Cholinergic alterations following alcohol exposure in the frontal cortex of Aldh2-deficient mice models.

We investigated the effects of alcohol (EtOH) and acetaldehyde (ACe) on choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in the frontal cortex of Aldh2-/- (KO) mice. KO mice were used as models of Aldh2-deficient humans to examine ACe effects. Brain samples were analyzed at 40 and 120 min after 2- and 4-g/kg intraperitoneal EtOH administration by RT-PCR and Western blot. Wild-type (WT) mice exhibited a remarkable decrease in ChAT and AChE mRNA expression at both time points only after 4-g/kg EtOH treatment compared with the naive control, whereas KO mice showed a considerable reduction in cholinergic markers after 2- and 4-g/kg EtOH treatment. The 4-g/kg EtOH-induced decrease in ChAT and AChE RNA expression at both time points was significantly greater than that in obtained with the administration of 2-g/kg at 40 min in WT mice. KO mice showed a significant difference in ChAT mRNA at 40 min between the EtOH groups. The findings regarding the ChAT mRNA levels are consistent with the results of Western blot in both types of mice, with some exceptions. EtOH-induced ChAT and AChE expression in KO mice was significantly lower than that in WT mice. This genotype effect occurred mostly at 40 min after EtOH dosing. Only ACe was quantified in the brains of KO mice, whereas EtOH was detected in both types of mice in vivo. These results suggest that EtOH and ACe combined or high EtOH alone alters cholinergic markers expression via changes in presynaptic and postsynaptic processes in the mice frontal cortex, thus indicating that central cholinergic neurons may be sensitive to EtOH and ACe.

Medico-legal investigation of chicken fat clot in forensic cases: immunohistochemical and retrospective studies.

Chicken fat clot (CFC), a fibrin-like substance, is sometimes found in the heart and large blood vessels in some autopsy cases. Reports of detailed histological findings of CFC are scant. We therefore examined CFC histologically in 53 autopsy cases and its correlation with ante-mortem or post-mortem evidence. We found three microscopic patterns of CFC: (1) wavelike fibrin fibers (WFF), (2) short fibrin fibers (SFF), and (3) short fibrin fibers mixed with wavelike fibrin fibers (SFF+WFF). WFF were found in the cases that survived less than 3 h after poisoning, burns, asphyxia, intracerebral hemorrhage, etc. SFF were found in the cases that survived more than 1 day after malignant neoplasms and acute or chronic inflammatory diseases, etc. SFF+WFF were found in the cases that died of inflammatory diseases, chronic heart failure, hemorrhagic shock, drowning, etc. About two-thirds of the SFF+WFF cases survived more than 1 day, with the rest surviving less than that. Our study confirmed three CFC patterns and their relation with survival interval. Therefore, these findings can be used as an index of the survival interval of a few acute and most chronic medico-legal death cases.

Catalase mediates acetaldehyde formation in the striatum of free-moving rats.

Using brain microdialysis, we measured both ethanol (EtOH) and acetaldehyde (AcH) levels in the striatum of free-moving rats following the inhibition of EtOH oxidation pathways. Rats received intraperitoneal EtOH (1g/kg) alone or in combination with 4-methylpyrazole (MP, 82 mg/kg, an alcohol dehydrogenase inhibitor), and/or catalase inhibitor sodium azide (AZ, 10mg/kg) or 3-amino-1,2,4-triazole (AT, 1g/kg), and/or cyanamide (CY, 50mg/kg, an aldehyde dehydrogenase inhibitor). Results revealed that both EtOH and AcH concentrations reached a plateau at 30 min after a dose of EtOH, and then gradually decreased for 4h. AcH was identified in the CY+EtOH, CY+AT/AZ+EtOH, and CY+4-MP+EtOH groups. The CY+EtOH-induced peak AcH level was 195.2+/-19.4 microM, and this level was significantly higher than the values in other groups studied. The catalase or ADH inhibitor in combination with CY lowered considerably the AcH concentration in the brain. The EtOH level reached a maximum of 25.9+/-2.3 mM in the CY+4-MP+EtOH group, and this level was markedly higher than in the EtOH group. No significant difference in brain EtOH levels was seen in any of the other groups examined. The findings strongly support the assumption that the enzyme catalase plays a significant role in AcH formation directly in the rat brain.

Ethanol and acetaldehyde: in vivo quantitation and effects on cholinergic function in rat brain.

First, ethanol (EtOH) and acetaldehyde levels were determined simultaneously in the striatum of free-moving rats after administration of their major oxidative enzyme inhibitors followed by EtOH. The results showed that acetaldehyde was present in the cyanamide (CY) + EtOH, CY + 4-methylpyrazole (4-MP) + EtOH and CY + sodium azide + EtOH groups. The CY + EtOH-induced peak acetaldehyde level was 195.2 +/- 19.4 microM, and this value was significantly higher than those in the other groups. The peak EtOH level was 25.9 +/- 2.3mM in the CY + 4-MP + EtOH group, and this level was considerably higher than the value in EtOH. No significant difference in brain EtOH levels was found in any of the other groups studied. Second, the effects of EtOH and acetaldehyde on choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were investigated in the frontal cortex and hippocampus of high acetaldehyde-producing rats using RT-PCR and Western blot. The results showed that EtOH and acetaldehyde decreased ChAT expression at 40 and 240 min after EtOH dosing in the brain. The acetaldehyde-induced reduction in ChAT expression was significantly higher than that induced by EtOH. No remarkable alteration of AChE expression was observed. The study suggested that catalase made a significant contribution to acetaldehyde formation in the rat brain, and that EtOH and acetaldehyde decreased ChAT expression at 40 and 240 min after EtOH dosing.

Effect of direct infusion of acetaldehyde on dopamine and dopamine-derived salsolinol in the striatum of free-moving rats using a reverse microdialysis technique.

The effects of acetaldehyde (ACD) on dopamine (DA) and DA-derived salsolinol (SAL) levels were investigated in the striatum of freely moving rats. Dialysate levels of DA and SAL were determined using in vivo reverse microdialysis coupled with high-performance liquid chromatography with an electrochemical detector. Perfusion with 1,000 microM ACD decreased DA levels significantly, as compared to baseline value, whereas 250 and 500 microM ACD perfusion did not result in any significant alteration of the DA levels in the striatal dialysates. SAL levels in the dialysates were determined first at 30 or 40 min after ACD perfusion, reached a peak at 150 min, followed by no alterations for 240 min with doses of 250, 500, and 1,000 microM ACD. Our in vivo study suggested that 1,000 microM ACD led to significant decreases in DA levels in the striatum with greater SAL formation, and the examined ACD concentrations induced a dose-dependent elevation in SAL levels in the striatum of freely moving rats.

Simultaneous determination of methamphetamine and its metabolite, amphetamine, in urine using a high performance liquid chromatography column-switching method.

We describe here a simple, precise, and highly sensitive method for the simultaneous determination of methamphetamine (MA) and amphetamine (AM) in urine using a high performance liquid chromatography (HPLC) column-switching method. A PK-2A (Shodex) column was used for extraction and deproteinization, and a CAPCELL PAK SCX semi-micro, polymer-coated cation-exchange column was employed for separation. The urine sample was mixed with an equal volume of borate buffer (0.1M, pH 9.4), and then 100 microl of the mixture was injected into the HPLC column. The column was switched for 6 min, and then 10 min later detection was performed at 210 nm. Recovery yields of the MA and AM spiked in the urine were 93.0-100.4% with a coefficient of variation of less than 1%. The calibration curves of MA and AM were in the range of 0.1-10 microg/ml with good linearity (r(2)=0.999), with the limit of qualification being 0.005 microg/ml. This method of using HPLC with column-switching can be used for both qualification and quantification of MA and its metabolite, AM, in urine, especially in forensic cases.

Autopsy and postmortem examination case study on genetic risk factors for cardiac death: polymorphisms of endothelial nitric oxide synthase gene Glu298Asp variant and T-786C mutation, human paraoxonase 1 (PON1) gene and alpha2beta-adrenergic receptor gene.

BACKGROUND/AIM: The Glu298Asp variant in exon 7 and T-786C mutation in the 5'-flanking region of the endothelial nitric oxide synthase (eNOS) gene, paraoxonase I gene (PON1), and alpha2beta-adrenergic receptor gene (alpha2beta-AR) have been reported to be genetic risk factors for coronary heart disease (CHD). The aim of this study was to investige the effects of these four genetic polymorphisms on the probability of death due to CHD, using data obtained from medico-legal autopsies. METHODS: Blood samples from three groups: healthy controls, dead cases with CHD and without CHD (the latter as a control for dead cases) were used. After DNA extraction, genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) test. RESULTS: The frequency of the T allele in Glu298Asp variant in the dead cases with CHD was significantly higher than that in the healthy control (p < 0.001, OR = 4.47) and that in the dead cases without CHD (p < 0.001, OR = 7.62). The gene frequency of PON1 was significandy different (p = 0.007) between dead cases with and without CHD, and was also significantly different (p = 0.025) between the healthy control and dead cases without CHD. The gene frequency of PON1 was not significantly different (p = 0.401) between the healthy controls and dead cases with CHD. Hence this gene was not associated with death due to CHD. The other polymorphisms (T-786C mutation, alpha2beta-AR) also showed no effect on death due to CHD. CONCLUSION: The polymorphism of Glu298Asp eNOS gene in dead cases may be useful for determining the cause of death in CHD cases in the Japanese population.

Differential regulation of proopiomelanocortin (POMC) mRNA expression in hypothalamus and anterior pituitary following repeated cyanamide with ethanol administration.

BACKGROUND/AIM: We have investigated proopiomelanocortin (POMC) mRNA expression in the arcuate nucleus of the hypothalamus (ARC) and the anterior lobe of the pituitary (AL) following repeated cyanamide-ethanol reaction (CER). METHODS: Adult male Sprague-Dawley rats (250-290 gr) were housed in a temperature and humidity controlled environment with free access to food and water. Four experimental groups were used as follows: saline (as control), cyanamide alone, ethanol alone and ethanol with cyanamide. The animals recived daily intraperitoneal injections (i.p.) of cyanamide (10 mg/kg, 60 min before ethanol dosing) with or without ethanol (1g/kg) for 5 consecutive days, and were sacrificed 60 min after the last dosing of ethanol. The results were presented as the mean +/- SEM for each group. All groups within each deta set were compared by one-way ANOVA followed by Fisher PLSD test for multiple comparisons. A value of p < 0.05 was considered significant. RESULTS: The POMC mRNA levels in ARC were significantly decreased with cyanamide compared to the control and ethanol alone (p < 0.05 and p < 0.05 respectively), but increased in AL following repeated CER. CONCLUSION: We speculate that this differential regulation of POMC mRNA expression may be partially involved in the preventive effects on alcohol intake in response to CER.

Inhibition of acetaldehyde metabolism decreases acetylcholine release in medial frontal cortex of freely moving rats.

The effect of high acetaldehyde (ACe) on acetylcholine (ACh) release was studied in vivo in the medial frontal cortex (mfc) of freely moving rats using brain microdialysis coupled with high performance liquid chromatography and an electrochemical detector. Ethanol (EtOH) and ACe concentrations were quantified simultaneously in the mfc of awake rats by in vivo microdialysis followed by head-space gas chromatography. Rats were treated intraperitoneally with saline, EtOH (1 and 2 g/kg) or cyanamide (CY, 50 mg/kg, a potent aldehyde dehydrogenase inhibitor) plus EtOH (1 and 2 g/kg). No significant effect on ACh levels was observed in saline groups, as compared to baseline value. The basal level of ACh in the dialysate was about 0.30 +/- 0.04 pmol/20 microl, and this value was reduced significantly in the EtOH (1 and 2 g/kg) and CY + EtOH (1 and 2 g/kg) groups for 240 min after EtOH administration. The time courses of ACh release continued to decrease significantly after EtOH administration in the CY + EtOH (1 and 2 g/kg) groups compared to the values in the saline and EtOH (1 and 2 g/kg) groups. A significant decrease in ACh release was observed from 140 to 240 min after EtOH dosing in the EtOH (1 and 2 g/kg) groups, as compared to saline groups. EtOH and ACe concentrations in the mfc were first determined at 15 min after a dose of EtOH, reached a peak at 30 min and then gradually decreased in the CY + EtOH (1 and 2 g/kg) groups. The present study suggests that both EtOH and ACe concentration in the brain can decrease in vivo ACh release in the mfc of free-moving rats, and the ACe-induced decrease in ACh levels was significantly higher than EtOH.

Failure of ethanol and acetaldehyde to alter in vivo norepinephrine release in the striatum and hippocampus of rats.

The effect of ethanol (EtOH) and acetaldehyde (AcH) on norepinephrine (NE) release was examined in the striatum and hippocampus of freely moving rats by means of in vivo microdialysis coupled with high-performance liquid chromatography and an electrochemical detector. Rats were treated intraperitoneally with EtOH (1 g/kg) or cyanamide (CY, 50 mg/kg, a potent aldehyde dehydrogenase inhibitor) plus EtOH (1 g/kg). No significant difference in NE levels in the dialysates was observed in the striatum and hippocampus in either the EtOH or CY+EtOH groups. NE levels in the hippocampal dialysates were about fivefolds higher than those in the striatum. The concentration of EtOH and AcH in the striatal dialysate reached a peak at 30 min after EtOH dosing and then gradually decreased in the CY+EtOH group. In the EtOH group the striatal concentration of EtOH also reached a peak at 30 min after EtOH dosing, and then gradually decreased while AcH was not detected. The present study suggests that there is no in vivo effect of brain EtOH or AcH on NE release in the striatum and hippocampus of awake rats.

Less contribution of the nicotinic cholinergic and alpha2-adrenergic action of high acetaldehyde concentration on the inhibition of intestinal ethanol absorption.

In the present study, we investigated the effects of hexamethonium, a ganglionic nicotinic receptor blocking agents and yohimbine, an alpha2-adrenergic antagonist, on reduction of ethanol absorption in presence of high acetaldehyde concentration. Hexamethonium had no effect, whereas yohimbine by itself reduced ethanol absorption, but no additional effects were observed with presence of high acetaldehyde. Propionaldehyde had an inhibitory action on intestinal 1-propanol absorption. As both yohimbine and propionaldehyde are associated with vagus nerve activation, these results indirectly support the hypothesis that a cholinergic mechanism through vagus nerve activation is responsible for the inhibition of intestinal ethanol absorption by acetaldehyde.

Microdialysis for the determination of acetaldehyde and ethanol concentrations in the striatum of freely moving rats.

The subject of the present paper is the simultaneous determination of ethanol (EtOH) and acetaldehyde (AcH) concentrations in the striatum of freely moving rats using an in vivo microdialysis followed by head-space gas chromatography (GC). Major operation conditions of GC were as follows: column, injector and detector temperatures 90, 110 and 200 degrees C, respectively; Supelcowax wide bore capillary column (60 m length, 0.53 mm i.d., 2 microm film thickness); carrier gas, nitrogen; flow rate, 20 ml/min. The recovery of EtOH and AcH at a concentration 40 mM and 250 microM, respectively, by microdialysis showed a maximum of 83.8+/-12.2 and 51.2+/-6.5%, respectively, at a flow rate of 0.8 microl/min. A good linear calibration curve in the concentration range of 5-50 mM for EtOH (r=0.998), and 10-250 microM for AcH (r=0.988) was observed. Microdialysates were collected for 10 min each after insertion of probe into the striatum. Rats were treated with cyanamide (100mg/kg, a potent aldehyde dehydrogenase inhibitor) and 60 min later with EtOH (1g/kg) intraperitoneally. A 10 min sample was about 8 microl. This volume was mixed with 40 microl of 0.002% t-butanol as an internal standard in 0.6N perchloric acid, and then analyzed by head-space GC method. The peak EtOH and AcH concentrations in the striatal dialysates reached maximum at 30 min, and then gradually decreased. This method represents a reasonable tool to quantify in vivo both AcH and EtOH levels simultaneously in rat brain.

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.

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.

Effect of different doses of cyanamide on striatal salsolinol formation after ethanol treatment.

To assess the dose-dependent effect of cyanamide (CY, a potent aldehyde dehydrogenase inhibitor) on salsolinol release in the striatum, rats were treated with CY (25, 50 and 100 mg/kg) plus ethanol (EtOH,1 g/kg) intraperitoneally. Striatal salsolinol was detected using in vivo microdialysis coupled with high-performance liquid chromatography with an electrochemical detector in free-moving rats, and blood acetaldehyde (AcH) and EtOH were detected using the head-space gas chromatographic method. With the increase in the doses of CY following EtOH, the peak concentrations of striatal salsolinol and blood AcH were increased significantly. Our study indicated that the magnitude of striatal salsolinol levels may depend on the concentration of blood AcH, and that there is a correlation between the blood AcH and striatal salsolinol.

Evidence of hexavalent chromium ingestion.

We describe the application of histochemical demonstration of chromium in a case of fatal ingestion of potassium dichromate in a suicide attempt. Using 2-(8-quinolylazo)-4,5-di-p-tolylimidazole (QTI), we could demonstrate chromium in the erythrocyte of the victim, in situ. This finding provides a means of proving the hexavalent chromium ingestion.

In vivo formation of salsolinol induced by high acetaldehyde concentration in rat striatum employing microdialysis.

AIMS: The in vivo formation of salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquionoline), an endogeneous condensation product of dopamine (DA) with acetaldehyde (AcH), was examined following the administration of cyanamide (CY) plus ethanol (EtOH) using microdialysis-high-performance liquid chromatography with electrochemical detection. METHODS: After the insertion of a microdialysis probe into the striatum, rats were treated with CY (a potent inhibitor of aldehyde dehydrogenase, 50 mg/kg), 4-methylpyrazole (4-MP, a strong inhibitor of alcohol dehydrogenase, 82 mg/kg), and CY + 4-MP, followed 1 h later by EtOH (1 g/kg), CY and 4-MP only by intraperitoneal administration. RESULTS: In the CY + EtOH group, salsolinol was detected in striatal dialysates and high AcH concentrations were found in the blood. The time course of changes in salsolinol concentrations correlated with blood AcH concentrations. In the other experimental groups, salsolinol in the dialysates and high AcH concentrations in the blood were not detected. CONCLUSIONS: These observations indicate that: (1) high AcH concentrations induce the formation of salsolinol in the rat striatum; (2) there is no effect of EtOH or AcH on striatal dialysate concentrations of DA and 5-hydroxytryptamine.

Effects of acetaldehyde on c-fos mRNA induction in the paraventricular nucleus following ethanol administration.

AIMS: The effect of acetaldehyde on c-fos mRNA expression in the paraventricular nucleus (PVN) of the rat was examined using in situ hybridization histochemistry. METHODS: Increases in acetaldehyde concentrations were induced using cyanamide (a potent inhibitor of aldehyde dehydrogenase), in the presence of two different doses of ethanol. Concentrations of blood ethanol and acetaldehyde were determined by head space gas chromatography. RESULTS: Neither cyanamide alone nor the low dose of ethanol (1 g/kg) alone increased c-fos expression in the PVN. However, the combination of cyanamide and low dose ethanol resulted in a significant and maximal increase in c-fos mRNA in the PVN. High dose ethanol (3 g/kg) resulted in a significant increase in c-fos mRNA. This stimulation also appeared maximal as there was no further increase in c-fos expression in the presence of cyanamide. CONCLUSIONS: These data suggest that acetaldehyde accumulation in blood has an important stimulatory effect on c-fos expression in the PVN at low ethanol concentrations. Furthermore, this stimulation of c-fos mRNA appears to be an either/or response: not activated in response to low dose ethanol, but maximally to high dose ethanol. These data also provide further evidence for a dissociation between the activation of c-fos and corticotrophin-releasing factor (CRF) mRNA in the PVN, as we have previously demonstrated that this dose of cyanamide alone is sufficient to evoke a sustained increase in plasma corticosterone and an increase in CRF mRNA.