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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 .

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.

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.

Previous studies in our laboratory have shown that L-carnitine suppresses seizures and alterations of brain energy metabolism in mice caused by hyperammonemia. The present study was done to exclude the effects of seizures on brain energy metabolism. When sublethal dose of ammonium acetate (12 mmol/kg b.wt.) was injected to mice, all mice survived without developing seizures, while clear increase of brain ammonia and alterations of brain energy metabolites were seen. In L-carnitine-treated animals, the levels of ammonia, AMP and lactate were lower and those of ATP and phosphocreatine were higher than in untreated animals. Treatment with D-Carnitine also preserved the phosphocreatine level. This indicates that the improvement of brain energy metabolism by L-carnitine in hyperammonemia is not simply a result of the suppression of seizures, and that the “physiological” function of carnitine may not be the sole mechanism underlying this effect.

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.

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

Brains of the newly discovered neurological mutant of the mouse, twitcher, were analysed from birth to the terminal stage of the disease for major tissue constituents and the lipid composition. A genetic deficiency of galactosylceramidase is the underlying cause of this mutant. The affected mice failed to gain body weight after 20 days (30 per cent of normal at 42 days) but the brain weight was much less affected (85 per cent of normal at 42 days). The water content and the four major fractions, chloroform-methanol insoluble residue, chloroform-methanol soluble protein, upper phase solids and total lipid, were essentially unaltered except that the total lipid was reduced slightly towards the terminal stage. Among lipids, only galactolipids, galactosylceramide and sulphatide were abnormal in the twitcher brain. Galactosylceramide was decreased at 37 and 42 days whereas decrease in sulphatide occurred earlier from 25 days, resulting in an increased ratio of galactosylceramide to sulphatide. The analytical abnormalities found in the twitcher brain are qualitatively similar to and quantitatively milder than those in the brain of human patients with globoid cell leukodystrophy (Krabbe disease), of which the twitcher is an enzymatically authentic animal model. The secondary abnormalities observed in brains of human patients as the result of tissue devastation were generally not present in the twitcher brain. Mouse brains contain relatively little myelin. As the pathology in twitcher is greatest in the spinal cord and brainstem (Duchen el al ., 1980), the fact that the changes in twitcher brains are quantitatively milder than those of human patients may partly reflect the relative amount of myelin in the two species. While more detailed studies of some specific constituents, such as galactosylsphingosine, and of other tissues, such as the peripheral nervous system and the kidney, will have to be performed, the present results serve as the reference for comparison between the human and murine galactosylceramidase deficiency states and for future experiments with the twitcher mutant, which is an invaluable tool for studies of globoid cell leukodystrophy.

UDP-galactose:ceramide galactosyltransferase activity was assayed in the spinal cord and kidney of the recently discovered neurological mutant, the twitcher mouse, which is an enzymatically authentic model of human globoid cell leukodystrophy (Krabbe disease). The activity in the spinal cord was essentially normal during the early myelination period up to 15 days. There was a slight reduction at 20 days. At 25 and 33 days, the galactosyltransferase activity was drastically reduced compared to controls. In contrast, the galactosyltransferase activity in the kidney of twitcher mice remained normal throughout the developmental stages examined. Activity of the control enzyme UDP-glucose:ceramide glucosyltransferase was always normal in both the spinal cord and kidney. Thus, reduction of galactosylceramide synthesis occurs in the CNS secondarily to the pathological alteration of the oligodendroglia. No such reduction occurs in the kidney, at least for the last step of galactosylceramide synthesis. Reduced synthesis as the result of metabolic regulation in the presence of the catabolic block is therefore unlikely to be the cause of the lack of abnormal accumulation of galactosylceramide in the kidney of patients with globoid cell leukodystrophy.

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.

The kidney tissue of the twitcher mice, a neurological mutant caused by a genetic deficiency of galactosylceramidase, contains enormously increased amounts, up to 50 times normal, of galactosylceramide. The finding is in sharp contrast with those in the enzymatically equivalent human disease, globoid cell leukodystrophy (Krabbe disease), in which no specific abnormal accumulation of galactosylceramide occurs despite the same genetic block in the catabolic pathway. This indicates that the same genetic defect can result in entirely different consequences in different species. Caution must be exercised even when "authentic animal models" are utilized for studies of human diseases.

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.

The glycolipid composition of the spinal cord, sciatic nerve, kidney, liver and lung of twitcher mice, neurological mutants caused by a genetic deficiency of galactosylceramidase activity, was analyzed during development. No abnormal increase of galactosylceramide or sulfatide was detected in the spinal cord and the sciatic nerve at any age. Galactosylceramide and sulfatide in the whole tissue were in fact decreased in the spinal cord toward later stages of the disease. These findings were qualitatively similar but quantitatively milder than those in the genetically and enzymatically equivalent human disease, globoid cell leukodystrophy (Krabbe disease). Lactosylceramide was abnormally increased in both the spinal cord and the sciatic nerve, but glucosylceramide was increased only in the sciatic nerve. In contrast, there was a massive accumulation of galactosylceramide in the systemic organs. The hydroxy-fatty acid-containing (HFA-) galactosylceramide was predominant in all organs. In the kidney at 42 days. HFA-galactosylceramide was 50-fold and the non-hydroxy-fatty acid-containing (NFA-) galactosylceramide 5-fold higher than the normal levels. Although the absolute amount of galactosylceramide in the liver and lung was much smaller, it was also increased greatly in the twitcher mouse. Lactosylceramide was slightly to moderately increased in the systemic organs but there was no abnormality in glucosylceramide. The analytical findings in the twitcher kidney were in sharp contrast to those in the human disease, in which no abnormal accumulation of galactosylceramide occurs. The abnormal accumulation of the substrate for the deficient enzyme is more consistent with the concept of "lysosomal storage disease". Even though the underlying genetic defect is the same, its consequences show clear species differences.

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.

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.

Progressive accumulation of a cytotoxic metabolite, galactosylsphingosine (psychosine), was found in the brain of the twitcher mouse, a mutant caused by genetic deficiency of galactosylceramidase. Similar abnormal accumulation was also found in the brain of the genetic galactosylceramidase deficiency disease in the dog and in human patients (globoid cell leukodystrophy or Krabbe disease). Galactosylphingosine was absent in the brains of normal and heterozygous mice. The finding provides support for the psychosine hypothesis as the biochemical pathogenetic mechanism of globoid cell leukodystrophy. Analogous mechanisms may be important in the pathogenesis of other genetic lysosomal diseases.

All five members of a family developed subacutely mental confusion and/or truncal ataxia. Symptoms and signs of polyneuropathy were seen later. The well water in the patients' home contained 400 ppm acrylamide. The present cases are unique in that they are cases of acrylamide poisoning induced by oral intake and percutaneous penetration, and that central nervous system symptoms were prominent.

A sensitive and specific analytical procedure has been developed for determination of galactosylsphingosine (psychosine) in the brain. The method takes advantage of two unusual properties of psychosine--the strong positive charge and the reactivity of the free amino group. It involves lipid extraction, separation from other lipids on a cation-exchange column (AG-50W), elimination of the last trace of galactosylceramide by silicic acid chromatography, dansylation of psychosine, Florisil and DEAE-Sephadex chromatography, and finally, fluorescent densitometry of dansylated psychosine separated by thin-layer chromatography. The detection limit is 5-10 ng/100 mg brain tissue when the standard procedure is followed exactly. Reliable determination can be made for 50 ng/100 mg or higher in the presence of 200,000-fold excess of other lipids and in the presence of 40,000-fold excess of galactosylceramide. The sensitivity can be increased fivefold by using a larger aliquot for the final determination. This analytical procedure has been successfully applied to demonstrate abnormal accumulation of psychosine in the brain in human, canine, and murine genetic galactosylceramidase deficiencies (globoid cell leukodystrophy).

Acrylamide administration to rats caused clear alteration of blood thiamine content, which appeared to reflect suppressed intake and/or utilization of the vitamin in the body. Decreased utilization of thiamine does not seem to be induced by decreased binding of thiamine pyrophosphate (TPP) and apotransketolase, because acrylamide did not affect the Km of TPP for transketolase activity.