a significant equal decrease in the survival rate. The number of dead offspring from Group A was significantly higher. Our results indicate that exposure to 1-BP during pregnancy and lactation has comparable effects on survival rate, but exposure during lactation has a more adverse effect on growth of offspring than that during pregnancy. Moreover, exposure during lactation is associated with reduced early survival of third generation (F2) rats.

The morphological and histological characters of the male external genitalia of the house musk shrew, Suncus murinus.

External genitalia are the reproductive organs necessary for efficient copulation and internal fertilization in various mammalian species. Their morphogeneses display significant morphological and developmental differences among species. The house musk shrew, Suncus murinus (hereafter described as suncus) is a species of the order Insectivora, which has been considered as primitive and one of the earliest eutheria phylogenetically. Comparative anatomical analyses of phylogenetically different mammals will contribute to the better understanding of morphological diversity of external genitalia. This study performed various anatomical and histological analyses concerning the organization of the external genitalia of male suncus. It was shown that the external genitalia of suncus possessed a muscular structure, which we proposed as musculus ischiocavernosus dorsalis of suncus. The musculus ischiocavernosus dorsalis is originated from the inner surface of the tuber ischiadicum and was allocated adjacent to the corpus cavernosum penis. In addition, a pair of alpha-smooth muscle actin positive muscles was located bilaterally to the urethra. This unique morphology of the external genitalia of suncus males may provide a unique model system to investigate genital morphogenesis.

Dose-dependent biochemical changes in rat central nervous system after 12-week exposure to 1-bromopropane.

1-Bromopropane is used as a cleaning agent or adhesive solvent in the workplace. The present study investigated the long-term effects of exposure to 1-bromopropane on biochemical components in the central nervous system (CNS) of rats. Four groups, each of nine male Wistar rats, were exposed to 200, 400, or 800 ppm 1-bromopropane or fresh air only, 8h per day, 7 days a week for 12 weeks. We measured the levels of neuron-specific gamma-enolase, glia-specific beta-S100 protein, creatine kinase (CK) subunits B and M, heat shock protein Hsp27 (by enzyme immunoassay), enzymatic activity of CK and levels of glutathione (GSH), oxidized glutathione (GSSG) and sulfhydrul (SH) base in the cerebrum, cerebellum, brainstem and spinal cord. gamma-Enolase decreased dose-dependently in the cerebrum, which showed a decrease in wet weight, at 400 ppm or over, but no change was noted in beta-S100 protein in any brain region or spinal cord. Hsp27 decreased in the cerebellum, brainstem and spinal cord. Protein-bound SH base, non-protein SH base and total glutathione decreased in every brain region. CK activity decreased dose-dependently at 200 ppm or over, and the ratio of CK activity to CK-B concentration tended to decrease in all regions. The decrease in gamma-enolase in the cerebrum suggests the involvement of biochemical changes in neurons with decrease in the wet weight of the cerebrum. Glutathione depletion and changes in proteins containing SH base as a critical site might be the underlying neurotoxic mechanism of 1-bromopropane. The biochemical changes in the cerebrum indicate that long-term exposure to 1-bromopropane has effects on the CNS.

Three-dimensional distribution patterns of PRL, GH and ACTH cells in the house musk shrew ( Suncus murinus).

Despite a multitude of reports on the classification and distribution of anterior pituitary cells, no previous study has attempted to obtain the three-dimensional (3D) and computer-graphic distribution pattern of each cell type in the whole pituitary. Therefore, we mapped the anterior pituitary cells of the house musk shrew ( Suncus murinus) and found a distinct cellular distribution pattern. Serial horizontal sections of whole shrew pituitaries were stained immunohistochemically for prolactin (PRL), growth hormone (GH), and adrenocorticotropic hormone (ACTH) cells. The contours of positive cells and the anterior, intermediate, and posterior lobes in each section were digitized for 3D visualization by a volume rendering method. The reconstructed images and virtual frontal and sagittal slices were examined in detail. On the 3D reconstructed images, the PRL and GH cells had similar distribution patterns, although the former were concentrated in the dorsolateral and ventrocentral portions, and the latter in the dorsocentral portions of the anterior lobe. On both sides of the pituitary stalk, there lay portions that were conspicuous by scarcity of PRL and GH cells. ACTH cells were widely scattered throughout the whole anterior lobe, but they were very few in the above portions and the dorsocentral portions where GH cells were concentrated. No sex difference in the distribution patterns of each cell type was observed. However, PRL cells in females were more numerous than in males, whereas the opposite was true for GH and ACTH cells. We discuss the relationship between the formation of the spatial distribution patterns and anterior pituitary ontogeny.

Biochemical changes in the central nervous system of rats exposed to 1-bromopropane for seven days.

1-Bromopropane is used widely as an alternative to ozone-depleting solvents. The neurotoxic effects of this agent have been described in humans and experimental animals. Here we investigated the underlying mechanisms of the neurotoxic effects of 1-bromopropane by examining the initial biochemical changes in the central nervous system. Four groups of 9 Wistar male rats each were exposed to 200, 400, or 800 ppm 1-bromopropane or only fresh air, 8 h per day for 7 days. At the end of the experiment, the cerebrum, cerebellum, brain stem and lumbar enlargement of the spinal cord were dissected out from each rat (n = 8) for biochemical analyses. Morphological examinations of the nervous system were performed in the remaining rat of each group. 1-Bromopropane dose-dependently decreased neurospecific gamma-enolase, total glutathione, and nonprotein sulfhydryl groups in the cerebrum and cerebellum. Creatine kinase activity decreased dose-dependently in the brain and spinal cord. Histopathological examination showed swelling of preterminal axons in gracile nucleus and degeneration of myelin in peripheral nerves. Our results of low levels of gamma-enolase suggested that 1-bromopropane might primarily cause functional or cellular loss of neurons in the cerebrum and cerebellum. Glutathione depletion or modification to functional proteins containing a sulfhydryl base as a critical site might be the underlying mechanism of 1-bromopropane neurotoxicity.

Aplasia of the Optic Nerve in Microphthalmic Offspring of Prenatally X-irradiated Rats.

A considerable number of postnatally-viable microphthalmic offspring with optic nerves completely absent were obtained by X-irradiation at a dose of 100 R in pregnant rats on gestational day 10.5. Thirteen of 15 mi-crophthalmic eyes examined displayed histological features characteristic of aplasia of the optic nerve: complete absence of optic papilla, nerve fiber layer and retinal blood vessels, and great reduction in the number of ganglion cells. The remaining 2 eyes showed the histological features of hypoplasia of the optic nerve. The present experimental system may afford suitable materials for postnatal patho-genetic studies and also for various physiological and behavioral studies of aplasia of the optic nerve.

Cell surface changes of the presumptive ectoderm following neural-inducing treatment by concanavalin A.

Scanning electron microscopic studies revealed that Concanavalin A (ConA) induces characteristic changes of the cell surface and the cell architecture of the presumptive ectoderm associated with differentiation into neural tissues. In Con A-treated cells, the filopodia with which cells were connected to each other disappeared from the interior (blastocoelic) surface and the cellular adhesivity decreased significantly. Thereafter, the cells underwent from those of the control explants. After cultivation for 60 h, a certain pattern of cell arrangement, which resembled the architecture of neural tissues, was observed among randomly arranged cells in the explants treated with Con A. The morphological changes specifically observed in Con A-treated explants were different from those found in explants treated with succinyl Con A (S-Con A) orDolichos biflorus agglutinin (DBA), which is unable to induce formation of the neural tissues. The molecular organization of the plasma membrane appears to be important in the mechanism of neural induction.