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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.

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.

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

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

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

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.

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

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.