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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.

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.

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.

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.

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.

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 (

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.

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.

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.

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.

Acetylcholinesterase (AchE) in erythrocytes is one of the typical extraneural AchEs. Its activity is high in human erythrocytes. It has been known that the enzyme is present only in the membrane in erythrocytes and that it is localized on the outer side of the membrane. Recent studies have disclosed much of the primary structure of AchE and the membrane anchor structure. However, the physiological functions of erythrocyte AchE are still totally unknown. Nevertheless, the enzyme may be regarded as a model of AchE in the nervous system. In addition, it may be used to examine the status of the erythrocyte membrane. The enzyme activity seems useful as an indicator of the effects of AchE inhibitors, such as pesticides. It should be noted, however, that the activity of AchE in erythrocytes is not always a good indicator of intoxication with AchE inhibitors.

In vitro, both acrylamide and acrylic acid inhibited creatine kinase (CK) activity in rat brain homogenates, and acrylic acid was more potent than acrylamide. In vivo, however, when given i.p. 50 mg/kg per day for 8 days to rats, only acrylamide inhibited CK activity in the brain and caused apparent neurological signs. 14C in the brain 24h after the injection of 14C-labelled chemicals was more than 7 times greater with acrylamide than with acrylic acid. The inhibition of CK activity by acrylamide varied in eight regions of the brain; from 54% in hypothalamus to 27% in cerebellar vermis. The regional difference of CK inhibition, however, did not agree well with either 14C distribution or with the distribution in regions which appear clinically or pathologically vulnerable to acrylamide.

The effects of methylmercury chloride (MMC) on creatine kinase (CK) activity in the rat brain were studied. Male Wistar rats were injected subcutaneously with 10 mg MMC/kg body weight/day for 7 consecutive days, and sacrificed on the 15th day when all rats showed a crossing phenomenon of the hind limbs. CK activity was mildly inhibited in the anterior, mid and posterior cerebral cortex. Aspartate aminotransferase and lactate dehydrogenase activities were also inhibited in some parts of the cerebral cortex, almost to the same extent as the CK activity. No definite inhibition of the enzyme activity was found in the striatum and the cerebellum. From this study we concluded that the mild inhibition of CK activity does not seem to play an important role in the genesis of neurotoxicity of MMC.

On applying [3H]galactosylsphingosine (psychosine) and albumin to a gel filtration column, either as a pre-incubated mixture or as separate solutions, the lipid and albumin co-eluted. When albumin and [3H]galactosylsphingosine were incubated with Sephadex G-50, the concentration of galactosylsphingosine outside the gel increased as the concentration of albumin increased. These observations indicate that albumin binds galactosylsphingosine. By using an equilibrium gel procedure, it was estimated that one molecule of albumin has 7.4 binding sites for galactosylsphingosine, and that the dissociation constant is 3.9 X 10(-6) M. These values appear compatible with the potency of albumin to suppress galactosylsphingosine-induced hemolysis.

Exposure of rats to 500 ppm ethylene oxide for six hours a day, three times a week, for 12 weeks, lowered serum creatine kinase activity by more than 40%. The only other change was a slightly decreased triglyceride concentration. After four weeks of exposure, neither aspartate aminotransferase nor lactate dehydrogenase activity in brain, spinal cord, and muscle was affected but creatine kinase activity was clearly inhibited. In vitro, ethylene oxide inhibited creatine kinase activity in brain homogenate and in a purified muscle enzyme preparation. Dithiothreitol did not counteract the effect of ethylene oxide. Though the amount of sulphydryl groups in purified creatine kinase was decreased considerably by exposure to ethylene oxide, the enzyme still showed moderate activity. Thus ethylene oxide inhibits creatine kinase activity in vivo and in vitro and the inhibition appears to be unrelated to the disruption of sulphydryl groups in the enzyme.

Sir, Erythrocytes have acetylcholinesterase (AchE), and its activity is often used as an indicator of intoxication with cholinesterase inhibitors. The physiological function(s) of AchE in erythrocytes, however, have not been clarified. The enzyme is entirely membrane bound and located in the outer side of the membrane (see Igisu et al. 1981). On the other hand, the outer leaflet of the erythrocyte membrane is rich in choline-containing phospholipids, i.e., phosphatidylcholine and sphingomyelin (Rothman and Lenard 1977). Hence, one may speculate that AchE might play a role in the metabolism of phospholipids of the membrane, especially those containing choline. Furthermore, in the brain, cholinergic activity and phosphatidylcholine content appears related (Buyukuysal and Wurtman 1990). We therefore examined whether or not repeated exposure to a potent inhibitor of cholinesterase (fenitrothion) affects phospholipid composition of erythrocyte membrane. Male Wistar rats weighing 150-190 g were used. The animals were exposed to fenitrothion (Sumithion) powder for 2 h a day, 5 times a week for 4 -6 weeks. The fenitrothion powder was generated by an apparatus which can produce powder with a fairly constant concentration over a long period (Tanaka and Akiyama 1984). The exposure concentration, aerodynamic median diameter of the powder, and geometric standard deviation were 12.9+ 17.9 (mg/m3, mean _+ SD), 4.9 Bm, and 2.3, respectively. Three hours after the final exposure, blood was obtained by cardiac puncture under ether anesthesia. Preparation of the erythrocyte membrane and measurement of AchE activity were done as described previously (Igisu et al. 1981 ). Lipids were extracted by the method of Folch et al. (1957) and partitioned with water. Lower-phase lipid was applied to a high performance thin-layer chromatogram (HPTLC) plate (Merck 5641) and developed in a mixture of chloroform-methanol-water (65 : 25 : 4, v/v/v). After visualization by spraying cupric acetate and heating (Fewster et al. 1969), the plate was scanned by a Shimadzu Fig. 1. Thin-layer chromatograms of lipids of erythrocyte membrane from rats of control (1), 4-week exposure (2) and 6-week exposure to fenitrothion (3). Spots of cholesterol (Ch), ethanolamine phospholipids (E), phosphatidylcholine (Pc), serine phospholipid (+ phosphatidylinositol) (S) and sphingomyelin (Sp) are seen

Erythrocyte membrane in myotonic dystrophy (MyD) was studied with respect to acetylcholinesterase (AchE), an enzyme localized on the external surface of the membrane. The activity was determined over the temperature range of 7–41 °C (in hemolysates) and 25–41 °C (in ghosts). The activity, the transition temperature in the Arrhenius plots, and the activation energy either above or below the transition temperature did not differ between MyD and controls. The Hill coefficient for the inhibition by fluoride was approximately 1 in MyD and in controls at 13, 25 and 37°C. The dose of fluoride for 50% inhibition of the enzyme activity differed between different temperatures but not between MyD and controls. There seems to be no gross abnormality in AchE or its environment in erythrocyte membrane in MyD.

Pi uptake and acetoacetate formation were suppressed by psychosine (galactosylsphingosine) in rat liver mitochondria. Besides, reduced form of cytochrome c increased in the reaction mixture which contained psychosine. Using reduced form of cytochrome c as substrate, less than 5 μM of psychosine (0.1 μmoles/mg of mitochondrial protein) inhibited cytochrome c oxidase by more than 50%. The inhibition was completely reversed by 1% human serum albumin. Thus, a lipid which is produced in the brain has a powerful and yet reversible inhibitory effect on an enzyme of cellular respiration.

Galactosylsphingosine, glucosylsphingosine and sphingosine all inhibited cytochrome c oxidase activity in mitochondria from rat liver; more than 50% inhibition was caused by 5 microM lipid (0.1 mumol/mg mitochondrial protein). However, these lysosphingolipids did not suppress the activity of purified cytochrome c oxidase. When the enzyme was "reconstituted" with phosphatidylcholine, the lysosphingolipids clearly inhibited the activity. On the other hand, galactosylsphingosine, glucosylsphingosine and sphingosine all hemolyzed erythrocytes, indicating that lysosphingolipids can disrupt the membrane. Thus, it appears that the inhibition of cytochrome c oxidase, a membrane-bound enzyme in mitochondria, is due to perturbation of the environment of the enzyme and that the primary attacking site of the lysosphingolipids is the membrane. Because the potency to inhibit cytochrome c oxidase and to hemolyze erythrocytes did not differ among these lysosphingolipids and because galactosylceramide caused neither inhibition of cytochrome c oxidase nor hemolysis, the free amino group in the lysosphingolipids seems to be essential to give the effects. In addition, both inhibition of cytochrome c oxidase and hemolysis caused by lysosphingolipids were completely abolished by albumin, suggesting that toxic effects of lysosphingolipids may not be apparent in blood.

Effects of repeated exposure to Fenitrothion (Sumithion) powder on erythrocyte membrane acetylcholinesterase (AChE) and plasma cholinesterase (PChE) were studied. Rats (Wistar, male) were exposed to fenitrothion powder for 2 hrs/day, 5 days/week for up to 3 months. The suppression of AChE and PChE increased as the exposure period and exposure concentration increased, although no suppression of AChE was found after a single 4-hr exposure. The persistence of AChE inhibition after the termination of exposure increased with the increase of the exposure period and was longer than that of PChE. Therefore, the AChE activity seems to be useful in examining the response to the repeated exposure to fenitrothion powder.

Propranolol enhanced utilization and generation of ATP in erythrocytes while ouabain suppressed both of them. After incubation of erythrocytes with propranolol, no change was noted in membrane ATPase activity while incubation with ouabain caused suppression of membrane ATPase. The effect of ouabain on erythrocyte ATP and lactate content is compatible with its inhibiting action of membrane ATPase activity. Propranolol may activate ATP-utilizing system(s) in the cytosol, or it may activate membrane ATPase activity only when combined with factor(s) lost during the preparation of the membrane. The above effect of propranolol may be unrelated to the β-adrenergic receptor blocking action since isoproterenol did not affect the erythrocyte ATP or lactate content and it did not counteract propranolol.

Effects of fenitrothion (Sumithion) powder on red cell acetylcholinesterase (AChE) and plasma cholinesterase (PChE) were studied in vitro and in vivo. Experiments in vitro revealed that fenitrothion has almost the same potency to inhibit AChE (in pre-washed red cell membrane) and PChE. Rats (Wistar, male) were exposed to fenitrothion powder for 4 hours using a new apparatus which can generate powder constantly over a long period. In this experiment, however, no apparent suppression of AChE was found while PChE was markedly inhibited. This inhibition persisted for 3 days after the exposure. This discrepancy was interpreted to be due to the inevitable procedure in preparing red cell specimens, i.e., washing of the red cells. After acute exposure in vivo, fenitrothion appears to be easily removed from the red cells by washing. Thus, PChE, rather than AChE, seems to be a good indicator of an acute exposure to fenitrothion powder in vivo.

Effects of taurine on the total ATPase activity inhibited by ouabain were investigated in the human erythrocyte membrane. The enzyme activity was not activated by taurine in the absence of calcium, but was significantly activated by taurine (15–60 mM) in the presence of calcium (5 mM) by which the enzyme is inhibited. Since taurine does not form a chelate complex with calcium ion, this activation may be due to an action of taurine in the presence of calcium through competition with these ions on the membrane.

We studied the Na+ + K+ ATPase activity of erythrocyte membranes from patients with Duchenne muscular dystrophy (DMD). Samples from patients and controls were obtained at almost the same time on the same day and processed simultaneously. Difference of anticoagulants, extent of washing the red cells, mechanical or osmotic disruption of the cells, presence or absence of EDTA in the washing solution of the ghosts, intentional removal of the peripheral proteins of the membranes, addition of DMD plasma to the assay system, and different temperatures of membrane storage caused no significant differences between DMD and control in Na+ + K+ ATPase or in response of the enzyme to ouabain.