Junzoh Kitoh

Testicular and Hematopoietic Toxicity of 2-Bromopropane, a Substitute for Ozone Layer-Depleting Chlorofluorocarbons

Testicular and Hematopoietic Toxicity of 2‐Bromopropane, a Substitute for Ozone Layer‐Depleting Chlorofluorocarbons: Gaku Ichihara, et al. Department of Hygiene, Nagoya University School of Medicine—In 1995r unexpected amenorrhea, oligozoospermia and anemia were discovered in Korean workers exposed to solvents containing 2‐bromopropane which was a substitute for chlorofluorocarbon. We aimed to determine experimentally the testicular and hematopoietic toxicity of 2‐bromopropane in male rats. Thirty‐six Wistar male rats were divided into four groups of nine each. The rats were exposed to 300r 1,000 and 3,000 ppm 2‐bromopropane or only fresh air, respectively, 8 hr a day, 7 days per week. The 300 ppm and 1,000 ppm groups were exposed for 9 weeks, but the 3,000 ppm groups exposure was discontinued and three rats in this group were dissected after 9‐11 days’ exposure because of serious illness. The others were dissected at the end of the experiment. At 300 ppm or over, the testicular and epididymal weights per body weight, epididymal sperm count, motile sperm percentage and the number of erythrocytes and platelets had decreased compared to the control. Histopathologically, all types of germ cells decreased in the 300 ppm group. Germ cells were absent but Sertoli cells still remained in the 1,000 ppm and 3,000 ppm groups at the end of the experiment. Spermatogonia were absent and the number of spermatocytes decreased in the 3,000 ppm group rats sacrificed after 1 1 days’ exposure. Sertoli cell vacuolations were marked in two of these three rats. Bone marrow was hypocellular in the 1,000 ppm group and in all the rats in the 3,000 ppm group. These results clearly showed that 2‐bromopropane had a testicular and hematopoietic toxicity in male rats.

Obstructed migration of Purkinje cells in the developing cerebellum of the reeler mutant mouse

It has been considered that cortical malformation in the brain of the reeler mutant mouse is due to a defect in the process of neuroblast migration during development. We examined the process of Purkinje cell migration in the cerebellar primordium of the reeler mutant immunohistochemically and electron-microscopically, employing a specific marker for Purkinje cells and markers for radial glia. To facilitate the recognition of the homozygote of the reeler mutation (r1) at the embryonic stage, we introduced the chromosome carrying the autosomal semi-dominant mutation, hammer-toe (Hm), by crossbreeding and backcross into the heterozygote of the reeler mutation, which is an autosomal recessive and located on the homologous chromosome. Using this double heterozygous strain (+/rl-Hm/+), the homozygote of rl can be selected from littermates by the normal appearance of the feet. Both the heterozygous rl embryos and non-carriers harbor the Hm locus and show the Hm phenotype as a deformity of the feet that can be recognized from the 15th day of gestation. In the cerebellar primordium of control mice, Purkinje cells migrated radially from the ventricular zone towards the cortex. In contrast, most of the migratory Purkinje cells remained in the intermediate zone, and their migration towards the cortex was obstructed in the cerebellum of the reeler mutant. A disorganized arrangement of both the processes and cell bodies of the radial glia was demonstrated in the cerebellar primordium of the reeler by labeling them with the antibody against tenascin, a neuron-glial adhesion molecule, and the monoclonal antibody 1D11, a marker for immature astroglia. Electron-microscopic observations revealed apposition of the migratory cells to the radially oriented glial processes in the intermediate zone of the control cerebellum. In contrast, the apposition of leading processes of the migratory neuroblasts to disorganized processes of the radial glia was observed in the intermediate zone of the reeler cerebellum. These findings suggest that the obstructed migration and disordered cortical alignment of Purkinje cells in the reeller cerebellum is due to dysgenesis and abnormal development of radial glia, resulting in disturbance of contact guidance in the process of Purkinje cell migration.

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.

Ovarian toxicity of 2-bromopropane in the non-pregnant female rat

Ovarian Toxicity of 2‐Bromopropane in the Non‐Pregnant Female Rat: Michihiro Kamijima, et al. Department of Hygiene, Nagoya University School of Medicine—A cluster of patients with amenorrhea, oligospermia and anemia were found among workers in an electronics factory in the Republic of Korea. 2‐Bromopropane was suspected to cause the disorders. This study aimed to clarify its ovarian toxicity in the virgin rat. Female Wistar rats were daily exposed to 0, 100, 300, or 1,000 ppm 2‐bromopropane for eight hours a day for 9 weeks. During the experimental period, vaginal smears were taken everyday to monitor ovarian cyclicity. Tissues were histopathologically examined and plasma concentrations of luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) were determined at the end of experiment. The vaginal smear test showed that the number of normal cycles decreased significantly both in the 300 and 1,000 ppm groups. The histopathological examination revealed dose‐dependent ovarian follicle atresia accompanied by a decreased number of normal antral and growing follicles in the 300 and 1,000 ppm groups. Especially, in the ovaries of rats with persistent estrous smears in the 1,000 ppm group, most of the follicles were atretic and there were no newly formed corpora lutea. There still remained normal antral follicles and corpora lutea in the ovaries of the remaining rats of the group and of the 300 ppm group with constant diestrous and occasional estrous smears. Hormonal examination revealed no statistically significant change in LH or FSH concentrations between any groups. The results showed that 2‐bromopropane has ovarian toxicity in rats, indicating that the secondary amenorrhea in human cases was due to 2‐bromopropane exposure.

Distribution of taste buds on the lips and inside the mouth in the minnow, Pseudorasbora parva

The distribution and abundance of taste buds were quantitatively examined by observing silver impregnated serial sections. The taste buds were widely dispersed on the skin, the lips, the mucosa in the oro-pharyngeal cavity, the esophagus, and the branchial apparatus. The great majority of them was found on the lips and inside the mouth. The external buds were concentrated especially on the outer lips and the adjacent skin, while their number diminished in a caudal direction. Very few were found on the scaled skin. The total number of external buds in a specimen of 6 cm in length was 1,486. The number of taste buds inside the mouth was 6,600. On the inner lips and the palatal organ densities were found to reach over 140 per mm2. High concentrations of taste buds were also found on the gill arches and rakers. These taste buds varied to some extent in size and shape, depending on the thickness of the epithelial layer. It is suggested that the minnow may use the lips, gills and palatal organ as its main taste organs.

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.

Fatty liver and hyperlipidemia in iddm (insulin-dependent diabetes mellitus) of streptozotocin-treated shrews

Severe IDDM (insulin-dependent diabetes mellitus) was produced in the musk shrew (Suncus murimus, Insectivora) by a high dose (a single intraperitoneal injection of 100 mg/kg Body Weight) of streptozotocin (STZ) injection. All shrews that were administered a high dose of STZ exhibited hyperglycemia (449 +/- 16 mg/dl vs 73 +/- 4 mg/dl in controls) and hypoinsulinemia(0.25 +/- 0.07 ng/ml vs 10.96 +/- 1.97 ng/ml in controls) with ketosuria 10 days after injection. Their livers were enlarged and exhibited ayellowish-brown color with marked triglyceride (TG) accumulation (63.25 +/- 7.10 mg/g Liver vs 2.11 +/- 0.19 mg/g Liver in controls). It is probable that the increased influx of fatty acids into the liver induced by hypoinsulinemia and the low capacity of excretion of lipoprotein secretion from liver in the musk shrew resulting from a deficiency of apolipoprotein B synthesis play important roles in fatty liver formation. Hyperlipidemia was another feature in shrews with severe IDDM. The blood TG level was especially high in these shrews (899 +/- 178 mg/dl vs 23 +/- 5 mg/dl in controls). These results indicate that the IDDM shrew, induced by high doses of STZ, is a unique model characterized by fatty liver and hyperlipidemia and may be useful for studying lipid metabolism of IDDM.

Etiology of idiopathic scoliosis : Computational study

A review of the literature on the mechanical aspects of the etiology for idiopathic scoliosis reveals that the buckling hypothesis has been presented as a purely mechanical phenomenon. In an attempt to confirm the buckling hypothesis, a numerical simulation of growth and the resulting buckling phenomena was done by means of finite element analysis. It previously was observed that growth was induced in the T4 to T10 vertebrae. Only the sacrum was assumed to be stationary. From the growth analysis, a deformation process that mitigated thoracic kyphosis was obtained as observed in healthy children during early adolescence. From the buckling analysis, the first to the fourth buckling modes that correspond to the first side bending, first forward bending, first rotation, and second side bending modes were obtained. The shape of the fourth buckling mode (second side bending mode) was in good agreement with the clinical shape. Considering the potential for controlling these modes by posture change, it is concluded that the second bending mode in the coronal plane is one of the most likely etiologic candidates in the mechanics of thoracic idiopathic scoliosis.

High levels of quinolinic acid in brain of epilepsy-prone E1 mice

Quinolinic acid (QUIN) may act.as an excitotoxin when it is abundant in the brain. We have shown previously that the activity of 3-hydroxyanthranilate 3,4-dioxygenase, a QUIN-synthesizing enzyme, was abnormally high in the brains of epilepsy-prone E1 mice as compared with that of ddY mice. Here, we estimated the QUIN contents in the brains of these mice. The results showed that the basal QUIN content in the cerebral cortex of E1 mice was twice as high as that of ddY mice. Systemic injection of 400 mumol/kg body weight of L-tryptophan (L-Trp) increased the cortical levels of QUIN in both E1 mice and ddY mice by 189% and 118%, respectively. Administration of 400 mumol/kg each of L-threonine and D,L-methionine had no appreciable effect on the L-Trp-caused increase in the cortical QUIN levels. Co-administration of 5-fluorotryptophan or 5-methyltryptophan, tryptophan analogs, with L-Trp did not reduce but rather enhanced the cortical QUIN levels (by 18% and 92%, respectively). No significant change in the cortical QUIN concentrations was observed with injection of 2 mg/kg body weight of E. coli lipopolysaccharide (LPS) in E1 mice. However, injection of L-Trp in the LPS-treated E1 mice produced a more marked increase in the cortical QUIN levels than that injected with L-Trp alone. These results suggest that the brain QUIN contents of E1 mice are dependent not only on the activity of QUIN-synthesizing enzyme but also on the rate of flux of its substrate, L-Trp or its metabolite(s), in the brain.

Mechanical sensitivity of the facial nerve fibers innervating the anterior palate of the puffer,Fugu pardalis, and their central projection to the primary taste center

Summary1.Mechanical and chemical sensitivity of the palatine nerve, ramus palatinus facialis, innervating the anterior palate of the puffer,Fugu pardalis, and their central projection to the primary taste center were investigated.2.Application of horseradish peroxidase (HRP) to the central cut end of the palatine nerve resulted in retrogradely labeled neurons in the geniculate ganglion but no such neurons in the trigeminal ganglion, suggesting that the palatine nerve is represented only by the facial component.3.Tracing of the facial sensory root in serial histological sections of the brain stem suggested that the facial sensory nerve fibers project only to the visceral sensory column of the medulla.4.Peripheral recordings from the palatine nerve bundle showed that both mechanical and chemical stimuli caused marked responses. Mechanosensitive fibers were rather uniformly distributed in the nerve bundle.5.Intra-cranial recordings from the trigeminal and facial nerves at their respective roots revealed that tactile information produced in the anterior palate was carried by the facial nerve fibers.6.Elimination of the sea water current over the receptive field also caused a marked response in the palatine nerve bundle or facial nerve root while this did not cause any detectable responses in the trigeminal nerve root.7.Single fiber analyses of the mechanical responsiveness of the palatine nerve were performed by recording unit responses of 106 single fibers to mechanical stimuli (water flow), HCl (0.005M), uridine-5′-monophosphate (UMP, 0.001M), proline (0.01M), CaCl2 (0.5M), and NaSCN (0.5M). All these fibers responded well to one of the above stimuli; however, most taste fibers did not respond well to the inorganic salts. The palatine fibers (n=36), identified as mechanosensitive, never responded to any of the chemical stimuli, whereas chemosensitive fibers (n=70) did not respond to mechanical stimuli at all. The chemosensitive units showed a high specificity to the above stimuli: they tended to respond selectively to hydrochloric acid, UMP, or proline.8.The responses of the mechanosensitive units consisted of phasic and tonic impulse trains and the sensitivity of the units varied considerably.9.The results reveal that the facial nerve fibers innervating the anterior palate of the puffer contain two kinds of afferent fibers, chemosensory and mechanosensory respectively, and suggest that the convergence of the tactile and gustatory information first occurs in the neurons of the primary gustatory center in the medulla.