mM Of Ethanol Research Articles

Effects of ethanol on ethylmorphine metabolism in isolated rat hepatocytes: characterization by means of a multicompartmental model.

Hepatic cytochrome P-450 enzymes mediate at least two important biotransformation pathways of codeine and ethylmorphine starting with either N-demethylation or O-dealkylation, producing polar metabolites which are then subsequently glucuronidated. The present study was designed to characterise the acute effects of ethanol on the metabolism of ethylmorphine and to compare it with the effects on codeine in suspensions of freshly isolated rat hepatocytes. Isolated rat hepatocytes from male Wistar rats were prepared by a collagenase perfusion method. Ethylmorphine, codeine and their metabolites were quantified by HPLC with UV detection. The total ethylmorphine elimination rate was reduced by 12% at 5mM and 38% at 100 mM ethanol. The corresponding percentages for codeine were 16 and 43%. In the presence of ethanol the concentrations of several intermediate and end products of ethylmorphine and codeine changed markedly from the control situation. The experimental data were applied to a mathematical compartmental linear model to estimate the influence of ethanol on the separate reaction rates in the two main metabolic pathways. The ratios between reaction rate constants in the ethylmorphine experiments at 100 and 0 mM ethanol were 0.65 for ethylmorphine-->norethylmorphine, 0.63 for norethylmorphine-->normorphine, 0.56 for ethylmorphine-->morphine, 0.49 for morphine-->normorphine, 0.31 for normorphine-->normorphine-3-glucuronide and 0.49 for morphine-->morphine-3-glucuronide. Almost similar effects of ethanol on codeine metabolism were found. In additional experiments, norethylmorphine or norcodeine (50 microM) was incubated with 5 mM to 100 mM of ethanol and the metabolism of both norethylmorphine and norcodeine was found to be inhibited by ethanol in a concentration-dependent manner. The glucuronidation of morphine and normorphine added in separate experiments was also inhibited by ethanol, from 22 to 36% for morphine-3-glucuronide and 30 to 60% for normorphine-3-glucuronide, respectively, in the presence of 5 mM to 100 mM of ethanol. It was concluded that all steps in the metabolism of ethylmorphine (and codeine) leading to the end products morphine-3-glucuronide and normorphine-3-glucuronide were inhibited by ethanol, and that the glucuronidation process were the ones most affected by ethanol.

High intracolonic acetaldehyde values produced by a bacteriocolonic pathway for ethanol oxidation in piglets.

Human colonic contents and many colonic microbes produce considerable amounts of acetaldehyde from ethanol in vitro. To examine in piglets if acetaldehyde is produced in the colon also in vivo, and if so, what is the fate of intracolonically formed acetaldehyde. Seventeen native, non-fasted female piglets (20-25 kg) were used. Six piglets received either 1.5 g/kg bw or 2.5 g/kg bw of ethanol intravenously. In seven piglets, 0.7 g or 1.75 g of ethanol/kg bw was administered intravenously, followed by a subsequent intragastric ethanol infusion of 1.8 g/kg bw and 4.5 g/kg bw, respectively. The samples of colonic contents for the assessment of ethanol and acetaldehyde concentrations were obtained up to seven hours. In four additional piglets, the intracolonic values of ethanol, acetaldehyde, and acetate were observed for 60 minutes after an intracolonic infusion of acetaldehyde solution. A raised intracolonic, endogenous acetaldehyde concentration (mean (SEM); 36 (9) microM) was found in all piglets before ethanol infusion. After the infusion of ethanol, intracolonic ethanol and acetaldehyde values increased in parallel, reaching the peak values 57 (4) mM of ethanol and 271 (20) microM of acetaldehyde in the group that received the highest dose of ethanol. A positive correlation (r = 0.45; p < 0.001) was found between intracolonic ethanol and acetaldehyde values. Acetaldehyde administered intracolonically was mainly metabolised to acetate but also to ethanol in the colon. Significant endogenous intracolonic acetaldehyde values can be found in the normal porcine colon. Furthermore, our results suggest the existence of a bacteriocolonic pathway for ethanol oxidation. Increased amounts of acetaldehyde are formed intracolonically from ingested ethanol by this pathway.

Role of protein synthesis on ethanol regulation of adenylyl cyclase activity in wild-type S49 murine lymphoma cells.

Adenylyl cyclase activity was determined in membranes from wild-type S49 murine lymphoma cells that had been exposed to ethanol for 4 hr. Mn-, NaF-, and forskolin-stimulated adenylyl cyclase activities of cells-pretreated with cycloheximide, puromycin, or serum deprivation-were significantly decreased by treatment with 50 mM of ethanol. As demonstrated for Mn-stimulated activity, the decrease was dose-dependent on ethanol and was temporal; a normal activity recovered after 16-24 hr treatment, even in the presence of cycloheximide and ethanol. Studies with a cell-free membrane system of S49 cells revealed a similar activity decrease after treatment of the membranes with ethanol. In contrast, cells treated with 50 mM of ethanol in a regular culture condition showed no decrease in adenylyl cyclase activity over 24 hr. These results indicate that ethanol regulation of adenylyl cyclase activity in S49 cells depends on reduced or impaired protein synthesis.

Effects of ethanol on glucose transporter expression in cultured hippocampal neurons.

Glucose transport was studied in primary hippocampal neuron cultures exposed to ethanol. Immunofluorescent staining with antibodies against neuron-specific enolase and glial fibrillary acidic protein identified approximately 95% of the cultured cells as neurons. Western blot analysis was conducted with polyclonal antisera to glucose transporter isoforms GLUT1 and GLUT3. As previously seen in astrocytes, GLUT1 protein was regulated by the culture medium glucose content. Exposure to 50 and 100 mM of ethanol for 5 hr induced dose-dependent reductions in GLUT1 and GLUT3 protein. In contrast, GLUT1 mRNA abundance was increased relative to controls under the same conditions. Glucose uptake, measured with the nonmetabolized analog, 2-deoxy-D-glucose, was reduced by 50 and 100 mM of ethanol in four experiments. These results indicate a direct effect of ethanol on neuronal glucose transporter expression, which may play a role in the neurotoxic effects of alcohol.

Acetaldehyde: genotoxicity and cytotoxicity in human lymphocytes.

We evaluated the DNA damaging effects of ethanol and its major metabolite, acetaldehyde. Freshly isolated human lymphocytes from two healthy donors were incubated with 0, 1.56, 6.25, 25, and 100 mM of ethanol or acetaldehyde in complete medium for 1 h. We used a newly developed, sensitive, alkaline microgel electrophoresis technique to quantitate single- and double-strand DNA breaks or alkali-labile sites in individual cells. Ethanol did not induce DNA strand breaks. However, acetaldehyde induced both single-strand (even at the lowest concentration of 1.56 mM) and double-strand breaks (only at the highest concentration of 100 mM). Following exposure to acetaldehyde, cells were incubated in complete medium for 30, 60, and 120 min. During this incubation period, most cells were unable to repair DNA single- and double-strand breaks caused by acetaldehyde. We also observed a significant cell loss after exposure to acetaldehyde. To our knowledge, this is the first study demonstrating DNA single- and double-strand breaks by acetaldehyde.

Reagentless enzyme-based sensor using a gas-permeable membrane for determination of alcohols

The sensor described consists of an anode mounted with immobilized alcohol dehydrogenase from Gluconobacter oxydons, a cathode and an electrolyte containing hexacyanoferrate(III). Its surface was covered with a gas-permeable membrane. Measurements were made without consumption of reagent by pumping a sample solution through the flow cell for 1 or 2 min. The calibration plot of the sensor using a porous PTFE gas-permeable membrane was linear up to 0.08 mM of ethanol with a relative standard deviation of 0.5%. The linear range was extended to 1.8 mM if a silicone rubber membrane was used. The selectivity was satisfactory; the penetration of interfering compounds was prevented by the gas-permeable membrane. Results obtained with the sensor and by gas chromatography agreed within 3.4% for alcohol beverages. The sensitivity decreased to 85% of its initial value after continuous use for 9 days. Responses of the sensor to 1-propanol and 1-butanol were examined.

BIPHASIC PROTEIN KINASE C TRANSLOCATION IN PC12 CELLS IN RESPONSE TO SHORT-TERM AND LONG-TERM ETHANOL EXPOSURE

Short-term and long-term effects of ethanol on protein kinase C (PKC) activity and PKC translocation from cytosol to membrane were examined in PC12 cells, a clonal cell line of neural crest origin. Treatment of PC12 cells with ethanol (30-100 mM) for 2 hr had no effect on PKC activity and PKC translocation. When PC12 cells were treated with 100 mM ethanol for 18, 44 and 74 hr, there was a biphasic effect on PKC translocation. At 18 and 44 hr ethanol treatment, PKC translocation was significantly (P < 0.001) increased, at 74 hr ethanol treatment, there was a significant decrease (P < 0.05). Less than 100 mM of ethanol had no effect on PKC activity and PKC translocation. Cyclic AMP and cyclic GMP-dependent protein kinase had no effect on PKC translocation. These findings indicate that biphasic PKC translocation from cytosol to membrane forms the basis of acute and chronic effects of ethanol on neurotransmission.

Ethanol decreases the velocity of spike propagation along a fast motor axon

Intracellular recordings were made from the fast bender excitor motor axon in autotomized crab limbs bathed in normal saline, and in salines made with up to 240 mM of ethanol. The presence of ethanol reduced the amplitude, the rise time and the decay time of the evoked action potential, and decreased the velocity at which the spike was conducted down the axon. There was a linear relationship between each of these four parameters and the concentration of ethanol in the saline. The close relationship between spike rise time and conduction velocity suggests that ethanol slows the rate of membrane depolarization by the spike and thus decreases the velocity at which action potentials are propagated along the axon.

Effects of ethanol on the excitability of hippocampal granule neurons

The effects of low dose ethanol treatment on neuronal firing threshold were studied in dentate granule neurons using intracellular recording. A higher threshold for orthodromic activation and lower threshold for somatic activation were observed in 50 mM of ethanol, based on an examination of the excitatory postsynaptic potentials, strength duration and repetitive firing properties. Experiments involving blockade of glutamate-receptor subtypes suggest that an N-methyl-D-aspartate (NMDA) receptor mediated mechanism is implicated in this action of ethanol.

Effect of ethanol on endothelin-1 release from gastric vasculature

The effects of ethanol on gastric vasculature in isolated vascularly perfused rabbit stomach was investigated. The isolated stomach was perfused with Krebs-Henseleit solution containing 3% dextran bubbled with 95% O2 and 5% CO2 at a rate of 12 ml/min. After mixture and perfusion of 10 mM to 400 mM of ethanol, perfusion pressure and endothelin-1 concentration in effluent from gastric vasculature were measured. Perfusion pressure and endothelin-1 concentration in effluent increased in a dose-dependent manner with increasing ethanol concentrations. In conclusion, the data suggest that ethanol may stimulate the release of endothelin from gastric vasculature and may cause gastric ischemia due to vasoconstriction resulting in acute gastric mucosal injury.