Irritative action of alcoholic beverages in rat stomachs: A comparative study with ethanol

The mucosal irritative action of alcoholic beverages such as white wine, Japanese sake and whisky was examined in rat stomachs in vivo and in vitro, in comparison with ethanol. The concentration of ethanol in these alcoholic beverages was 15%. Mucosal application of ethanol (15%) and whisky in the chambered stomach caused a decrease in gastric potential difference (PD), while that of Japanese sake and white wine caused a slight increase but not decrease in PD. Likewise, both ethanol and whisky markedly reduced the cell viability of RGM1 cells after 5 min incubation, whereas neither Japanese sake nor white wine had any effect. In addition, supplementation of glucose, one of the non-alcoholic ingredients of white wine and Japanese sake, antagonized a reduction in both PD and cell viability caused by ethanol. These results suggest that the mucosal irritative action of Japanese sake and white wine is much less than that of ethanol or whisky and that these properties may be, at least partly, due to the glucose contained in these alcoholic beverages.     

Effects of l-tryptophan and ethanol on sleep parameters in the rat

Sleep parameters were monitored following (1) a single 2 g/kg oral dose of ethanol, (2) an oral dose of l-tryptophan (600 mg/kg), and (3) administration of both drugs simultaneously. Ethanol reduced REM and increased slow wave significantly. The effects of l-tryptophan were apparent only in the case of one parameter, REM latency. Administration of both drugs resulted in a significantly shorter REM latency than that observed for ethanol administered alone. Results are discussed in terms of possible changes in the biosynthesis of 5-HT.     

Effects of ethanol in vitro on some parameters of the immune response

The action of ethanol (ETH) on murine lymphocyte subpopulations and on human peripheral blood mononuclear cells (PBMC) stimulated in vitro by mitogens was studied. ETH caused a concentration-dependent decrease in DNA synthesis in the different murine cell types. ETH was more immunosuppressive for T lymphocytes than for B lymphocytes. An enhancement of the blastogenic response was observed for B cells at 0.5% ETH. Interleukin 2 synthesis by murine splenocytes was inhibited by ETH in a concentration-related manner; the lowest concentrations of ETH caused an increase in interleukin 2 synthesis. The highest concentrations of ETH tested decreased the size of the cell clusters formed in cultures of mitogen-stimulated lymphocytes, whereas an increase in PBMC cluster size was observed in the presence of 0.5 and 1% ETH.     

In vivo/in vitro study in rat embryos on indium-caused tail malformations

Pathogenesis of indium-caused tail malformations was investigated by in vivo and in vitro experiments. In the in vivo experiment, pregnant Wistar rats received single intravenous administration of indium trichloride at 0.4 mg/kg on day 10 of gestation, and their embryos were examined on days 11, 12 and 13. Embryos in the indium group showed caudal hypoplasia from day 11. Increased apoptosis was observed in their tailbud on day 11. Similar effects were observed in the in vitro experiment, when day 10 rat embryos were cultured in the presence of indium trichloride at 50 microM for 24 h and for further 24 h in the absence of indium. It was considered from these results that caudal hypoplasia probably due to excessive cell loss by increased apoptosis in the tailbud accounted for indium-caused tail malformations in rat fetuses, and that indium-caused embryotoxic effects were direct effects on the conceptus.     

In vitro study of pesticide hematotoxicity in human and rat progenitors

Some pesticides are hematotoxic and cause aplastic anemia, agranulocytosis, neutropenia, and thrombopenia. In order to evaluate hematopoietic progenitor cultures in the exploration of pesticide hematotoxicity, human and rat colony-forming unit-granulocyte and macrophage (CFU-GM) were cultured in the presence of different concentrations of pesticides, known to be either hematotoxic or innocuous for blood cells. The results were compared to the control culture of the same sample. Four insecticides (lindane, azinphos, mevinphos, parathion methyl), three herbicides, (2,4,5, T, bromacil, MCPA), and two fungicides (fosethyl-aluminum, DNOC) were tested and exhibited the following data: 1) Pesticides, known to be hematotoxic, inhibited the development of progenitors. Different phenomena were observed and suggested different mechanisms: cell destruction, block in mitosis, decrease or delay in mitosis. 2) Difference in sensitivity to molecules between human and rat progenitors was observed. Human progenitors were more sensitive to pesticides, except for 2,4,5 T.     

In vitro co-metabolism of ethanol and cyclic ketones

The uridine uptake inhibition assay is a sensitive microassay for measuring cytotoxicity. This assay is normally performed with Hela S3 cells, which lack metabolic activity. In an earlier study, we adapted the test to HepG2 cells, a human hepatoma cell line that retains many hepatocyte characteristics, such as functional metabolic enzymes. This study describes a new automated protocol for the assay that makes it much more rapid. In the previous protocol, after the cells were treated with the test compounds and allowed to take up uridine for 30 min, samples were taken manually one by one and spotted onto 3MM Whatman paper. After drying, the paper sheet was then chromatographed in 5% (P/V) TCA for 2 h in order to precipitate and measure the total amount of RNA. In the new method, instead of paper chromatography, samples are transferred onto a 96-well microplate equipped with GF/C glass filters. Then, RNA precipitation by TCA is carried out with a manifold system, and the amount of radiolabeled uridine taken up by the cells is counted directly with a radioactivity microplate reader. This method makes it possible to screen many compounds simultaneously for cytotoxicity. To evaluate its sensitivity, we compared the IC(50) values obtained with new and original protocol for each eight toxic compounds. We found an excellent correlation between the two methods (r(2)=0.99). With the automated protocol, the uridine uptake inhibition assay is both sensitive and rapid enough for high-throughput daily screening.     

Effect of Vita Glucan on some antioxidant parameters of the human blood. In vitro study

Recently, beta-glucan has been postulated to modulate antioxidant enzyme activity (superoxide dismutase-SOD) as well as to inhibit lipid peroxidation in studies concerning rats or rabbits. There are very few reports on antioxidant effect of beta-glucan in the human blood. The study was aimed to estimate influence of Vita Glucan (VG) on SOD and catalase (CAT) activities as well as on total antioxidant power measured as ferric reducing activity and ascorbate concentration (FRASC) in the human blood in vitro. SOD activities were measured according to Fridovich's method, CAT activity by Aebi's and FRASC value by Benzi's one. Results of this study have shown that Vita Glucan at concentrations 42.5, 85, 170, and 340 mg x 100 mL(-1) increased markedly activities of antioxidant enzymes and FRASC values in human red blood cells hemolysates.     

In vivo acetaldehyde in the brain of the rat treated with ethanol

Ethanol, 4.5 g/kg, was administered intragastrically to rats to determine if acetaldehyde could be detected in brain interstitial fluid. Samples from both blood and brain were collected at half-hour intervals. Brain interstitial fluid samples were collected from both the caudate nucleus and the thalamushypothalamus region using the push-pull perfusion technique. The ethanol and acetaldehyde concentrations in these samples were determined by a head space gas Chromatographie technique. Blood ethanol levels typically ranged from 200 to 400 mg/100ml, while acetaldehyde levels ranged from 15 to 40 μM in blood and 5 to 20 μM in brain fluid. When disulfiram was given to the rats 20 hr prior to ethanol administration, blood acetaldehyde increased to 70–280 μM and brain interstitial fluid acetaldehyde increased to between 25 and 120 μM. Whole brain acetaldehyde levels were also measured after an ethanol dose was given. No acetaldehyde could be detected in whole brain unless the animal had first been treated with disulfiram. These data demonstrate that acetaldehyde does enter the brain, coming into direct contact with the brain cells bathed in the interstitial fluid. The acetaldehyde concentration in the interstitial fluid is higher than that in the brain cells, probably due to its rapid oxidation in the cells catalyzed by aldehyde dehydrogenase.     

In vitro metabolism study of edaravone in Wistar and hairless rat skin

We investigated the skin metabolism of edaravone as a radical scavenger in Wistar and hairless rat skin. Approximately 1 g of abdominal skin was excised from 10-week-old Wistar and hairless rats, homogenized in 10 ml saline, and centrifuged at 10000 g for 20 min. The supernatant fluid was used for the examination of edaravone metabolism in the skin, and we also used supernatant fluid that was heated at 80 degrees C. Edaravone solution (0.05 ml, 2.4 micromol/ml) was added to 0.95 ml Wistar rat and hairless rat skin homogenate supernatant fluids. In Wistar rats, the residual amount of edaravone in skin homogenate supernatant fluid at 37 degrees C after 0, 5, 10, 20 and 30 min was 61.58+/-1.65, 41.84+/-8.52, 35.54+/-8.62, 19.73+/-5.99 and 13.89+/-4.40%, respectively. In hairless rats, the residual amount of edaravone in skin homogenate supernatant fluid at 37 degrees C after 0, 5 and 10 min was 50.19+/-14.17, 6.71+/-5.82 and 0.89+/-0.80%, respectively, and edaravone was not detected after 20 min. Although it was thought that metabolic enzyme activity in skin homogenate supernatant fluid was lost following heat treatment at 80 degrees C, the residual amount of edaravone in our skin homogenate supernatant fluid decreased with time. It is suggested that edaravone metabolism in the skin is necessary for non-enzymatic reactions.     

The effect of acute ethanol ingestion on in vitro metabolism of choline and ethanol derivatives in rat liver

The effect of acute ethanol ingestion on hepatic phospholipid metabolism has been investigated in the rat. No significant increase in the content of either lecithin or phosphatidylethanolamine occured in the liver after 12 hr from treatment. The triglyceride content of the liver increased three-fold. An increase of lecithin and phosphatidylethanolamine synthesis by the Kennedy pathway was found in vitro in the homogenates and microsomal fractions prepared from the ethanol-treated rats. A higher rate of conversion of phosphorylcholine and phosphorylethanolamine to CDP-choline and CDP-ethanolamine was also found in the experimental animals. The breakdown of CDP-choline to phosphorylcholine was noticeably decreased in the liver fractions of the ethanol-treated rats. No changes in the sequential methylation pathway for lecithin synthesis in the liver were observed after acute ethanol ingestion.     

In vitro metabolic study of EGYT-3615 using rat liver microsomes

In vitro metabolism of EGYT-3615, a prospective new antidepressant was studied by using rat liver microsomes. The metabolite (M) found in earlier in vivo studies was unanimously found to be the product of a microsomal enzymatic process. The Michaelis-Menten constant of the reaction was calculated.     

In vivo quantification of ethanol kinetics in rat brain.

Proton magnetic resonance spectroscopy was used at 3T to measure the uptake and clearance of brain ethanol in rats after bolus intraperitoneal (i.p.) or intragastric (i.g.) alcohol injection, and to estimate the effects of acute alcohol on brain metabolites. The observation duration was 1-1.5 h with temporal resolution of alcohol sampling ranging from 4 s-4 min. The observed time course of alcohol brain concentration followed a consistent pattern characterized by a rapid absorption, an intermediate distribution, and a slower clearance that approached a linear decay. In a sample of eight healthy Wistar rats, the intercept of the linear clearance term, extrapolated back to the time of injection, correlated well with the administered dose per unit of lean body mass. Alcohol concentration estimation based on spectroscopically measured clearance was compared with blood alcohol levels from blood samples at the end of observation, and were in good agreement with the administered dose. Serial proton spectroscopy measurements provide a valid in vivo method for quantifying brain alcohol uptake and elimination kinetics in real time.     

In vitro metabolism of cadaverine in the pregnant rat

The in vitro metabolism of 14C-cadaverine by diamine oxidase was investigated in various tissues of pregnant and non-pregnant rats. The metabolites formed were: delta 1-piperideine, delta-aminovaleric acid, carbon dioxide and some unidentified compound(s). In most of the tissues investigated delta 1-piperideine was the predominant metabolite, but considerable amounts of delta-aminovaleric acid and the unidentified compound(s) were also formed. The oxidative products of cadaverine might be of importance in various physiological and pathophysiological connections associated with elevated diamine oxidase levels.     

In vitro effect of ethanol on cell-mediated cytotoxicity by murine spleen cells

The effects of ethanol on murine spleen cell-mediated lysis have been studied. Concentrations of 5.5-176 mM ethanol produced progressive inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC). Binding of spleen cells to antibody-sensitized target cells was not inhibited by comparable concentrations of ethanol. Kinetic analysis revealed decreased rates of lysis with increasing concentrations of ethanol. Changes of effector to target cell ratios revealed an inhibition of maximum lysis and decreased lytic efficiency in the presence of 88 mM ethanol. Preincubation experiments showed the inhibitory effect of ethanol to be reversible. Macrophage-depleted spleen cells appeared to be as susceptible to inhibition by ethanol as unfractionated spleen cells. Ethanol also inhibited natural killer and alloimmune cytotoxic T cell activity. The ADCC data were analysed by using a mathematical model which incorporates the kinetics of lysis, dose-response relationships, heterogeneity of the lytic effectors, reversibility of inhibition and ethanol loss during incubation. An inhibition constant (KI) of 373 mM-2 when two ethanol molecules interact with the site of inhibition was calculated. 50% inhibition of lysis is produced by 52 mM (0.24%) ethanol. The results are consistent with a model which assumes that lysis is due to a critical number of interactions which ultimately trigger the lytic event. Alcohol interferes with lysis by reacting with sites which are required for triggering the lytic event. Although the molecular details of the mechanism of inhibition are as yet undefined, we infer that ethanol inhibits ADCC at the programming for lysis or the lethal hit stages.     

In vitro study on the pulmonary cytotoxicity of amiodarone

Abstract

Context: Amiodarone (an iodinated benzofuran) is a Class III antiarrhythmic drug that produces significant pulmonary disease. Proposed mechanisms of this cytotoxicity include necrosis, apoptosis, mitochondrial dysfunction and glutathione depletion.

Objective: This study was designed primarily to explore whether amiodarone impairs lung tissue cellular bioenergetics in BALB/c and Taylor Outbred mice.

Materials and methods: Cellular respiration (mitochondrial O₂ consumption), ATP, caspase activity and glutathione were measured in lung fragments incubated in vitro with 22?µM amiodarone for several hours.

Results: Without amiodarone, lung tissue cellular mitochondrial O₂ consumption decayed exponentially with time, showing two distinct phases sharply separated at t?≥?150?min. The rate of cellular respiration was 6–10-fold higher in the late phase compared to the early phase (p?<?0.0001). Lung tissue ATP also decayed exponentially with time, suggesting “uncoupling oxidative phosphorylation” was the responsible mechanism (low cellular ATP with high mitochondrial O₂ consumption, resulting in rapid depletion of cellular metabolic fuels). Although intracellular caspase activity increased exponentially with time, the uncoupling was not prevented by the pancaspase inhibitor zVAD-fmk (N-benzyloxycarbonyl-val-ala-asp (O-methyl)-fluoromethylketone). The same profiles were noted in the presence of amiodarone; but cellular ATP decayed 50% faster. Cellular glutathione for untreated tissue was 560?±?287?pmol?mg^?1 (n?=?12) and for treated tissue was 490?±?226?pmol?mg^?1 (n?=?12, p?=?0.5106).

Conclusion: Uncoupling oxidative phosphorylation was demonstrated in untreated mouse lung tissues. Amiodarone lowered cellular ATP. Further studies are needed to explore the susceptibility of the lung to these deleterious insults and their relevance to human diseases.     

Effects of triethyl lead on various cholinergic parameters in the rat brain in vitro

The effects of triethyl lead acetate (triethyl Pb) on the cholinergic system in the brain of the rats were investigated in vitro. Triethyl Pb, at concentrations below 10(-4) M, inhibited the depolarized release of acetylcholine (ACh) from slices of cortex and they synthesis of ACh in such slices, while it potentiated in a dose-dependent manner the non-depolarized release of ACh. In contrast, lead inhibited noncompetitively the high-affinity uptake of choline into synaptosomes with a Ki of 4.03 X 10(-6) M and the activity of choline acetyltransferase with a Ki of 4.07 X 10(-5) M. Triethyl Pb has an inhibitory effect (IC50 not equal to 5 X 10(-5) M) on the binding of [3H]quinuclidinyl benzilate to muscarinic ACh receptors. Triethyl Pb inhibited acetylcholinesterase activity slightly at 5 X 10(-5) and 10(-4) M. It is suggested that ACh transmission, in particular the synthesis of ACh and the release of ACh, is susceptible to organolead neurotoxicity.     

头孢菌素对凝血功能影响的体外研究

目的 探讨头孢菌素类抗生素对凝血功能的影响.方法 测定正常混合血浆和各浓度梯度含抗生素血浆的PT、APTT、FIB、TT及FaⅡ.比较各浓度含抗生素血浆组与正常混合血浆组的测定结果之间的差异,并对凝血检测结果与抗生素的浓度作线性回归.结果 血浆中头孢硫脒浓度达100、200、800、1600、100mg/L时,或头孢西丁钠浓度达800、400、1600、3200、200mg/L时,或头孢噻肟钠浓度达800、3200、1600、1600、200mg/L时,PT、APTT、FIB、TT及Fa Ⅱ的测定结果与正常混合血浆结果之间的差异具有统计学意义.五项测定结果与血浆中的抗生素浓度存在线性关系.结论 在体外实验中,头孢菌素类抗生素可以影响凝血检测结果.     

In vitro effect of methanol on folate-deficient rat hepatocytes

Methanol is primarily metabolized by oxidation to formaldehyde and then to formic acid. These processes are accompanied by formation of superoxide anion and hydrogen peroxide. This paper reports the in vitro antioxidant effect of vitamin E on isolated hepatocytes of folic acid deficient rats rendered so as to emulate a human hepatocyte model. These hepatocytes were treated with 320 microM of methanol per million cells and incubated for 30 min. The microsomal fraction of these hepatocytes showed a decreased level of superoxide dismutase (SOD), with increase in lipid peroxidation (LPO) shown by increase in recorded levels of malondialdehyde (MDA). Catalase activity was shown to be increased. Levels of reduced glutathione (GSH) were decreased and the activity of glutathione peroxidase (GSH-Px) and of glutathione reductase (GSSG-R) were not altered. The hepatocytes of folate deficient rats pretreated with vitamin E, when subjected to methanol treatment, showed no significant change in SOD levels and a significant decrease in MDA levels. The catalase activity in this group of animals showed a highly significant decrease. These animals had normal levels of GSH, while a significant fall in GSH-Px and GSSG-R levels were observed. These results suggest that Vitamin E exerts a protective effect on hepatocytes by acting as a free radical scavenger, proving its usefulness in treating methanol toxicity.     

In vitro metabolism study of saikosaponin d and its derivatives in rat liver microsomes

1.?Saikosaponins, one of the representative bioactive ingredients in Radix Bupleuri, possess hepatoprotective, anti-inflammatory, antiviral, antitumor, and other pharmacological activities. Up to now, few studies focused on the further metabolism of saikosaponins and their secondary metabolites absorbed into the circulatory system.

2.?To understand the in vivo efficacy of saikosaponin d, the in vitro metabolism of saikosaponin d, and its two derivatives formed in the gastrointestinal tract, prosaikogenin G and saikogenin G was investigated in rat liver microsomes, respectively.

3.?Fifteen metabolites were detected using high-performance liquid chromatography hybrid ion trap and time-of-flight mass spectrometry and triple-quadrupole mass spectrometry, and the predominant metabolic reactions were hydroxylation, carboxylation and combinations of these steps on the aglycone moiety.

4.?The metabolic pathways of saikosaponin d, prosaikogenin G, and saikogenin G were proposed in vitro and the results contribute to the understanding of saikosaponins in vivo metabolism.