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Measurement of released lactate dehydrogenase (LDH) activity, a commonly used marker of lethal cell injury in both in vitro and in vivo screenings, has been used to assess the cytotoxicity of nanoparticles (NPs), chemical compounds, and environmental factors. We have recently demonstrated that titanium dioxide (TiO2) particles bind to several serum proteins. In the present study we investigated the binding of TiO2 NPs to LDH. Purified LDH was incubated with TiO2 NPs at 37°C for 1 h. The particles were then sedimented by centrifugation, and the activity and quantity of LDH in the supernatant and precipitated fraction were analyzed. Incubation with TiO2 reduced the LDH activity in the supernatant in a dose-dependent manner, while LDH activity in the precipitated fraction increased in a dose-dependent manner. Moreover, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed a TiO2 dose-dependent reduction in the quantity of LDH protein in the supernatant and an increase of LDH in particulate re-suspensions. These findings, although based on a purified form of LDH, suggest that TiO2 NPs bind to LDH, and consequently, TiO2 NP-induced toxicity could be underestimated by the LDH activity assay.

When human neuroblastoma cells (SH-SY5Y) were exposed to 0.5 - 5 mM acrylamide for 18 hr, the levels of heat shock proteins (HSPs) of 90, 70 and 27 kDa (Hsp90, Hsp70, and Hsp27, respectively) were elevated in the incubation media depending on the dose of acrylamide whereas only the Hsp70 level increased within cells. U0126, a specific inhibitor of extracellular signal-regulated protein kinase kinase and a potent suppressor of the cytotoxicity of acrylamide, suppressed the increase in the levels of all HSPs in the incubation media but not their expression within cells. Total protein concentrations in the incubation media increased depending on the dose of acrylamide, and this increase was associated with the increasing number of bands detected by silver staining after SDS-polyacrylamide gel electrophoresis. One of the clearest bands was identified as Hsp90 by peptide mass fingerprinting. Thus, acrylamide causes release of proteins, including that of HSPs, from SH-SY5Y cells. HSP in extracellular fluid may be a good indicator of cytotoxicity of acrylamide.

Using human neuroblastoma SH-SY5Y cells, effects of acrylamide on p53 protein and intracellular signal transducting pathways were examined. Acrylamide increased p53, phosphorylated p53, and p53-associated protein murine double minute 2 (MDM2). The phosphorylation of p53 was specific for the Ser15 site. Among mitogen-activated protein kinases (MAPKs), acrylamide caused phosphorylation of extracellular signal-regulated protein kinase (ERK) and p38 but not c-Jun NH(2)-terminal kinase. Nevertheless, blocking p38 pathway by LL-Z1640-2 did not suppress the phosphorylation of p53 at Ser15. In contrast, a specific inhibitor of ERK kinase (U0126 or PD98059) could abolish the accumulation as well as the phosphorylation of p53 at Ser15. Elevation of MDM2 was also abolished by U0126. An inhibitor of phosphatidylinositol 3-kinase-related kinase (PIKK) pathway (wortmannin) suppressed the increase of p53 and its phosphorylation at Ser15. Hence, acrylamide increases p53 protein and its phosphorylation at Ser15 through ERK and/or PIKK pathways. On the other hand, U0126 and PD98059 suppressed to some extent the cytotoxicity of acrylamide evaluated by trypan blue exclusion and lactate dehydrogenase (LDH) leakage, whereas neither LL-Z1640-2 nor wortmannin was effective in suppressing the toxicity. Thus, ERK pathway seems to play a role both in causing the phosphorylation of p53 at Ser15 and in the cytotoxicity of acrylamide in SH-SY5Y cells.

Acrylamide inhibits creatine kinase (CK) activities in the brain of rats or mice. However, its effects on genetic expression of CK have not yet been studied. CK mRNA and protein level were examined by RT-PCR and Western blotting, respectively. Neither cytosolic CK (B subunit) and mitochondrial CK (ubiquitous form) mRNA nor B subunit protein was clearly changed in the cerebellum from rats intoxicated with acrylamide (50 mg/kg/day i.p. for 8 days) and showing clinical neurotoxic signs.

We examined effects of asbestos exposure on the phosphorylation of p53 protein in human pulmonary epithelial type II cells (A549), which express wild-type p53. In cells exposed to two different types of asbestos, chrysotile (approximately 1-6% iron content) and crocidolite (approximately 27% iron content) fibers, at the doses of 1, 5, and 10 microg/cm2 for 24 hr, the levels of p53 phosphorylated at Ser15 and p53 protein were correlated with the dose. On a per-weight basis, chrysotile was more potent in inducing Ser15 phosphorylation and accumulation of p53 protein than was crocidolite. After exposure to 10 micro g/cm2 chrysotile, the levels of p53 phosphorylated at Ser15 and of p53 protein increased after 18 hr. Among serines in p53 protein immunoprecipitated from A549 cells treated with chrysotile, only Ser15 was markedly phosphorylated. In contrast, no clear phosphorylation was observed at Ser6, Ser9, Ser20, Ser37, Ser46, or Ser392. Blocking of the extracellular signal-regulated protein kinase pathway with U0126 or inhibition of p38 activity with SB203580 did not suppress chrysotile-induced Ser15 phosphorylation. On the other hand, treatment with wortmannin, an inhibitor of DNA-activated protein kinase and ataxia-telangiectasia mutated, suppressed both chrysotile-induced Ser15 phosphorylation and accumulation of p53 protein. Treatment with either catalase or N-acetylcysteine failed to suppress chrysotile-induced Ser15 phosphorylation, suggesting that reactive oxygen species do not play a major role in the phosphorylation of p53 protein. The present results show that asbestos, particularly chrysotile, induces phosphorylation of p53 protein at Ser15 in A549 cells depending on a DNA damage-signaling pathway.

When Jurkat human T cells were incubated with 20 microM of pentachlorophenol (PCP) or its metabolite, tetrachlorohydroquinone (TCHQ), for 10 hr, flow cytometric analyses revealed marked increase in the number of apoptotic cells. DNA fragmentation was also observed in these cells. TCHQ was more potent than PCP in causing apoptosis. After incubation with 20 microM TCHQ for 1 hr, all mitogen-activated protein kinases (MAPKs) examined [i.e., extracellular signal-regulated protein kinase (ERK), p38, and c-Jun NH(2)-terminal kinase (JNK)] were phosphorylated, whereas no clear phosphorylation was induced by PCP. TCHQ-induced apoptosis was markedly suppressed by treatment with a p38 inhibitor (SB203580) and mildly (but significantly) suppressed by treatment with a MAPK/ERK kinase inhibitor (U0126). When cells were treated with both inhibitors at the same time, TCHQ-induced apoptosis disappeared almost completely. PCP-induced apoptosis was also suppressed by SB203580 and/or U0126. Nevertheless, treatment with LL-Z1640-2, which inhibits JNK phosphorylation, did not suppress the apoptosis caused by either TCHQ or PCP. Thus, p38 and ERK appear to be important signal transduction pathways leading to apoptosis in a human T-cell line exposed to a ubiquitous pollutant or its metabolite in the general and occupational environment.

Rats were exposed to 290 or 495 ppm methyl bromide gas for 6 h/day, 3 times/wk for 4 to 8 wk. Creatine kinase (CK), aspartate aminotransferase (ASAT), and lactate dehydrogenase (LDH) activities and bromide ion concentrations were measured in eight regions of the brain. Methyl bromide gas inhibited CK activities in all regions of the brain, though the inhibition tended to be smallest in the cerebellum (hemisphere and vermis) and largest in the brainstem (hypothalamus, midbrain, and medulla oblongata). The dose of methyl bromide to inhibit CK activities was lower than that to damage the central nervous system histologically. No inhibition of ASAT or LDH activities was seen except for a slight inhibition of these in striatum. Inhibition of CK activities did not increase clearly on increasing dose (290 to 495 ppm) or on prolonging exposure period (4 to 8 wk). Although 50% recovery of CK activities and the half-life of bromide ion agreed well in the medulla oblongata, changes in CK activities and bromide ion concentrations did not correlate otherwise. Thus, inhibition of CK activities in brain appears to be a sensitive indicator of methyl bromide intoxication, and may be related to genesis of its neurotoxicity. The inhibition seems to be caused by methyl bromide itself rather than by bromide ion. When effects on enzyme activities in brain homogenate were examined in vitro by bubbling with methyl bromide gas, CK inhibition was seen within 15 s of exposure. Dithiothreitol suppressed the CK inhibition, whereas N-acetylcysteine did not. These observations suggest that methyl bromide may attack sites in the CK molecule different from those attacked by ethylene oxide or acrylamide.

When CCRF-CEM cells were incubated with 5-40 microM CdCl(2,) apoptosis was observed most clearly at 10 microM. Prior to the development of apoptosis, mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, were activated with different sensitivity to CdCl(2) exposure. ERK and p38 MAPK were phosphorylated with incubation of 1 microM CdCl(2,) but higher than 20 microM CdCl(2) was required for the clear phosphorylation of JNK. In the time-course study, ERK and p38 MAPK were phosphorylated earlier than JNK after CdCl(2) exposure. The in vitro activities of MAPKs also increased in response to CdCl(2) exposure. Pretreatment with an intracellular Ca(2+) chelator, 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), suppressed almost completely CdCl(2)-induced phosphorylation of JNK and p38 MAPK, but not ERK phosphorylation, indicating that the activation of JNK and p38 MAPK depends on the intracellular Ca(2+) but that of ERK does not. On the other hand, treatment with a MAPK/ERK kinase (MEK) inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene ), suppressed CdCl(2)-induced ERK activation and the apoptosis as well. The inhibition of p38 MAPK activity with SB203580 (4-[4-fluorophenyl]-2-[4-methylsulfinylphenyl]-5-[4-pyridyl]1H- imidaz ole) did not protect cells from apoptosis. The present results showed that the activation of ERK, JNK, and p38 MAPK is differently regulated in CCRF-CEM cells exposed to CdCl(2,) and that the ERK pathway seems to be responsible for the induction of apoptosis by CdCl(2) exposure in this human T cell line.

Rat brain was examined with 4.7 T proton magnetic resonance (MR). On administering hexachlorophene (HCP) 30 mg/kg/day for 5 days, myelin-rich structures stood out in T2-weighted images. Apparent diffusion coefficient (ADC) was markedly suppressed in all regions examined except for cerebral cortex. Seven days after terminating the exposure to HCP, without enhancement in T2-weighted images, ADC was still decreased in corpus callosum, optic nerve and trigeminal nerve. Rat administered with HCP and followed with high magnetic field proton MR seems to provide a good model for cytotoxic brain edema, and it may also be useful to visualize heavily myelinated structures.

Suppression of PentylenetetrazolInduced Seizures and c-fos Expression in Mouse Brain by L-Carnitine: Yoshihisa IRYO, et al. Department of Environmental Toxicology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health—Seizure development in ddY mice was recorded on a videotape for 20 min after injection of pentylenetetrazol (PTZ) (50 mg/kg) and analyzed in detail. In addition, expression of the c-fos gene in the brain was semi-quantified by reverse transcription-polymerase chain reaction analysis. When saline, L-carnitine (10 mmol/kg), or Dcarnitine (10 mmol/kg) was administered 30 min prior to PTZ, the seizures were suppressed in mice given Lcarnitine; the seizure scores were significantly lower in mice treated with L-carnitine at several time points, and the overall seizure scores were 31.43 ± 2.49, 10.57 ± 2.86 and 24.71 ± 3.05 (expressed as the mean ± S.E.M., n=7), in saline, L-carnitine and D-carnitine groups, respectively. The latency to the first clonictonic seizure was also prolonged in mice treated with L-carnitine. The level of c-fos mRNA in the brain was lower in the animals treated with L-carnitine than in those treated with saline or D-carnitine. Thus, Lcarnitine shows not only anticonvulsive effects in one of the most widely used animal models of chemically induced seizures but also the potential to suppress the seizure-associated expression of an immediate early gene in the brain.

In response to various environmental stresses including heavy metals, the c-Jun N-terminal kinase (JNK) is phosphorylated and then it phosphorylates c-Jun protein. In the present study, effects of mercury chloride (HgCl2) on JNK signalling pathway were examined in LLC-PK1 cells. When exposed to 10 or 20 microM HgCl2, the level of phosphorylated JNK and the activity of JNK assayed in vitro using glutathione-S-transferase-c-Jun as substrate increased markedly. The level of phosphorylated JNK increased 30 min after HgCl2 exposure and remained elevated even at 8 h. On the other hand, no changes were found in the total amount of JNK protein. Consistent with the activation of JNK, c-Jun proteins phosphorylated on Ser63 and Ser73 were accumulated in cells exposed to HgCl2. Concomitantly, the levels of c-jun mRNA and c-Jun protein were elevated. When compared to other heavy metal compounds such as manganese chloride, zinc chloride, cadmium chloride, and lead chloride, HgCl2 could phosphorylate JNK more markedly. Neither intracellular Ca2+ nor sulfhydryl groups appeared to play a major role in the activation of JNK by HgCl2 exposure in this porcine renal epithelial cell line.

Washed human erythrocytes were incubated with titanium dioxide (TiO2) particles at 37 degrees C for 1 hr and hemolysis was determined by the percentage of hemoglobin released (optical density at 540 nm; OD540) from the cells. Effects of TiO2 on OD540 were corrected and dose-response curves were analyzed by the Hill plot. Judging from the estimated dose to cause 50% hemolysis, the anatase form of micron-scale (5000 nm) particles was 73 and 11 times more potent than the amorphous (50 nm) and rutile (5000 nm) forms, respectively, whereas it was 1.3 times more potent than the nano-scale (25 nm) anatase particles. Plasma abolished the hemolysis due to anatase and rutile forms. Thus, hemolytic effects of TiO2 can be greatly different depending on the polymorph but not on the primary size (nano- or micron-scale) of particles. TiO2-induced hemolysis is unlikely to occur in vivo because of the presence of plasma.

The level of phosphorylated c-Jun NH2-terminal kinase (JNK) in LLC-PK1cells treated with CdCl2increased after 30 min and remained elevated even at 8 hr. And the activity of JNK assayed using glutathioneS-transferase-c-Jun as substrate increased dose-dependently. Consistent with the JNK activation, marked increases in the levels of c-Jun and c-Jun phosphorylated on Ser63 and Ser73 were observed in cells treated with CdCl2. The pretreatment with an intracellular Ca2+chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), abolished cadmium-induced JNK phosphorylation. However, pretreatment with a cell permeable chelator of heavy metals,N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), did not. The present results showed that cadmium induces persistent activation of JNK pathway in a renal epithelial cell line, and that intracellular Ca2+is necessary for the activation.

Pentachlorophenol (PCP) increased oxygen consumption and lowered the respiratory control ratio (RCR) in mitochondria from rat liver. These effects of PCP were lessened by 1 mM L-carnitine but not by D-carnitine. In contrast, up to 150 mM of pentylenetetrazol (PTZ) added at state 4 of respiration did not accelerate oxygen consumption. When mitochondria were incubated with 3.3 mM of PTZ, oxygen consumption, RCR and ADP/O ratio were all decreased. Moreover, these could not be suppressed even by high concentrations (-20 mM) of L-carnitine. Thus, while L-carnitine could suppress effects of PCP, it could not counteract PTZ in mitochondria. It appears that anticonvulsive effects of carnitine in PTZ-induced seizures may not be due to mitochondrial protection.

The c-fos, a member of the immediate early genes, has been reported to be expressed in the renal proximal tubule in response to ischemic and toxic injury. In the present study, effects of mercury chloride (HgCl2) on the expression of c-fos were examined in LLC-PK1 cells. The reverse transcription polymerase chain reaction (RT-PCR) analysis for the semi-quantification of mRNA showed that the treatment of 20 microM HgCl2, markedly increased c-fos mRNA levels. The level of c-fos mRNA began to increase after a 30-min exposure, peaked at 1 h and then returned to the control level at 8 h. The HgCl2-induced c-fos expression was abolished completely by actinomycin-D, indicating it was due to transcriptional activation of the gene. Western blotting immunodetection revealed accumulation of c-Fos protein after 1 h exposure to 20 microM HgCl2. The cytotoxicity of HgCl2 as assayed by mitochondrial dehydrogenase activity (MTT conversion) was observed after 18 h exposure but not at 0.5-8 h. Also, the decrease in cell viability was accompanied with DNA fragmentation, which is characteristic of apoptosis. The present results showed that HgCl2 could induce the early expression of c-fos gene in a renal epithelial cell line.

Hexachlorophene (HCP), pentachlorophenol (PCP), 2,4,5-trichlorophenol (2,4,5-TCP) and 2,4,6-trichlorophenol (2,4,6-TCP) all hemolyzed washed human erythrocytes and inhibited acetylcholinesterase (AchE) activities in erythrocyte membrane. HCP was much more potent in either effect than any other compound examined. The inhibition of AchE activities by HCP was reversed on adding albumin. The dose-response curves by HCP and PCP were sigmoidal, indicating cooperative inhibition, while those by 2,4,5-TCP and 2,4,6-TCP were not. Furthermore, the cooperativity of the inhibition by HCP was greater than by PCP. Differing from that by PCP, the cooperativity of inhibition increased depending on the temperature (13, 25, 37 degrees C) and decreased when the membrane was treated with Triton X-100. The cooperativity was also decreased in the presence of albumin. On a Scatchard plot analysis, erythrocyte membranes appeared to have multiple binding sites of different affinities for HCP; binding of HCP to the low affinity site [dissociation constant (Kd) 4.7 x 10(-5) M] seemed to be responsible for the observed cooperative inhibition of AchE activities. Neither neostigmine nor fenitrothion altered the cooperativity. HCP seems to be the most potent cooperative inhibitor of AchE in human erythrocyte membranes known to date. HCP may be useful to examine AchE and milieu in human erythrocyte membranes.

Lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) activities and total protein concentrations were examined in the postrinsing solutions from rat lungs preserved in phosphate-buffered saline (PBS), Euro-Collins (E-C) solution, or low-potassium E-C solution for 8 h at 4 degrees C, 10 degrees C, or 20 degrees C. The LDH and AST activities were higher when the organs were preserved in PBS for 8 h at 20 degrees C than at 4 degrees C or 10 degrees C, while the total protein concentration did not differ according to the temperature or solution. The activities were also higher when the lungs were preserved in Euro-Collins (E-C) solution than in PBS or the low-potassium E-C solution. On examining pulmonary functions utilizing an ex vivo reperfusion model, the lungs preserved at 20 degrees C showed poorer gas exchange than those preserved at 4 degrees C or 10 degrees C. Moreover, the organs preserved in E-C solution showed poorer function than those preserved in any other solution. These findings suggest that some enzymatic activities in the postrinsing solution could be indicators of lung viability after preservation.

The effects of tributyltin on the expression of immediate early genes, c-fos and c-jun, were examined in PC12 cells using reverse transcription polymerase chain reaction analysis. Tributyltin at concentrations of more than 0.4 μM induced the expression of c-fos after 15 min exposure. The induction of c-fos was accompanied with c-jun expression. Tributyltin-induced c-fos expression was abolished completely by actinomycin D, indicating it was due to transcriptional activation of the gene. Chelation of intracellular Ca(2+) suppressed the expression of c-fos markedly, while removal of external Ca(2+) did not. These results suggest that Ca(2+) mobilized from intracellular stores played a major role in the tributyltin-induced transcriptional activation of c-fos in PC12 cells.

OBJECTIVES--To examine whether the activities of creatine kinase (CK) correlate with neurological disturbances caused by acrylamide. METHODS--The activities of CK and other enzymes reported to be inhibited by acrylamide in the brain and plasma, and landing foot spread (LFS) were measured in mice and rats intoxicated with acrylamide. RESULTS--Activity of CK was suppressed by acrylamide in the brain of mice in parallel with the neurological dysfunction measured by LFS. No clear alterations were found in glyceraldehyde-3-phosphate dehydrogenase, neuron-specific enolase, and lactate dehydrogenase activities over the experimental period (eight days for the exposure and 43 days for the recovery). In rats, among the plasma enzymes examined, suppression of CK activity was most notable, but thyroid activity was not affected. CONCLUSIONS--Among the enzymes so far examined, the CK activities in the brain and blood seem to be the most sensitive indicators of acrylamide intoxication.

We describe herein a new experimental model in which an isolated rat lung was ventilated with a mixture of 95% nitrogen and 5% carbon dioxide to decrease the oxygen and increase the carbon dioxide in the perfused blood to create and maintain a gas composition similar to that of venous blood. By utilizing this system as a "deoxygenator," pulmonary functions, including gas exchange, could be measured for at least 60 min in isolated and preserved lungs on reperfusion. When the effects of glucose in the flushing and storage solution were examined, 5 mM glucose in the solution resulted in better preservation of the lung, as shown by a higher uptake of oxygen and a lower intratracheal pressure, than when no glucose was given. However, the presence of 50 mM glucose was not beneficial, but rather increased the wet/dry weight ratio of the tissue.

Hypoglycemia and hyperglycemia were induced in mice by fasting and by injecting with glucose, respectively. These and normally fed (normoglycemic) animals were exposed to 0.5% carbon monoxide (CO) for 10 min. This altered concentrations of energy metabolites in the brain, including decreases in phosphocreatine (PCr) and increases in creatine and lactate. The only difference between normoglycemic and hypoglycemic mice was lower lactate in the latter. In hyperglycemic mice, PCr and ATP were better preserved during CO exposure and lactate was lower than in normoglycemic mice. Blood glucose concentrations correlated well with glucose but not with lactate in the brain. Thus, moderate hypo- or hyperglycemia seems not to exacerbate CO-induced alterations of brain energy metabolism.

After the rat lung was flushed with 40 mmol/L phosphate-buffered saline solution containing 160 mEq/L of sodium and the heart-lung block was immersed in the same solution for 6 hours, the lung was reperfused with diluted autologous blood and ventilated for 30 minutes. Pulmonary artery pressure, airway pressure, difference in oxygen tension between inflow and outflow perfusate, and wet to dry weight ratio of the tissue were determined. Lungs treated at pH 7.75 showed a significantly lower pulmonary artery pressure and wet to dry weight ratio than those treated at pH 7.26 or 7.96. Organs preserved at 10 degrees C showed a significantly lower pulmonary artery pressure than those preserved at 5 degrees or 15 degrees C. When the effect of smaller temperature variations was examined, the 12 degrees C group showed a significantly lower pulmonary artery pressure than the 10 degrees C group. Thus, the isolated rat lung reperfusion model appears to provide an efficient screening system to examine the preservation solution or other conditions for lung preservation. Under the present experimental conditions, the optimal pH and temperature for rat lung preservation seem to be 7.75 and 12 degrees C, respectively.

Objectives: To determine the capacity of human serum proteins to bind to titanium dioxide (TiO2) particles of different polymorphs and sizes. Methods: TiO2 particles were mixed with diluted human serum, purified human serum albumin (HSA) or purified human serum gamma-globulin (HGG) solutions. After incubation at 37°C for 1 h, the particles were sedimented by centrifugation, and proteins in the supernatant, as well as those bound to the particles, were analyzed. Results: The total protein concentration in the supernatant was lowered by TiO2, whereas the albumin/globulin ratio was elevated by the particles. Incubation with TiO2 also lowered the immunoglobulin, pre-albumin, beta2-microglobulin, ceruloplasmin and retinol-binding protein levels, but not ferritin levels, in the supernatant. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins in the supernatant, especially HGG, were observed to decrease, while those released from the particles (after adding 1% SDS and heating) increased, depending on the dose of TiO2. Purified HGG and HSA were also bound to TiO2, although the former appeared to have a higher affinity. All the proteins tested showed the highest binding potency to the amorphous particles (

Although l -carnitine has been reported to have protective effects against ammonia toxicity, conflicting results have also been presented and the mechanisms underlying the protection, if any, are not clear. In the present study, we examined the effects of l -carnitine, d -carnitine and acetyl- l -carnitine on the neurotoxicity of ammonia. Administration of ammonium acetate (15 mmol/kg) to mice caused Seizures, elevation of blood ammonia and urea concentrations, and marked alterations of brain energy metabolites. Pretreatment with either l -carnitine, d -carnitine or acetyl- l -carnitine reduced the frequency of the Seizures, prolonged the time until the first fit, lowered the levels of ammonia in the blood and brain, and suppressed the alterations of brain energy metabolites caused by hyperammonemia. There was no significant difference between l - and d -carnitine in the potency to inhibit the Seizures. In addition, there was no difference between the two chemicals in the potency to decrease the ammonia contents in the blood and brain, or to suppress the alterations of energy metabolites in the brain. When compared with l -carnitine, however, acetyl- l -carnitine better preserved ATP in the brain, while it lowered ammonia in the blood and brain less markedly. These results show that l -carnitine and its analogues do have the potential to suppress the neurotoxicity of ammonia. Moreover, the results suggest that the protective effects of carnitine against the toxicity of ammonia are systemic, that the action of acetyl- l -carnitine may differ from that of l - or d -carnitine, and that the “classical” function of carnitine is not the sole mechanism underlying the suppression of the neurotoxicity of ammonia.

The effects of ethylene oxide (EO), acrylamide, N,N′ -methylene-bis-acrylamide (bis-acrylamide), and methyl mercury chloride (MMC) on brain creatine kinase (CK) activity were examined in vivo . EO and acrylamide, both of which cause central–peripheral distal axonopathy, inhibited CK activity in the brain and spinal cord. On the other hand, neither bis-acrylamide, a nonneurotoxic analogue, nor MMC which causes neuronopathy affected brain CK activity significantly. The inhibition of CK may play a role in the pathogenesis of distal axonal degeneration in the central and peripheral nervous systems.

Psychosine (galactosylsphingosine) potently inhibits cytochrome c oxidase (COX) activity and hemolyzes washed erythrocytes in vitro. However, no hemolytic anemia is seen in patients with Krabbe disease or its animal models in which a psychosine-degrading enzyme is missing. This "discrepancy" may be caused by albumin because, 1) albumin is synthesized only in liver and it does not enter cells once it is released into the blood stream; 2) albumin can bind psychosine and suppress its effects effectively; 3) psychosine cannot exert its toxicity in the blood, should it be present there. On the other hand, an artificial substance, pentachlorophenol (PCP), which is widely used for wood preservation, causes mitochondrial dysfunction and hemolysis in vitro, both of which can be suppressed by albumin. Hence, it appears reasonable that hemolytic anemia is extremely rare, and that a dramatic improvement was seen in patients severely intoxicated with PCP. Thus, differences in the effects of these toxins seen in vitro and in vivo seem to be well explained by the effects of albumin. These also suggest that attention to the possible effects of albumin may be rewarding in monitoring and treating some intoxications.

A common mechanism appears to play a pathogenetic role in acrylamide intoxication, which is due to an exogenous cause, and in giant axonal neuropathy, an intrinsic disorder. In giant axonal neuropathy, a genetic defect in the processing of an intrinsic acrylamide or acrylamide-like substance may be postulated. On the other hand, the devastating pathology in Krabbe disease (a genetic disorder affecting almost exclusively the nervous system) appears to be due to the effects of psychosine, a toxic lipid produced in the nervous system. These suggest that the "toxicological approach" can be more diverse and of greater use than usually considered.

When rats were injected intraperitoneally with acrylamide (50 mg/kg per day) for 8 days, all animals developed ataxia and weakness in the hindlimbs. On examining their brain with an ultrahigh-field (4.7 T) magnetic resonance (MR) spectrometer, the lateral ventricles on both sides and the third ventricle were dilated. The aqueduct and cisterns were also enlarged. The size of the cerebral cortex was quantified in three MR image slices covering the cerebrum. Compared with the images of the brain of body weight-matched controls, the cerebral cortex of rats intoxicated with acrylamide was found to be smaller in the primary motor area in all slices, and in the primary or secondary sensory area in two slices. Taken together with previous enzymatic analyses, rats intoxicated with acrylamide (50 mg/kg per day for 8 days) seem to represent an animal model of acrylamide encephalopathy not only biochemically but also structurally.

In our previous study, we found that caspase-dependent apoptosis played a role in the genesis of toxicity of acrylamide in human neuroblastoma (SH-SY5Y) cells (Sumizawa and Igisu in Arch Toxicol 81:279-282, 2007). In the present experiment, we examined whether carboxyfullerene may suppress the cytotoxicity of acrylamide because carboxyfullerene has been reported to protect nerve cells from various pathologic processes including apoptosis. Carboxyfullerene lowered lactate dehydrogense leakage and elevated cell viability in SH-SY5Y cells exposed to acrylamide. It also lowered caspase-3 activities and cell population in the sub-G(1) phase induced by acrylamide. Nevertheless, carboxyfullerene enhanced cellular uptake of [(14)C]acrylamide. On the other hand, acrylamide markedly decreased glutathione (GSH)-content in cells and carboxyfullerene blocked the decrease. The toxicity of acrylamide was suppressed by adding GSH or GSH monoethyl ester, whereas it was not lowered by carboxyfullerene when GSH synthesis was inhibited by L: -buthionine-(S,R)-sulfoximine. Thus, the cytotoxicity of acrylamide including apoptotic processes is closely related to GSH level in SH-SY5Y cells and carboxyfullerene suppresses the toxicity by maintaining GSH content. Neither tricarboxylic acids without fullerene moiety nor hydroxylated fullerene showed comparable effects of carboxyfullerene (60 microM) against 1-5 mM acrylamide, suggesting the importance of the three malonic acid groups at specific positions in a fullerene molecule for the effects.

Administration of ammonium acetate to mice caused seizures and alterations of brain energy metabolites. Pretreatment of animals with L-carnitine suppressed the frequency of the seizures and prolonged the latency to the first fit. When examined using the 'freeze clamp' method, brain energy metabolites were well preserved and the elevation of ammonia was less marked on administration of L-carnitine. Thus, L-carnitine suppresses ammonia-induced seizures and biochemical alterations of the brain in mice.

Acrylamide (1-5 mM) dose-dependently decreased cell viability in human neuroblastoma cells (SH-SY5Y). The caspase-3 activity and cell population in sub-G(1) phase were elevated and peaked on exposure to 3 mM acrylamide, while both were less so at higher dose (4 and 5 mM). Z-VAD-fmk, a pan-caspase inhibitor, lowered the apparent cytotoxicity of acrylamide. U0126, a specific inhibitor of extracellular signal-regulated protein kinase (ERK) kinase, suppressed the elevation of caspase-3 activities as well as that of sub-G(1) population. Thus, although mechanisms other than caspase-dependent apoptosis may be involved, apoptotic process seems to take place in the genesis of toxicity of acrylamide in SH-SY5Y cells through ERK pathway and activation of caspase-3.

Examinations of brain of rats intoxicated with hexachlorophene or acrylamide with ultrahigh-field (4.7 T) proton magnetic resonance (MR) showed alterations consistent with clinical pictures in humans and morphological findings in experimental animals. On the other hand, conventional biochemical analyses have revealed that ethylene oxide, methyl bromide, and acrylamide inhibit creatine kinase (CK; an enzyme catalyzing the reaction: ATP+creatine--ADP+phosphocreatine) activities in the brain of animals. Thus, 31P MR combined with magnetization transfer may be utilized to monitor living humans (or animals) intoxicated with these chemicals by determining CK activities in the target organ.

c-Jun NH 2 -terminal kinase (JNK), also known as stress-activated protein kinase (SAPK), is activated primarily by inflammatory cytokines and environmental stresses including toxic metal exposure. To reveal the upstream kinase responsible for JNK activation by toxic metals, the phosphorylation status and the activity of JNK were examined in mouse embryonic stem (ES) cells lacking MKK4 or MKK7 following exposure to CdCl 2 or HgCl 2 . Treatment with CdCl 2 or HgCl 2 induced the phosphorylation of JNK in a dose- and time-dependent manner in wild-type ES cells. In both mkk4 −/− and mkk7 −/− ES cells, CdCl 2 - or HgCl 2 -induced phosphorylation and activation of JNK were suppressed significantly. However, in mkk7 −/− ES cells treated with CdCl 2 and HgCl 2 , JNK activation was not abolished (suppressed by 56% and 78%, respectively). These findings suggest that the full activation of JNK by toxic metal exposure requires both MKK4 and MKK7, and these upstream kinases might contribute differentially in JNK activation between mouse ES cells exposed to CdCl 2 and HgCl 2 .

This descriptive study reports the results of a systematic survey on occupational safety and health hazards and risks to exposed workers at companies co-operating with a medical university, and investigates the effective methods for their management. Varieties of works at all these co-operating companies have been categorized into 14 types of businesses. The survey was performed in two-fold; an individual interview of managers and a comprehensive inspection of workshops, and a questionnaire to all workers (response rate 95.9%). All of the co-operating companies were small-scale enterprises with less than 50 employees. The occupational safety and health risks to be controlled were accidental contact with biomedical wastes, heat in the kitchen, laundry and boiler room, unfavorable ventilation at workplace close to medical facility, and physical fatigue of guards and janitors. A higher number of occupational health physicians and a higher rate of health examinations were observed compared to other small-scale enterprises in Japan. About 80% of all workers were conscious of working on the premises of medical facilities. To improve the occupational safety and health activities of co-operating companies, we propose the use of professionals at the medical university, the promotion of joint activities among the co-operating companies, and the strengthening of mutual connection and coordination between the co-operating companies and the medical university.

The signaling pathways leading to cellular protection or cell death following exposure to heavy metals have not been fully clarified. Mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK transmit extracellular signals into the nucleus, and have been shown to participate in a diverse array of cellular functions such as cell growth, differentiation and apoptosis. Treatment with cadmium, inorganic mercury or tributyltin can activate ERK, JNK and p38 MAPK, and induces the expression of c-fos and c-jun genes prior to the development of apoptosis. However, the members of the MAPK family appear to be differentially activated depending on the heavy metal and the cell type exposed. Consequently, various cellular responses may be caused by the distinct pattern of MAPKs activation. MAPKs may be one of the important cellular signal transduction pathways affected by various environmental pollutants, including heavy metals.

Pentachlorophenol (PCP) inhibited acetylcholinesterase (AchE) activity in human erythrocyte membranes with high cooperativity. The Hill coefficient for the inhibition was 4-5 in “untreated” membranes. Differences in the temperature (13, 25 and 37°) or treatment with 1% Triton X-100 did not clearly affect the cooperativity which, however, increased after the erythrocyte membranes were treated with 2-mercaptoethanol and iodoacetatic acid, suggesting that higher cooperativity in the inhibition of AchE by PCP may reflect conformational changes of AchE. Thus, PCP may be useful for the study of AchE in human erythrocytes.

When MCF-7 cells were incubated with 10 or 20 microM CdCl(2), p53 protein level increased after 18 h. Among serines in p53 protein immunoprecipitated from cells treated with CdCl(2), only Ser 15 was phosphorylated. No clear phosphorylation was found on Ser 6, 9, 20, 37, and 392. Accumulation of p53 protein phosphorylated at Ser 15 was also found after 18 h exposure. While phosphorylation of extracellular signal-regulated protein kinase, c-Jun NH2-terminal kinase and p38 was found in cells treated with CdCl(2), treatment with U0126, LL-Z1640-2, or SB203580 did not suppress Ser 15 phosphorylation. On the other hand, treatment with wortmannin or caffeine suppressed CdCl(2)-induced Ser 15 phosphorylation and accumulation of p53 protein. The present results showed that cadmium induces phosphorylation of p53 at Ser 15 in MCF-7 cells depending on phosphatidylinositol 3-kinase related kinases, but not on mitogen-activated protein kinases.

Ischemia (decapitation) caused marked changes of energy metabolites in the mouse brain. However, the changes were clearly less severe, when the mouse was pre-treated with l -carnitine. This suggests that l -carnitine may protect the brain from the ischemic attack. Since d -carnitine showed a similar effect, the effects may be due to mechanism(s) other than the ‘classical’ function of carnitine.

Exposure of HgCl2 to CCRF-CEM human lymphoblastoid cells induced phosphorylation of mitogen-activated protein kinases (MAPKs); extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. LL-Z1640-2, a macrocyclic nonaketide, inhibited HgCl2-induced JNK phosphorylation at 5-100 ng/ml. It also inhibited phosphorylation of ERK and p38 but only at 100 ng/ml. The same doses of radicicol did not suppress MAPKs activation. LL-Z1640-2 (at 100 ng/ml) inhibited HgCl2-induced JNK phosphorylation in NIH 3T3 fibroblasts but not in LLC-PK(1) renal epithelial cells. Thus, LL-Z1640-2 is a potent inhibitor of HgCl2-induced MAPKs activation, especially that of JNK, in CCRF-CEM cells.

Effects of tributyltin chloride (TBT) and other organotin compounds on mitogen-activated protein kinases (MAPKs) were examined in CCRF-CEM human T lymphoblastoid cells. In response to the incubation with 0.25-2 microM TBT for 1 h, the levels of the phosphorylated form of extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 MAPK increased in a dose-dependent manner. The phosphorylation was observed after 15 min and lasted for 4 h following exposure to 1 microM TBT, while the cell viability was not lowered significantly within 6 h. On the other hand, no clear changes were found in the total protein levels of ERK, JNK, and p38 MAPK. The in vitro activities of MAPKs also increased in response to TBT exposure. The potentials of MAPKs phosphorylation and of cellular damage were TBT > dibutyltin dichloride (DBT) > monobutyltin trichloride (MBT). When compared to other triorganotin compounds such as trimethyltin chloride (TMT), triphenyltin chloride (TPT), and triethyltin bromide (TET), TBT exposure induced the most marked phosphorylation of MAPKs. Chelation of intracellular Ca(2+) suppressed TBT-induced MAPKs phosphorylation almost completely, but removal of external Ca(2+) did not. The present results showed that tributyltin is a potent activator of ERK, JNK, and p38 MAPK pathways, and Ca(2+) mobilized from intracellular stores plays an important role for the phosphorylation of MAPKs in this human T cell line.

Cadmium has been known to induce the expression of the c- fos gene in various cell types including fibroblasts. To clarify the biological significance of c- fos induction by cadmium, mouse 3T3-like fibroblasts lacking c- fos were exposed to cadmium, and the resultant cellular damage was assayed by WST-8 (4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2 H -5-tetrazolio]-1,3-benzene disulfonate sodium salt) conversion, trypan blue exclusion, or lactate dehydrogenase leakage. The c- fos -deficient cells (f1 and f10) were affected more severely than the wild-type cells (NIH 3T3 and f20) with respect to both cell growth and cellular damage following exposure to 10 or 20 μM cadmium chloride. These results suggest that c- fos may play a protective role against the cytotoxic effects of cadmium at least in these 3T3-like fibroblasts.

In vitro, both acrylamide and N,N′-methylene-bis-acrylamide inhibited creatine kinase (CK) activity from brain or sciatic nerve with almost the same potency. In vivo, only acrylamide (50 mg/kg b. wt./day, 8 days) caused paralysis of hind limbs and suppressed CK activity in cerebrum, cerebellum, spinal cord and muscle in rats. Bis-acrylamide (50 mg or 100 mg/kg b. wt./day, 8 days) caused no paralysis nor inhibition of CK activity in any tissue examined. Definite inhibition of CK was not found in sciatic nerve from rats given either chemical. The inhibition of CK may play a role in the genesis of toxicity of acrylamide especially in the central nervous system.