(-)-Epigallocatechin gallate attenuates the induction of HSP27 stimulated by sphingosine 1-phosphate via suppression of phosphatidylinositol 3-kinase/Akt pathway in osteoblasts.

We previously reported that sphingosine 1-phosphate stimulates the induction of heat shock protein 27 (HSP27) through p38 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether (-)-epigallocatechin gallate (EGCG), the major polyphenol found in green tea, affects the induction of HSP27 in these cells and its mechanism, since it was previously reported that catechin, including EGCG, suppresses bone resorption. EGCG significantly reduced the induction of HSP27 stimulated by sphingosine 1-phosphate in a dose-dependent manner between 10 and 30 microM. Immunofluorescence microscopy studies revealed that sphingosine 1-phosphate certainly stimulated the induction of HSP27 in the cytosol of these cells, and that EGCG clearly suppressed its induction. However, sphingosine 1-phosphate-stimulated phosphorylation of p38 MAP kinase or MAPKAP-2 was not affected by EGCG. By contrast, EGCG markedly suppressed the phosphorylations of both Akt and glycogen synthase kinase-3beta stimulated by sphingosine 1-phosphate. These results strongly suggest that EGCG suppresses the induction of HSP27 stimulated by sphingosine 1-phosphate via attenuation of, not the p38 MAP kinase pathway, but of the phosphatidylinositol 3-kinase/Akt pathway in osteoblasts.

Alpha2 adrenoreceptor agonist regulates protein kinase C-induced heat shock protein 27 phosphorylation in C6 glioma cells.

Dexmedetomidine (Dexmd), a potent and highly specific alpha(2) adrenoreceptor agonist, is an efficient therapeutic agent for sedation. Dexmd has been recently reported to have a neuroprotective effect. Heat shock protein (HSP) 27, a low-molecular weight HSP has been shown to be expressed following cerebral ischemia in astrocytes but not in neurons. HSP27 expression is involved in ischemic tolerance of the brain. This study investigated the effect of Dexmd on HSP27 in rat C6 glioma cells. 12-O-tetradecanoylphorbol-13-actate (TPA), a direct activator of protein kinase C (PKC), stimulated the phosphorylation of HSP27 at Ser82, but not Ser15 in a time-dependent manner. Prostaglandin (PG) E(1) or PGE(2) which activates the adenylyl cyclase-cAMP system as well as forskolin and dibutyryl-cAMP, suppressed the TPA-induced phosphorylation of HSP27. Dexmd reversed the suppression of HSP27 phosphorylation by the adenylyl cyclase-cAMP system. Therefore, these results strongly suggest that Dexmd reverses the suppression of HSP27 phosphorylation by the adenylyl cyclase-cAMP system activation through the inhibition of its system in C6 cells. alpha(2) Adrenoreceptor agonists may therefore show a neuroprotective effect through the modification of HSP27 phosphorylation induced by PKC activation.

(-)-Epigallocatechin gallate reduces transforming growth factor beta-stimulated HSP27 induction through the suppression of stress-activated protein kinase/c-Jun N-terminal kinase in osteoblasts.

We previously reported that transforming growth factor-beta (TGF-beta) stimulates heat shock protein 27 (HSP27) induction through p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether (-)-epigallocatechin gallate (EGCG), the major polyphenol found in green tea, affects the TGF-beta-stimulated induction of HSP27 in these cells, and its underlying mechanism. EGCG significantly suppressed the HSP27 induction stimulated by TGF-beta in a dose-dependent manner between 10 and 30 microM without affecting the HSP70 levels. TGF-beta with or without EGCG did not affect the advanced oxidation protein products. The TGF-beta-induced phosphorylation of p38 MAP kinase and ERK1/2 was not affected by EGCG. SP600125, a specific inhibitor of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), markedly reduced the HSP27 expression induced by TGF-beta. EGCG significantly suppressed the TGF-beta-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of Smad2. EGCG attenuated the phosphorylation of both MKK4 and TAK1 induced by TGF-beta. These results strongly suggest that EGCG suppresses the TGF-beta-stimulated induction of HSP27 via the attenuation of the SAPK/JNK pathway in osteoblasts, and that this effect is exerted at a point upstream from TAK1.

(-)-epigallocatechin gallate inhibits prostaglandin D2-stimulated HSP27 induction via suppression of the p44/p42 MAP kinase pathway in osteoblasts.

We previously reported that prostaglandin D2 (PGD2) stimulates heat shock protein 27 (HSP27) induction through p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether (-)-epigallocatechin gallate (EGCG), the major polyphenol found in green tea, affects the induction of HSP27 in these cells and the mechanism. EGCG significantly reduced the HSP27 induction stimulated by PGD2 without affecting the levels of HSP70. The PGD2-induced phosphorylation of p38 MAP kinase or SAPK/JNK was not affected by EGCG. On the contrary, EGCG markedly suppressed the PGD2-induced phosphorylation of p44/p42 MAP kinase and MEK1/2. However, the PGD2-induced phosphorylation of Raf-1 was not inhibited by EGCG. These results strongly suggest that EGCG suppresses the PGD2-stimulated induction of HSP27 at the point between Raf-1 and MEK1/2 in osteoblasts.

Expression levels of heat shock protein 20 decrease in parallel with tumor progression in patients with hepatocellular carcinoma.

Heat shock protein (HSP) 20, a low-molecular-weight HSP, is constitutively expressed in various tissues, such as smooth muscle, skeletal muscle, and liver. However, the characteristics and function of HSP20 have not been precisely understood. In the present study, we investigated correlations of expression levels of HSP20 in hepatocellular carcinoma (HCC) tissues and the surrounding tissues with clinical and pathologic characteristics in 53 resected HCC specimens. Although HSP20 was detected in all 53 HCC tissues, the expression levels were reduced compared with those in the adjacent non-tumor tissues. The expression levels of HSP20 were inversely correlated with tumor stage by TNM classification (p<0.01), presence of microvascular invasion (p<0.05), and tumor size (p<0.05). Our findings strongly suggest that HSP20 may play a role against the progression of human HCC.

Prostaglandin D2 induces the phosphorylation of HSP27 in osteoblasts: function of the MAP kinase superfamily.

We previously reported that prostaglandin D(2) (PGD(2)) stimulates the induction of heat shock protein 27 (HSP27) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether PGD(2) stimulates the phosphorylation of HSP27 in MC3T3-E1 cells exposed to heat shock. In the cultured MC3T3-E1 cells, PGD(2) markedly stimulated the phosphorylation of HSP27 at Ser-15 and Ser-85 in a time-dependent manner. Among the mitogen-activated protein (MAP) kinase superfamily, p44/p42 MAP kinase and p38 MAP kinase were phosphorylated by PGD(2) which had little effect on the phosphorylation of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). The PGD(2)-induced phosphorylation of HSP27 was attenuated by PD169316, an inhibitor of p38 MAP kinase or PD98059, a MEK inhibitor. SP600125, a SAPK/JNK inhibitor did not affect the HSP27 phosphorylation. In addition, PD169316 suppressed the PGD(2)-induced phosphorylation of MAPKAP kinase 2. These results strongly suggest that PGD(2) stimulates HSP27 phosphorylation via p44/p42 MAP kinase and p38 MAP kinase but not SAPK/JNK in osteoblasts.

TNF-alpha decreases hsp 27 in human blood mononuclear cells: involvement of protein kinase c.

Treatment of PBMCs with TNF-alpha decreased the levels of heat shock protein (HSP) 27, but had little effect on the level of HSP70. Parallel to the decrease of HSP27, TNF-alpha increased the level of HSP27 in the incubation medium of the cells. The decrease of HSP27 induced by TNF-alpha was suppressed by the pretreatment of PBMCs with the specific protein kinase C (PKC) inhibitor, GF109203X. Furthermore, phorbol myristate acetate (PMA), a PKC stimulant, but not dibutyryl cyclic AMP, a protein kinase A stimulant, decreased the levels of HSP27. To investigate the effect of TNF-alpha on the oligomerization state of HSP27 in PBMCs, we performed sucrose density gradient centrifugation with subsequent fractionation and immunoassay. Extract of vehicle-treated PBMCs contained mainly dissociated forms of HSP27. The amounts of dissociated forms of HSP27 in PBMCs was decreased by TNF-alpha, while the amounts of aggregated form of HSP27 was little changed. In intact PBMCs, HSP27 is constitutively phosphorylated at Ser78, but not at Ser15 or at Ser82. The amount of phosphorylated HSP27 at Ser78 was decreased by TNF-alpha. These results indicate that TNF-alpha reduces HSP27 in PBMCs through PKC activation. This decrease may be due to efflux of dissociated form of HSP27, phosphorylated HSP27 at Ser78, from the cells.

Upregulation by retinoic acid of transforming growth factor-beta-stimulated heat shock protein 27 induction in osteoblasts: involvement of mitogen-activated protein kinases.

We investigated whether transforming growth factor-beta (TGF-beta) stimulates the induction of heat shock protein (HSP) 27 and HSP70 in osteoblast-like MC3T3-E1 cells and the mechanism underlying the induction. TGF-beta increased the level of HSP27 but had no effect on the HSP70 level. TGF-beta stimulated the accumulation of HSP27 dose-dependently, and induced an increase in the level of mRNA for HSP27. TGF-beta induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by TGF-beta was significantly suppressed by PD98059, an inhibitor of the upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase. PD98059 and SB203580 suppressed the TGF-beta-stimulated increase in the level of mRNA for HSP27. Retinoic acid, a vitamin A (retinol) metabolite, which alone had little effect on the HSP27 level, markedly enhanced the HSP27 accumulation stimulated by TGF-beta. Retinoic acid enhanced the TGF-beta-induced increase of mRNA for HSP27. The amplification of TGF-beta-stimulated HSP27 accumulation by retinoic acid was reduced by PD98059 or SB203580. Retinoic acid failed to affect the TGF-beta-induced phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. These results strongly suggest that p44/p42 MAP kinase and p38 MAP kinase take part in the pathways of the TGF-beta-stimulated HSP27 induction in osteoblasts, and that retinoic acid upregulates the TGF-beta-stimulated HSP27 induction at a point downstream from p44/p42 MAP kinase and p38 MAP kinase.

Hsp27-2D-gel electrophoresis is a diagnostic tool to differentiate primary desminopathies from myofibrillar myopathies.

Small heat shock proteins prevent abnormal protein folding and accumulation. We analyzed the expression of hsp27 and alphaB-crystallin in skeletal muscle specimens of patients with desminopathies, plectinopathies, myotilinopathy, and other myofibrillar myopathies by means of differential centrifugation, 2D-gel electrophoresis, Western blotting, and mass spectrometry. Hsp27-P82 and -P15 as well as alphaB-crystallin-P59 and -P45 are the major serine phosphorylation isoforms in normal and diseased human skeletal muscle. 2D-gel-electrophoresis revealed spots of hsp27 in a range of pH 5.3-6.4 in samples of all skeletal muscle specimens, except for the seven desminopathies. They indicated a shift of the main hsp27-spot to alkaline pH degrees, which may help to differentiate primary desminopathies from other myopathies with structural pathology of the desmin cytoskeleton.

Characterization of alpha-enolase as an interferon-alpha 2 alpha 1 regulated gene.

Interferons (IFNs) are multifunctional cytokines that after binding to the cell surface receptor induce the expression of a large number of genes, which in turn, mediate many biological processes including host defense, cell growth control, signaling, and metabolism. Here we show that IFN-alpha activates the mitogen-activated protein kinases (MAPK) ERK1/2 and the transcription factor CREB/ATF-1, which lead to the alpha-enolase (alpha-ENO) gene expression in fibroblasts. Alpha-ENO mRNA accumulation was apparent 6 h post-IFN stimulation and required both de novo protein synthesis and active gene transcription, which is typical of a secondary response gene. Alpha-ENO expression does not appear to be restricted to fibroblasts, since it was equally verified in peripheral blood mononuclear cells (PBMC). Furthermore, IFN-alpha stimulates the expression of the primary response genes c-fos and egr-1, which was followed by an increase in DNA binding activity of c-FOS and EGR-1 proteins, as verified by shift assays using the cis-acting elements AP-1 and EGR-1 localized at the alpha-ENO promoter. Finally, we also demonstrated that IFN treatment of PBMC cause an increase in both, alpha-ENO expression on the cell surface and plasmin generation followed addition of exogenous plasminogen.

Innervation-dependent phosphorylation and accumulation of alphaB-crystallin and Hsp27 as insoluble complexes in disused muscle.

Levels and phosphorylation states of the two small molecular chaperones, alphaB-crystallin and Hsp27, in disused rat soleus muscles were determined by Western blot analysis of extracts with antibodies recognizing each of the two proteins and their phosphorylated serine residues. Increased phosphorylation and relocalization to insoluble fractions were found within a few days of hind-limb suspension. High phosphorylation of alphaB-crystallin at Ser-59 (and to a certain extent, at Ser-45) and of Hsp27 at Ser-15 and Ser-85, along with phosphorylated, active states of p38 and p44/42 mitogen-activated protein kinases were maintained during hind-limb suspension but promptly returned to control levels within a 5-day recovery period. These results are similar to those observed with U373 MG glioma cells exposed to proteasome inhibitors (16). However, the responses of alphaB-crystallin and Hsp27 in suspended soleus muscles did not appear with ipsilateral transection of the sciatic nerve trunk, indicating mediation by nerve activity. The fact that ubiquitinated proteins accumulated in the insoluble fractions of suspended soleus muscle further suggests participation of alphaB-crystallin and Hsp27 in quality control of proteins in disused soleus muscle, with involvement of nerve activity-dependent processes.

Riluzole enhances expression of brain-derived neurotrophic factor with consequent proliferation of granule precursor cells in the rat hippocampus.

The dentate gyrus of the hippocampus, generating new cells throughout life, is essential for normal recognition memory performance. Reduction of brain-derived neurotrophic factor (BDNF) in this structure impairs its functions. To elucidate the association between BDNF levels and hippocampal neurogenesis, we first conducted a search for compounds that stimulate endogenous BDNF production in hippocampal granule neurons. Among ion channel modulators tested, riluzole, a neuroprotective agent with anticonvulsant properties that is approved for treatment of amyotrophic lateral sclerosis, was highly effective as a single dose by an intraperitoneal injection, causing a rise in BDNF localized in dentate granule neurons, the hilus, and the stratum radiatum of the CA3 region. Repeated, but not single, injections resulted in prolonged elevation of hippocampal BDNF and were associated with increased numbers of newly generated cells in the granule cell layer. This appeared due to promoted proliferation rather than survival of precursor cells, many of which differentiated into neurons. Intraventricular administration of BDNF-specific antibodies blocked such riluzole effects, suggesting that BDNF increase is necessary for the promotion of precursor proliferation. Our results suggest the basis for a new strategy for treatment of memory dysfunction.

Cleavage of calnexin caused by apoptotic stimuli: implication for the regulation of apoptosis.

Calnexin is an endoplasmic reticulum (ER)-resident molecular chaperone that plays an essential role in the correct folding of membrane proteins. We found that calnexin is subjected to partial cleavage in apoptotic mouse cells. Both ER stress-inducing and ER stress-non-inducing apoptotic stimuli caused the cleavage of calnexin, indicating that this event does not always occur downstream of ER stress. The inhibition of caspases that target the amino acid sequence DXXD abrogated calnexin cleavage in apoptotic stimulus-treated cells. In addition, disruption of one of two DXXD sequences located in the cytoplasmic domain caused calnexin to escape cleavage during apoptosis. Furthermore, calnexin was cleaved in vitro by recombinant caspase-3 or caspase-7. Finally, the overexpression of a presumed cleavage product of calnexin partly inhibited apoptosis. These results collectively suggest that caspase-3 or caspase-7 cleaves calnexin, whose cleaved product leads to the attenuation of apoptosis.

Inhibition of proteasomes induces accumulation, phosphorylation, and recruitment of HSP27 and alphaB-crystallin to aggresomes.

Molecular chaperones and the ubiquitin-proteasome pathway are known to participate in the quality control of proteins in cells. In this study, we examined the responses of small heat shock proteins to proteasome inhibitors to clarify their roles under conditions where misfolded proteins are abnormally accumulated. HSP27 and alphaB-crystallin accumulated in both soluble and, more prominently, insoluble fractions after exposure to MG-132, a proteasome inhibitor. Enhanced expression of mRNAs for HSP27 and alphaB-crystallin was observed, suggesting transcriptional activation. Phosphorylation of HSP27 and alphaB-crystallin in cells treated with MG-132 was enhanced concomitantly with activation of p38 and p44/42 MAP kinase pathways. Immunofluorescence analysis revealed that exposure to proteasome inhibitors induced the formation of aggresomes in U373 MG cells, to which HSP27 and alphaB-crystallin were recruited. However, phosphorylation was not required for this accumulation in aggresomes. Thus, HSP27 and alphaB-crystallin are increased, phosphorylated and localized in aggresomes when proteasome activity is inhibited.

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.

Methotrexate enhances prostaglandin D2-stimulated heat shock protein 27 induction in osteoblasts.

As for the pathogenesis of rheumatoid arthritis (RA), prostaglandins (PGs) act as important mediators of inflammation and joint destruction. Among them, PGD2 is well recognized as a potent regulator of osteoblastic functions. We previously showed that PGD2 stimulates the induction of heat shock protein 27 (HSP27) via protein kinase C (PKC)-dependent p38 mitogen-activated protein (MAP) kinase and p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. Therefore, it is a current topic to clarify how HSP27 plays a role for regulating osteoblastic functions in the lesion of RA. On the other hand, methotrexate (MTX) is one of the most effective medicines for the treatment of RA. Here, we examined the effect of MTX on PGD2-stimulated HSP27 induction in MC3T3-E1 cells. The cells were pretreated with various doses of MTX including therapeutic dosage for RA, and then stimulated by PGD2. MTX significantly enhanced the PGD2- increased levels of HSP27 in a dose-dependent manner, although MTX alone had no effect on the levels of HSP27. In addition, MTX amplified the PGD2-increased levels of HSP27 mRNA. On the contrary, MTX had little effect on PGD2-induced formation of inositol phosphates, PKC activation and phosphorylations of MAP kinases. Our results strongly suggest that MTX enhances PGD2-stimulated HSP27 induction at a point downstream from MAP kinases in osteoblasts.

Involvement of p38 mitogen-activated protein kinase in heat shock protein 27 induction in human neutrophils.

We investigated whether tumor necrosis factor-alpha (TNF-alpha) stimulates the induction of heat shock protein 27 (HSP27) in human neutrophils and the mechanism underlying this induction. In intact neutrophils, almost no HSP27 was detected. Stimulation of neutrophils by TNF-alpha increased the levels of HSP27 in the presence, but not in the absence, of cycloheximide. Reverse transcription-polymerase chain reaction (RT-PCR) experiments showed that TNF-alpha also induced HSP27 mRNA in the presence of cycloheximide. TNF-alpha induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by TNF-alpha was significantly suppressed by 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) or 4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole (PD169316); both are specific inhibitors of p38 MAP kinase, but not by 2'-amino-3'-methoxyflavone (PD098059, a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase). The accumulation of HSP27 induced by TNF-alpha plus cycloheximide was also suppressed by pretreatment with a specific protein kinase C (PKC) inhibitor. Furthermore, phorbol myristate acetate (PMA), a PKC stimulant, but not dibutyryl cyclic AMP, a protein kinase A stimulant, stimulated the accumulation of HSP27. Interestingly, SB203580 did not inhibit PMA-stimulated HSP27 induction. These results strongly suggest that TNF-alpha may act as the regulator of HSP27 induction in neutrophils. p38 MAP kinase (but not p44/p42 MAP kinase) and PKC take part in TNF-alpha-stimulated HSP27 induction in human neutrophils.

HSP20, low-molecular-weight heat shock-related protein, acts extracellularly as a regulator of platelet functions: a novel defense mechanism.

We previously showed that a dissociated form of a low-molecular-weight heat shock-related protein 20 (HSP20) but not an aggregated form of HSP20 suppresses platelet aggregation. In the present study, we investigated the behavior of HSP20 in response to endothelial injury and the possible mechanism of HSP20 in platelet functions. The levels of HSP20 in vessel wall after endothelial injury were markedly reduced. This observation was supported by the results of Western blotting analysis and immunohistochemical analysis. Additionally, the plasma levels of HSP20 in cardiomyopathic hamsters were markedly elevated. Centrifugation on sucrose density gradients allowed detection mainly of the dissociated form of plasma HSP20 in these hamsters. Human platelets showed specific binding sites for HSP20. Moreover, HSP20 markedly reduced thrombin-induced phosphoinositide hydrolysis by phospholipase C in human platelets. Taken together, our results strongly suggest that HSP20, which immediately responds to pathological events, acts extracellularly as a regulator of platelet functions.

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.