Inhibition of creatine kinase activity in rat brain by methyl bromide gas.

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.

Cadmium induces phosphorylation of p53 at serine 15 in MCF-7 cells.

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.

Involvement of the extracellular signal-regulated protein kinase (ERK) pathway in the induction of apoptosis by cadmium chloride in CCRF-CEM cells.

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.

Inhibition of HgCl2-induced mitogen-activated protein kinase activation by LL-Z1640-2 in CCRF-CEM cells.

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.

Brain of rats intoxicated with acrylamide: observation with 4.7 tesla magnetic resonance.

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.

Activation of mitogen-activated protein kinases by tributyltin in CCRF-CEM cells: role of intracellular Ca(2+).

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.

Hexachlorophene-induced brain edema in rat observed by proton magnetic resonance.

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.

Increased cytotoxicity of cadmium in fibroblasts lacking c-fos.

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)-2H-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 microM 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.

Mercury chloride activates c-Jun N-terminal kinase and induces c-jun expression in LLC-PK1 cells.

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.

Activation of c-Jun NH2-terminal kinase (JNK/SAPK) in LLC-PK1 cells by cadmium.

The level of phosphorylated c-Jun NH2-terminal kinase (JNK) in LLC-PK1 cells treated with CdCl2 increased after 30 min and remained elevated even at 8 hr. And the activity of JNK assayed using glutathione S-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.

Protection from cadmium cytotoxicity by N-acetylcysteine in LLC-PK1 cells.

N-acetylcysteine (NAC) has been known not only to stimulate synthesis of glutathione but also to affect the gene regulation. In our study, effects of NAC on the cytotoxicity of cadmium (Cd) were examined in LLC-PK1 cells. Preincubation and subsequent incubation with 1 mM NAC almost completely suppressed Cd-induced cellular damage evaluated either by trypan blue exclusion or lactate dehydrogenase leakage. This almost complete protection required the presence of NAC during Cd exposure. Treatment with 1 mM NAC increased the intracellular glutathione level approximately 2-fold. Inhibition of this increase by buthionine sulfoximine did not abolish the protection by NAC. One mM NAC also suppressed Cd-induced increase of c-Fos protein although NAC alone did not change the protein content. The inhibition of transcriptions by actinomycin D did not affect the protection by NAC. Thus, NAC-induced protection appeared to be independent of glutathione level or the transcriptional activation of genes including c-fos. However, treatment with NAC markedly lowered the uptake of Cd into cells although it did not affect the efflux clearly. Addition of NAC during the exposure to Cd suppressed Cd-induced cellular damage but the suppression decreased when the duration of the exposure without NAC increased. These results suggest that NAC-induced protection against Cd cytotoxicity is mainly due to the lowered uptake of Cd into the cells.

Effects of pentachlorophenol, pentylenetetrazol and carnitine on mitochondria.

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.

Suppression of pentylenetetrazol-induced seizures by carnitine in mice.

When ddY mice were pretreated with L-carnitine (5, 10 or 20 mmol/kg), clonic as well as tonic seizures induced by pentylenetetrazol (PTZ) were dose-dependently suppressed. A time/response study (PTZ was injected 1, 5, 15 or 30 min after L-carnitine) showed that the anticonvulsive effects were apparent when the interval between L-carnitine and PTZ administration was 15-30 min. Saline containing 43% sucrose prolonged the latency to the first clonic seizure but was less effective than 20 mmol/kg L-carnitine and did not suppress clonic or tonic seizures. Alterations in brain energy metabolites caused by PTZ including increase of lactate and decrease of ATP and phosphocreatine were also suppressed by L-carnitine. L-carnitine was more potent than D-carnitine in prolonging the latency to the first clonic seizure and in decreasing the frequency of clonic as well as tonic seizures. The anticonvulsive effects of L-carnitine in PTZ-induced seizures may be unrelated to the transport of long-chain acyl CoA since they were not interfered with by D-carnitine.

Cooperative inhibition of acetylcholinesterase activities by hexachlorophene in human erythrocytes.

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.