Hideji Tanii

Studies on the mechanism of acute toxicity of nitriles in mice.

Acute toxicity and metabolism of 21 nitriles in mice were studied in relation to their chemical structures. All the test nitriles liberated cyanide ions both in vivo and in vitro, with the exception of benzonitrile, although the extent of liberation and the effect of carbon tetrachloride (CCl4) pretreatment on the mortality of animals differed among nitriles. From these results, test compounds were tentatively divided into three groups. In group 1 (13 compounds), acute toxicity was greatly reduced by CCl4 pretreatment, in group 2 (seven compounds), toxicity was not significantly changed or was somewhat enhanced, and in group 3, benzonitrile only, toxicity was clearly enhanced. The amount of cyanide was higher (0.68–0.80 μg CN/g brain) at death in the brains of mice given group-1 compounds, the level being comparable to that found in mice killed by dosing with potassium cyanide. After oral doses of each nitrile, the time course for cyanide levels in the liver varied among the compounds. The difference between group-1 and -2 compounds lay in the dose-cyanide liberation relationship in liver, and in the kinetics for cyanide liberation in the hepatic microsomal enzyme system. Double-reciprocal plots of enzyme activity showed a linear relationship for nitriles of group 1 and a non-linear one for group 2. The relationship between log (1/LD50) and log P for the compounds in group 1 fitted a parabolic plot, while that for compounds in group 2 was linear.

Neurotoxicity of acrylamide and related compounds and their effects on male gonads in mice

Neurotoxicity of acrylamide and related compounds and their effects on the testis after repeated oral doses were studied in mice. Of fourteen analogues tested, five produced neuropathy. In decreasing order of potency as assessed by the rotarod performance test, these were as follows: acrylamide > N-isopropylacrylamide > N-methylacrylamide = methacrylamide > N-hydroxymethylacrylamide. The development of neurotoxicity was either greatly reduced or delayed by phenobarbital treatment. Acrylamide, N-hydroxymethylacrylamide, N-isopropylacrylamide, N-methylacrylamide and N,N′-methylene-bis-acrylamide produced testicular atrophy. Atrophy was either prevented by phenobarbital treatment, as in the cases of acrylamide and N-isopropylacrylamide, or reduced, as in the case of N-hydroxymethylacrylamide. Histological changes in the testis produced by the active compounds were degenerations of the epithelial cells of the seminiferous tubules, with the interstitial cells being normal.

Neurotoxicity of acrylamide and related compounds in rats. Effects on rotarod performance, morphology of nerves and neurotubulin.

Neurotoxic properties of acrylamide and seven related compounds in rats were studied with regard to the effects on rotarod performance, morphology of nerves and neurotubulin. Compounds used in the present study were acrylamide, N-hydroxymethylacrylamide, N-isopropylacrylamide, methacrylamide, N-methylacrylamide, crotonamide, diacetone acrylamide, and N-tert-butylacrylamide. Animals were given chemicals in their drinking water for 90 days. Deficit of rotarod performance was produced by five compounds; acrylamide, N-hydroxymethylacrylamide, N-isopropylacrylamide, methacrylamide, and N-methylacrylamide. Morphological changes in tibial and sural nerves, such as shrinkage and loss of myelinated fibres, myelin retraction, and corrugated myelin sheaths, were observed after treatment with these five compounds. Depression of the [3H]colchicinebinding to neurotubulin (the soluble protein) of sciatic nerves was detected after giving these five compounds. After acrylamide dosing, the depression progressed with time. A significant reduction of the colchicine-binding to neurotubulin was also detected in the spinal cord of both the cervical and the lumbar regions, but neither in the brain nor the cerebellum.

The influence of neuronal cells on the development of glutamine synthetase in astrocytes in vitro

The influence of neurons on the development of astroglial cells was examined in vitro using glutamine synthetase (GS) activity as an index of metabolic maturation. The GS activity in forebrain astrocytes was significantly increased (about 70%) when they were co-cultured with forebrain neuronal cells. A similar effect was also observed when astrocytes from the immature septum, hippocampus or cerebellum were co-cultured with neurons derived from the septal-diagonal band region. The magnitude of the effect was not uniform; the cerebellar astrocytes, with relatively low GS activity, showed a greater (about 290%) quantitative response to the subcortical nerve cells than did the septal (about 115%) or the hippocampal (about 120%) astroglial cells. The addition of conditioned medium derived from neuronal cultures or plating the cells on a substratum of heat-killed nerve cells, elevated the GS activity of astroglial cells by 33% and 39%, respectively. Our results indicate that a trophic factor secreted by neurons and direct contact with the nerve cell matrix, are both involved in the regulation of the differentiation of astrocytes.

Allylnitrile: A compound which induces long-term dyskinesia in mice following a single administration

A single oral dose of allylnitrile (ALN) in mice pretreated with CCl4 induced behavioral abnormalities such as circling, hyperactivity, and head twitching, which lasted for a 4-month observation period. Histopathologically hemorrhage, demyelinated fibers and necrotic neurons were observed in the midbrain and pons 40 to 50 days after the administration of ALN. The head twitching was either reduced by treatment with serotonin and dopamine antagonists or enhanced by a serotonin releaser, suggesting that both serotonin and dopamine systems are involved in the behavioral abnormalities by ALN. These disorders by ALN may be used as an animal model of the dyskinetic syndrome.

Influence of ethanol on the in vivo and in vitro metabolism of nitriles in mice.

The effect of ethanol on metabolism of 20 nitriles was studied in vivo and in vitro in mice. The hepatic microsomal metabolizing activity for nitriles was at a maximum 13 h after ethanol dosing (4.0 g/kg). Using microsomes from mice pretreated with ethanol under the above conditions, enhancement of the in vitro metabolism of nitriles was 1.00–1.83 compared with the glucose-treated control. When mice were orally given nitriles 13 h after dosing with either ethanol (4.0 g/kg) or glucose (7.0 g/kg), the hepatic metabolizing activity of nitriles for the ethanol-treated group was always higher than that for the glucose group, although no change in the content of hepatic microsomal P-450 was observed between the two groups. However, ethanol added to the incubation mixture inhibited the in vitro metabolism of most nitriles. The results in the present study suggest that ethanol can enhance the acute toxicity of nitriles.

Studies on in vitro metabolism of acrylamide and related compounds

The in vitro biotransformations of acrylamide and ten related compounds in the hepatic enzyme system of the mouse were studied in order to learn more about their toxic actions in vivo. Of nine analogues, which could be analyzed quantitatively by gas chromatography, seven compounds — N-tert-butylacrylamide, diacetone acrylamide, N,N-dimethylacrylamide, N-isobutoxymethylacrylamide, N-isopropylacrylamide, methacrylamide, and N-methylacrylamide — were metabolized in microsomal enzymes with a NADPH generating system. One or two metabolites from each of the seven compounds, except for N-isobutoxymethylacrylamide, were detected by gas chromatography. The metabolite of N-isopropylacrylamide was identified as acrylamide by gas chromatography-mass spectrometry. The metabolite of N,N-dimethylacrylamide showed a RT value identical with and a mass spectrum similar to N-methylacrylamide. No metabolites from the other four compounds have yet been identified. Acrylamide and crotonamide did not seem to be metabolized in the same system. Phenobarbital pretreatment of mice enhanced the metabolic reactions of the seven compounds, but did not elevate those of acrylamide and crotonamide. The Km value of N-isopropylacrylamide was 0.35 mM, which was the smallest of all the test analogues. All of the eleven analogues studied were found to be metabolized by hepatic glutathione S-transferases as well. This reaction was also elevated by the phenobarbital treatment of mice. The relationships between the in vitro metabolisms and the in vivo toxicities of acrylamide analogues are discussed.

Reduction in the local expression of complement component 6 (C6) and 7 (C7) mRNAs in oesophageal carcinoma.

Differential displays of tumour/normal pair specimens of human oesophagus identified complement component 7 (C7) as being enhanced in normal tissues, but remarkably reduced in carcinoma tissues. In situ hybridisation confirmed the localisation of C7 mRNA in normal oesophageal epithelial cells and its disappearance in tumour cells. When mRNA expressions of other components were examined by means of semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in 10 tumour/normal pair specimens, significant reductions in C6 and C7 mRNAs were observed, while C3 and C5 mRNAs were enhanced in both normal and tumour tissues. A similar reduction was observed in colon and kidney cancers using the tumour/normal expression array analysis. Gene deletion of C7 was not found in the cell lines by Southern blot analysis. Our findings suggest a possible relationship between oesophageal tumorigenesis and reduced expression of C6 and C7 mRNAs, which is probably caused by a change in gene expression regulation and not by genetic loss of the locus.

Behavioral syndrome induced by allylnitrile, crotononitrile or 2-pentenenitrile in rats

A single oral administration of allylnitrile, crotononitrile or 2-pentenenitrile in rats induced behavioral abnormalities, such as head-twitching, head weaving, hindlimb abduction, backward pedaling and pivoting. The head-twitching, which was most consistently observed, was suppressed by serotonin (5-HT) antagonists, cyproheptadine or methysergide or by the 5-HT depleter, dl-p-chlorophenylalanine but was accentuated by the 5-HT releaser, dl-p-chloroamphetamine. The results suggest that the 5-HT system is involved in producing the behavioral abnormalities. To discover the effects of allylnitrile, crotononitrile and 2-pentenenitrile on the metabolism of 5-HT and dopamine, 6 areas of the brain of the rat were examined on days 1, 6, 15 and 30 after injection. Each of the nitriles caused significant increases in the level of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) and in the ratio of 5-HIAA/5-HT, one day after injection. The increase in 5-HIAA was most remarkable, suggesting an enhancement of the serotonergic system. The three nitriles had no effect on the metabolism of dopamine, over a period of 30 days.

Neurofilament degradation in the nervous system of rats intoxicated with acrylamide, related compounds or 2,5-hexanedione

Degradation of neurofilament (NF) proteins by Ca2+-activated neutral protease (CANP) was studied in the nervous system of rats treated with neurotoxic or non-neurotoxic compounds. In the tibial nerve, the degradation of NF 68K was depressed by five neurotoxic compounds: acrylamide, N-hydroxymetylacrylamide, N-isopropylacrylamide, methacrylamide and 2,5-hexanedione. A non-neurotoxic compound, diacetone acrylamide, did not show any effect on the degradation. An immunoblot analysis confirmed the reduction in the degradation and revealed a difference in the degradation pattern between the control and acrylamide-treated rats. In the spinal cord, the degradation of the three subunits of NF was depressed in animals treated with acrylamide. Although the exact mechanism of the reduction in the degradation of NF is not yet known, the present results suggest that an inhibitory effect on CANP activity might be relevant to the mechanism of neurotoxic action of acrylamide derivatives.