Changes in cholinergic function in the frontal cortex and hippocampus of rat exposed to ethanol and acetaldehyde.

Our previous microdialysis study demonstrated that both ethanol (EtOH) and acetaldehyde (ACe) decrease in vivo acetylcholine (ACh) release in the medial frontal cortex of freely moving rats. To better understand the mechanisms of EtOH and ACe's effects on the cholinergic system in the brain, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) expression was examined at 40 and 240 min after a dose of EtOH (1 g/kg) in the rat frontal cortex and hippocampus. The control group was treated with 0.9% saline, and other groups received EtOH or cyanamide (CY, 50 mg/kg, a potent aldehyde dehydrogenase inhibitor) and 60 min later by EtOH intraperitoneally. Reverse-transcription polymerase chain reaction (RT-PCR) analysis revealed that ChAT mRNA levels were decreased by 72.8% and 71.6% in the EtOH and CY+EtOH groups, respectively, at 40 min after EtOH injection compared with saline in the frontal cortex. The hippocampal ChAT levels were reduced by 76.5% and 53.0% in the EtOH and CY+EtOH groups, respectively, at this time. CY+EtOH-induced depletion in ChAT mRNA levels was markedly higher than EtOH in the hippocampus. A similar decrease pattern of ChAT was observed at protein levels as determined by Western blot, but the reduced ChAT levels were significantly higher in the CY+EtOH group as compared with the EtOH group both in the frontal cortex and hippocampus. At 240 min after EtOH injection, the EtOH group had no effect on ChAT at mRNA levels, as compared with saline, whereas CY+EtOH group induced a significant decrease in ChAT mRNA expression to 62.0% and 65.5% in the frontal cortex and hippocampus, respectively. These data were consistent with the results of the Western blot analysis. AChE expression at mRNA levels was not changed at either 40 or 240 min after EtOH dosing in either of these groups in the frontal cortex and hippocampus. Within 40 and 240 min, a statistically significant difference in ChAT expression at mRNA and protein levels was found in the EtOH and CY+EtOH groups both in the frontal cortex and hippocampus. The data obtained from this study demonstrate that EtOH and ACe concentrations decreased ChAT expression at 40 and 240 min after EtOH administration in the frontal cortex and hippocampus, and this result suggests that reduced ChAT expression is strongly related to a decrease in ACh release in the rat brain.

IgG subclass distributions of anti-horse serum antibodies and natural venom-antibodies produced in response to antivenom injection or snake bite in humans.

The Japanese Mamushi (Agkistrodon halys blomhoffi, BOIE) is the most common snake in Japan. Bite victims treated with antivenom (horse serum) can produce antibodies against the horse serum and the snake venom. We studied distributions of the IgG subclasses of both these antibodies produced in response to antivenom injection and snake bite. We found that IgG1 and IgG4 of each antibody in the victims' serum were present for a long period of time.

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).

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.

Hypothalamo-pituitary-adrenal axis activation by administration of cyanamide: a potent inhibitor of aldehyde dehydrogenase.

In this report, we investigated the effects of cyanamide (a potent inhibitor of aldehyde dehydrogenase (ALDH: EC 1.2.1.3)) on hypothalamo-pituitary adrenal (HPA)-axis using in situ hybridization histochemistry and radioimmunoassay. Cyanamide administration resulted in a dose-dependent increase in plasma corticosterone concentrations, significant increases in not only corticotrophin releasing factor (CRF) mRNA, but also arginine vasopressin (AVP) mRNA in the paraventricular nucleus (PVN) and proopiomelanocortin (POMC) mRNA in the anterior pituitary. These results suggest that cyanamide is able to activate the HPA axis at all levels of the axis.

Acute fatal poisoning cases due to furathiocarb ingestion.

Seven cases involving acute fatalities due to ingestion of furathiocarb, a carbamate insecticide, are presented. Furathiocarb 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 a nitrogen-phosphorus detector (NPD). The fatal levels of furathiocarb in the blood ranged from 0.1 to 21.6 micrograms/ml.

Simultaneous determination of bromvalerylurea, bromodiethylacetylurea, and allylisopropylacetylurea in serum and urine by high-performance liquid chromatography with a multiwavelength UV detector and thin-layer chromatography.

A method for rapid detection and identification of bromvalerylurea (BVU), bromodiethylacetylurea (BDU), and allylisopropylacetylurea (AIU) in serum and urine by high-performance liquid chromatography (HPLC) with a multiwavelength UV detector after Sep-Pak C18 cartridge extraction is reported. A Jasco Finepak C18 reversed-phase column was used for the separation. Acetonitrile-distilled water (1:1, v/v) was used as a mobile phase. There was no significant absorption of the three hypnotics in the UV spectra (210-350 nm). However, the absorption of each was higher at the shorter wavelengths. The quantifications for the three hypnotics detected at 210 nm by the chromatogram were linear over the range 0.2-4 micrograms/mL and the detection limits of BVU, BDU, and AIU were 5, 10, and 10 ng as absolute amounts, respectively. The mean recovery yields of BVU, BDU, and AIU by Sep-Pak C18 cartridge extraction were 85.7 +/- 4.1, 98.6 +/- 2.2, and 95.1 +/- 3.5% (n = 5) in serum and 79.5 +/- 3.8, 95.7 +/- 1.8, and 93.0 +/- 4.2% (n = 5) in urine, respectively. An optimal system of thin-layer chromatography for the identification of the hypnotics is also discussed.

A rapid and sensitive quantitation of Dipterex in serum by solid-phase extraction and gas chromatography with flame thermionic detection.

A simple, sensitive, and rapid quantitative method for Dipterex in serum is described. A SepPak C18 cartridge for the extraction and gas chromatography with flame thermionic detection for determination are used. The detection limit is 2.5 ng/mL, and linearity is obtained in the range 5-500 ng/mL.

A rapid, simultaneous determination of paraquat and diquat in serum and urine using second-derivative spectroscopy.

A rapid, simple method based on second-derivative spectroscopy of the simultaneous analysis of paraquat and diquat in serum and urine is described. Paraquat and diquat in serum were deproteinized with sulfosalicylic acid, and those in urine were reduced with NaOH-dithionite solution. A qualitative and quantitative analysis of reduced paraquat and diquat was made at the amplitude peaks of 396-403 nm and 454-464 nm in the second-derivative spectra, respectively. The entire procedure was completed within about 10 minutes for a serum sample and within about 5 minutes for a urine sample. Application of the proposed method on a poisoned patient is also reported.

A rapid and sensitive quantitation of Amitraz in plasma by gas chromatography with nitrogen-phosphorus detection and its application for pharmacokinetics.

We report a simple, sensitive, and rapid quantitation of Amitraz in plasma after Extrelut-3 column extraction by gas chromatography with nitrogen-phosphorus detection (GC-NPD). The plasma sample was diluted four-fold with borate buffer (0.01M, pH 11), put into an Extrelut-3 column, left for 15 min, and then eluted with 15 mL of n-hexane. The n-hexane eluate was evaporated under nitrogen gas flow at room temperature. The residue was reconstituted with 0.1 mL of acetone containing nitrazepam as an internal standard. A 2-microL aliquot was injected into a wide-bore capillary column GC-NPD. The detection limit was 0.5 ng/mL and linearity was obtained in the range of 1-200 ng/mL. Amitraz in the buffer at pH 11 remained stable in a freezer for one week at -20 degrees C. The GC-NPD method was found useful in studying the pharmacokinetics of a single dose intravenous administration of Amitraz to a dog.

Genetic polymorphism of factor B (Bf) in Okayama Prefecture, Japan.

The genetic polymorphism of factor B (Bf) was investigated in Okayama Prefecture, Japan. Cellogel immunofixation electrophoresis was employed according to Martin and Ziegler (1981) with minor modifications. In 316 non-blood related Japanese, the Bf was: Bf S, 70.6%; Bf FS, 27.8%; and Bf F, 1.6%. No rare variants were observed. The gene frequencies of Bfs and BfF were 0.845 and 0.155, respectively. The gene frequencies in Okayama Prefecture were quite similar to those in other districts of Japan. Considering the phenotype distribution in Japan, the Bf system might be a useful marker for personal identification and in disputed paternity cases.

Lack of association between smoking and CYP2A6 gene polymorphisms in A Japanese population.

This study determined the genotypes of the CYP2A6 gene in 96 smokers and 141 non-smokers in a Japanese population. The frequencies of wild-type of the CYP2A6* 1 and those with a whole deletion of the CYP2A6 gene were 93 (96.9%) and 3 (3.1%) in 96 smokers, and 134 (95.0%) and 7 (5.0%) in non-smokers, respectively. In addition, neither the CYP2A6* 2 nor CYP2A6* 3 alleles were observed in the population studied. There were no significant differences in the CYP2A6 genotype frequencies between smokers and non-smokers, as well as in the number of cigarettes smoked and the nicotine amounts inhaled per day between the CYP2A6* 1 and the deletion of CYP2A6. These results suggest that either the deletion or non-deletion of the CYP2A6 gene shows no significant effect on smoking behavior for the Japanese population examined.

[Relationship between alcoholism and CYP2E1 genotypes].

The genotype of the CYP2E1 loci in 36 alcoholic and 42 non-alcoholic (healthy) Japanese were investigated to examine the relationship between the polymorphisms of CYP2E1 (C/D) and the susceptibility to alcohol dependence. There was a significant (df = 1, chi 2 = 4.39, p < 0.05) difference in CYP2E1 CD (heterozygote) genotype frequency between alcohol dependents (56%) and controls (33%), suggesting that the CD (heterozygote) genotype of CYP2E1 may have something to the risk of developing alcoholism in Japanese, whereas DD (homozygote) genotype was high among controls.

[The distribution of toluene in the brain and its effects on the brain catecholamines in acute toluene poisoning].

The regional distribution of toluene in the brain and its effects on the brain catecholamine levels in rats is reported. Separate sets of rats were exposed to the toluene via inhalation, at 1,500 ppm and at 10,000 ppm respectively, and via an ingestion (400 mg/kg). Given these three exposure conditions, the values of brain/blood toluene ratios were found to be the highest in the brain stem areas (2.85-3.22) such as the pons and medulla oblongata, in the middle range in the midbrain, the thalamus, the caudate-putamen, the hypothalamus and the cerebellum (1.77-2.12), and in the lowest ranges in the olfactory bulb, the hippocampus and the cerebral cortex (1.22-1.64). The values of the partition coefficient of toluene in the brain regions and in the blood of rats examined in vitro were also the highest in the brain stem and the lowest in the cerebral cortex. The highest values of brain/blood toluene and partition coefficients of toluene in the brain regions and blood were obtained in the lipid-rich regions. These results suggest that toluene, which has a high lipid solubility and no protein binding capability, distributes according to lipid contents of the brain. The inhalation of toluene (1,500 ppm and 10,000 ppm) caused decreases in the noradrenaline (NA) levels in the dorsal part of rat pons, rich in locus coeruleus, and in the dopamine (DA) levels in the hypothalamus and ventral part of the rat midbrain, rich in substantia nigra.(ABSTRACT TRUNCATED AT 250 WORDS)

Hymenoptera sting anaphylaxis: detection and clinical significance of individual bee and wasp venoms specific IgE and IgG4 antibodies.

In Japan, an average of 37 fatalities per year related to bee or wasp stings were reported during the years 1979-1988. To confirm fatal anaphylaxis serologically, we measured bee or wasp venom specific IgE (sIgE) and IgG4 (sIgG4) antibodies in the sera of 22 patients who visited hospitals with either allergic or anaphylactic reactions after bee or wasp stings by using Enzyme-Linked Immunosorbent Assay. Specific IgE or IgG4 antibodies against Polistes apachus (Paper wasp) and/or a mixture of Polistes annularis, P. exceramans, P. fuscatus, and P. metricus venoms of Polistes genus were detected in 11 patients and the detection frequencies were the highest among positive antibodies against bee or wasp venoms. The severity of allergic reactions was graded from 0-4 according to Mueller. The detection frequencies of sIgE in Mueller grade 0-3 patients were in the range of 33% to 67%, and in grade 4 were 100%. Whereas, the detection frequency of sIgG4 was high (67-100%) in grade 2-4 patients. Especially high levels of sIgE and sIgG4 were detected in patients experiencing the most severe clinical reactions. The detection of venom specific IgE and IgG4 antibodies appears to be useful when determining bee or wasp stings as the cause of fatalities.

[Experimental studies of postmortem diffusion of toluene in blood, brain, muscle and fat of rats exposed to toluene vapor].

This report describes the postmortem diffusion of toluene in blood, brain, muscle and fat of rats exposed to toluene vapor. After exposure to toluene vapor (4,000 ppm) for 15 and 60 min, rats were killed by CO2 exposure and left for 12 and 24 hours in a fresh air. Toluene concentrations in blood, brain, muscle and fat were determined by head space gas chromatography. Postmortem decrease of toluene concentration in blood and increase of that in fat were observed with postmortem interval in both exposure conditions. But an extremely larger postmortem changes in the short exposure than those in the long exposure were observed. These are due to the facts that the value of fat/blood toluene concentration ratio at the time of death in the short exposure (3.3) was extremely smaller than that in the long exposure (10.5). Postmortem increase in muscle was also observed slightly. Postmortem concentrations in brain in both exposure intervals were relatively constant during these experiments. This postmortem stability is attributable to its anatomical features. These results suggest that brain toluene concentration at the autopsy is useful for the estimation of blood toluene concentration at the time of death and by comparison between toluene concentration in blood and that in brain it may be possible to presume the circumstances of death.

[In vitro studies of the metabolism of paraquat and diquat using rat liver homogenates--isolation and identification of the metabolites of paraquat and diquat].

The metabolism of paraquat and diquat was studied in vitro using rat liver homogenates, and the resulting metabolites were identified. Rat liver was homogenized with three volumes of isotonic buffer, and aliquots of the homogenate were preheated in a boiling water bath for 5 min prior to use. One milliliter of a mixture including both paraquat and diquat in an isotonic buffer solution (10 micrograms ion/ml) was incubated with an equal volume of fresh or preheated homogenate for 1 to 60 min at 37 degrees C. Quantification of paraquat and diquat was carried out by high-performance liquid chromatography (HPLC). In the fresh homogenate, a gradual decrease of paraquat concentration (about a 30% decrease over 60 min of incubation) and a rapid decrease of diquat concentration (not detectable after 10 min of incubation) were observed, but the same phenomenon was not evident with the preheated homogenate. Analysis of the incubated mixture of fresh liver homogenate with paraquat and diquat revealed three unknown peaks on the HPLC chromatograms; these seemed to be breakdown products of paraquant and diquat. The products were isolated and purified from the mixture by Sep-Pak C18 cartridge extraction, HPLC, silica gel column chromatography and Sephadex LH-20 column chromatography. Analysis of the chemical structure of the purified compounds was performed by infrared spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy. These analyses determined that paraquat-monopyridone (1',2'-dihydro-1,1'-dimethyl-2-oxo-4,4'-bipyridylium ion) was derived from paraquat, and that diquat-monopyridone (6,7-dihydro-4-oxodipyrido [1,2-a':2',1'-c] pyrazinium ion) and diquat-dipyridone (6,7-dihydrodipyrido [1,2-a:2',1'-c] pyrazine-4,9-dione) were derived from diquat. These results indicate that paraquat and diquat are metabolized by rat liver homogenate, diquat more readily so than paraquat. As the toxicity of these metabolites has been reported to be much lower than those of the parental compounds, it would seem that there is a system capable of detoxifying paraquat and diquat in rat liver.

Forced double suicide by fire revealed by autopsy and toxicological examination: a case report.

We report here a forced double suicide by fire. Two burned bodies discovered in the debris of a house fire were identified as a father and his son by odontological findings. In the father, the concentration of carboxyhemoglobin (CO-Hb) in the left ventricle was over 90% and endrin, an organochlorine pesticide, was also detected in the stomach contents. However, 30.5% of CO-Hb in left ventricular blood and gasoline traces from an intratracheal puncture were detected in the son who had been undergoing treatment at a neuropsychiatric clinic. From these results, we determined this case to be a double suicide forced by the father in which the father set a fire with gasoline, thus burning his son to death, and then died in the fire himself after ingestion of Endrin. This case suggests the importance of integration of the results from the detailed forensic pathological and toxicological examinations and the scene investigation.