Kiyoshi Ameno

Regional brain distribution of toluene in rats and in a human autopsy.

Toluene concentrations in 9 brain regions of acutely exposed rats and that in 11 brain regions of a human case who inhaled toluene prior to death are described. After exposure to toluene by inhalation (2000 or 10000 ppm) for 0.5 h or by oral dosing (400 mg/kg), rats were killed by decapitation 0.5 and 4 h after onset of inhalation and 2 and 10 h after oral ingestion. After each experimental condition the highest range of brain region/blood toluene concentration ratio (BBCR) was in the brain stem regions (2.85–3.22) such as the pons and medulla oblongata, the middle range (1.77–2.12) in the midbrain, thalamus, caudate-putamen, hypothalamus and cerebellum, and the lowest range (1.22–1.64) in the hippocampus and cerebral cortex. These distribution patterns were quite constant. Toluene concentration in various brain regions were unevenly distributed and directly related blood levels. In a human case who had inhaled toluene vapor, the distribution among brain regions was relatively similar to that in rats, the highest concentration ratios being in the corpus callosum (BBCR: 2.66) and the lowest in the hippocampus (BBCR: 1.47)

Acute ethanol decreases NPY mRNA but not POMC mRNA in the arcuate nucleus.

The acute effects of ethanol and acetaldehyde on neuropeptide mRNA expression in the arcuate nucleus (ARC) was assessed. Acetaldehyde was increased in blood following ethanol with cyanamide (a potent inhibitor of aldehyde dehydrogenase) administration. Acetaldehyde is a toxin which can cause a variety of adverse effects following ethanol ingestion in some Oriental people with a genetic lower activity of aldehyde dehydrogenase. Neuropeptide Y (NPY) mRNA levels in ARC were significantly decreased in response to ethanol in the presence or absence of cyanamide compared to control. In contrast, proopiomelanocortin mRNA in ARC was not changed. These novel findings suggest that ethanol suppresses NPY gene expression in ARC and may play a role in ethanol-induced changes in neuronal function.

Analysis of paraquat, diquat and two diquat metabolites in biological materials by high-performance liquid chromatography

The determination of paraquat, diquat and two metabolites of diquat in biological materials was developed using high-performance liquid chromatography combined with UV and fluorescence detectors. Paraquat, diquat and internal standards (ethyl viologen and o-acetamidophenol) were detected by the UV detector. Diquat-monopyridone and diquat-dipyridone were monitored by the fluorescence detector. Paraquat, diquat, diquat-monopyridone and ethyl viologen were effectively extracted from blood, liver and brain, using a Sep-Pak C(18) cartridge. Diquat-dipyridone and o-acetamidophenol were extracted with methanol. Paraquat and diquat at a concentration range of 0.1-10 microg/ml (or g), and diquat-monopyridone and diquat-dipyridone at a concentration range of 0.01-1 microg/ml (or g) in biological material were determined with high accuracy and precision. The detection limits of paraquat, diquat, diquat-monopyridone and diquat-dipyridone were 1, 1, 0.02 and 0.02 ng, respectively, as an injection amount. This method was applied for toxicological examination of a case of suspected paraquat and diquat intoxication. The analysis of the metabolites of diquat was helpful for the estimation of the elapsed time from ingestion to death.

In Vivo Study of Salsolinol Produced by a High Concentration of Acetaldehyde in the Striatum and Nucleus Accumbens of Free-Moving Rats

BACKGROUND Salsolinol, a neuropharmacologically active compound, is formed by the condensation of acetaldehyde (AcH) with dopamine (DA) in the brain. The aim of our study was to examine the effect of a high concentration of AcH on salsolinol formation and to compare the release of DA, serotonin (5-HT), and salsolinol in the striatum and nucleus accumbens (NAc) in free-moving rats. METHODS After the insertion of a microdialysis probe, male Wistar rats (250-300 g) were treated with cyanamide (CY, a potent aldehyde dehydrogenase inhibitor) + ethanol (EtOH), CY + 4-methylpyrazole (4-MP, a strong alcohol dehydrogenase inhibitor) + EtOH, 4-MP + EtOH, CY, and 4-MP. Simultaneous quantitation of DA, 5-HT, and salsolinol in dialysates was performed by using in vivo microdialysis coupled with high-performance liquid chromatography with an electrochemical detector and blood EtOH and AcH by using a head-space gas chromatographic method. RESULTS Salsolinol was detected only in the CY + EtOH groups in both the striatum and NAc, and we also detected a high AcH concentration in the blood in those groups. A correlation was found between the dialysate levels of salsolinol and blood concentrations of AcH. The striatal levels of DA and 5-HT were approximately two times higher, whereas salsolinol levels were approximately three times higher compared with the usual level in the NAc. No significant difference of DA and 5-HT levels in the dialysates was observed in either the control or the other study groups. CONCLUSION Our observation suggested that the brain salsolinol formation may depend on the concentrations of DA and AcH in freely moving rats, and there is no effect of a high concentration of AcH on DA and 5-HT levels in the striatum and NAc.

Acetaldehyde adducts in the brain of alcoholics

Acetaldehyde binds to some proteins and becomes a Schiff base. It is assumed that after the consumption of ethanol the acetaldehyde binds to the proteins to form adducts, and such acetaldehyde adducts are associated with organ diseases. We investigated the detection of acetaldehyde adducts in the brain region of a human alcoholics. Brain samples collected from an alcoholic autopsied case were used. Determination of acetaldehyde adducts was performed using a fluorescence immmunohistochemical staining method with antibodies against acetaldehyde adducts. We demonstrated acetaldehyde adducts in the frontal cortex and the midbrain of an alcoholics. Our studies showed that an acetaldehyde adduct was produced in the brain of alcoholics.

Blood carbofuran concentrations in suicidal ingestion cases

We describe four fatal cases due to ingestion of carbofuran, a carbamate insecticide. Carbofuran was detected in the gastric contents using thin layer chromatography (TLC) and gas chromatography/mass spectrophotometry (GC/MS), and quantified in the blood using a gas chromatograph equipped with nitrogen-phosphorus detector (NPD). Fatal concentrations of carbofuran in blood ranged from 0.32 to 11.6 microg/ml.

Short-term ethanol exposure alters calbindin D28k and glial fibrillary acidic protein immunoreactivity in hippocampus of mice

The effects of a short-term ethanol treatment on hippocampus have been studied in mice exhibiting intoxication signs. The alterations of neurons and astrocytes as well as quantitative changes of calbindin D28k-immunoreactivity and glial fibrillary acidic protein-immunoreactivity (GFAP-IR) in selected regions of the dorsal hippocampus were examined using anti-calbindin and anti-GFAP monoclonal anti-body (mAb), respectively. The administration of 6% (v/v) ethanol during first week led to the neuronal death and decrease of the total number of calbindin-IR neurons in the examined brain regions. Moreover, the calbindin positive neurons were shown to have diminished processes following short-term ethanol exposure. These neuronal changes were associated with the increase of the GFAP-IR astrocytes. Hypertrophy of cell bodies and cytoplasmic processes of reactive astrocytes were also seen. In addition, dense, thick and highly-stained GFAP-IR cells with long processes in granular cell layer appeared entering into molecular layer of dentate gyrus. In agreement with the discrepancy percentage of neuronal cell loss and increase of reactive astrocytes detected by calbindin and GFAP-IR using image quantitative analysis, the regional differences in the vulnerability to the neurotoxic effects following short-term ethanol exposure were found: CA3>CA2>CA1>DG. These findings also illustrate the importance of correlation between calbindin and GFAP-IR when determining the morphological alteration of neuron and astroglial following short-term ethanol treatment. The disruption of calbindin and GFAP could affect neuronal-astroglial interaction, resulting in disturbance of behaviors dependent on hippocampus.

A fatal case of oral ingestion of toluene

A 51-year-old male ingested orally a large quantity of toluene and died about 30 min later. The presence of toluene in body fluids and tissues was confirmed by gas chromatography and gas chromatography/mass spectrometry. Tissue distribution of toluene showed that the liver detected the highest content of toluene (433.5 micrograms/g), except for the stomach contents, followed by pancreas (88.2 micrograms/g), brain (85.3 micrograms/g), heart (62.6 micrograms/g), blood (27.6 micrograms/g), fat (12.2 micrograms/g) and finally cerebrospinal fluid (11.1 micrograms/g).

Relationship between Alcoholism and CYP2E1 C/D Polymorphism

The genotypes of the CYP2E1 loci of 35 alcoholic and 130 nonalcoholic (healthy controls) Japanese were investigated to determine the relationship between CYP2E1 (C/D) polymorphism and susceptibility to alcohol dependence. There was a significant (d.f. = 1, χ2 = 11.0, p < 0.001) difference in the CYP2E1 C gene frequency between alcohol dependents (0.41) and controls (0.28), suggesting that the C gene of CYP2E1 may be related to the risk of developing alcoholism in Japanese, whereas the frequency of the D gene was higher among controls than alcoholics. Healthy Japanese controls showed a significantly (d.f. = 1, χ2 = 30.1, p < 0.001) higher frequency of the C allele than Swedish healthy controls (0.10) did.

Short-term exposure to ethanol causes a differential response between nerve growth factor and brain-derived neurotrophic factor ligand/receptor systems in the mouse cerebellum.

Alcohol ingestion affects both neuropsychological and motor functions. We hypothesized that one of the key factors involved in such functions are neurotrophins and their receptors. We have therefore examined the effects of short-term ethanol exposure on the mRNA expression and protein levels of neurotrophin ligands and receptors in the cerebellum using real-time RT-PCR and Western blotting techniques. Male BALB/C mice were fed a liquid diet containing 5% (v/v) ethanol. The pair-fed control mice were fed an identical liquid diet except that sucrose was substituted isocalorically for ethanol. The cerebellum of mice exhibiting intoxication signs of stage 1 or 2 were used in the present study. We found that exposure to ethanol resulted in elevated levels of nerve growth factor (NGF) and TrkA mRNA expression but a decreased level of brain-derived neurotrophic factor (BDNF) mRNA expression. The expression of TrkB and p73 mRNA was unchanged. Changes in the level of these proteins were found to mirror these mRNA expression levels. We conclude that exposure to ethanol for a short period can cause a differential responsive in the various neurotrophin ligand/receptor systems. The functional consequences of these changes are unknown at present.