Human Hepatic Alcohol Dehydrogenase and Human Erythrocyte Catalase Do Not Metabolize the Cytochrome P-4502E1 Substrate, Chlorzoxazone

Studies of cytochrome P-4502E1 (CYP2E1)-mediated oxidation of ethanol have been hampered by the lack of a suitable probe for in vivo human studies. Chlorzoxazone, a prescribed skeletal muscle relaxant, is metabolized to 6-hydroxychlorzoxazone by CYP2E1 and has been advocated as a specific probe of this enzyme on the basis of microsomal studies. The applications of this probe may include delineating the contribution of CYP2E1 to in vivo human ethanol metabolism. However, the activity of nonmicrosomal enzymes in metabolizing chlorzoxazone is unknown. Alcohol dehydrogenase (ADH), predominantly a hepatic cytosolic enzyme, may be more important than CYP2E1 in the oxidation of ethanol to acetaldehyde. The contribution of catalase in the in vivo oxidation of ethanol to acetaldehyde is controversial. To determine if either of these enzymes metabolizes chlorzoxazone and whether ethanol oxidation by either enzyme is inhibited by chlorzoxazone or its metabolite, multiple in vitro studies were performed. ADH enzyme kinetics were performed with human recombinant β₁β₁ and β₃β₃ ADH with ethanol and chlorzoxazone (0.5 to 2.5 mM). Neither ADH isoenzyme exhibited NAD⁺-dependent oxidation of chlorzoxazone, but displayed Michaelis-Menten kinetics for ethanol with K_m values of 89 μM and 34 mM, for β₁β₁, and β₃β₃, respectively. Typical in vivo concentrations of chlorzoxazone and its metabolite, 6-hydroxychlorzoxazone, did not alter β₁β₁, or β₃β₃ ADH-mediated oxidation of ethanol to acetaldehyde. Studies of human hepatic nonmicrosomal enzyme activity were expanded to include all nonmicrosomal NAD⁺-dependent hepatic enzymes by starch gel electrophoresis assessment. Human hepatic enzymatic activity in the presence of chlorzoxazone was similar to that observed in the control sample (no added substrate), suggesting a lack of metabolism by NAD⁺-dependent enzymes. Similarly, human erythrocyte catalase, in the presence of a hydrogen peroxide generating system, did not metabolize chlorzoxazone. Furthermore, neither chlorzoxazone nor 6-hydroxychlorzoxazone altered the catalase-induced formation of acetaldehyde from ethanol. These data are consistent with chlorzoxazone as a specific probe of CYP2E1 that may be useful to alcohol researchers.     

Characteristics of Japanese Alcoholics with the Atypical Aldehyde Dehydrogenase 2*2.I. A Comparison of the Genotypes of ALDH2, ADH2, ADH3, and Cytochrome P-4502E1 Between Alcoholics and Nonalcoholics

We examined the genotypes of the aldehyde dehydrogenase (ALDH)-2 , alcohol dehydrogenase (ADH)-2, ADH3 , and P-4502E1 loci of 53 alcoholics and 97 nonalcoholics. All of the subjects fulfilled the DSM-III-R criteria for alcohol dependence. The control group consisted of 97 subjects who were either hospital staff or students. We also compared the frequencies of homozygous ALDH2*1/1 and heterozygous ALDH2*1/2 genotypes in alcoholics. Our study revealed differences in the allelic frequencies of the ALDH2, ADH2 , and ADH3 loci between alcoholics and nonalcoholics. For alcoholics with both homozygous ALDH2*1/1 and heterozygous ALDH2*1/2 genotypes, it was found that ADH2 and ADH3 played important roles. Alcoholics with the heterozygous ALDH2*1/2 genotype showed a significantly higher frequency of ADH2*1/1 than ones with the homozygous ALDH2*1/1 genotype. We assume ADH2*1 plays an important role in the development of alcoholism in alcoholics with the heterozygous ALDH2*1/2 genotype.     

Genetic Polymorphism and Activities of Human Colon Alcohol and Aldehyde Dehydrogenases: No Gender and Age Differences

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isoenzyme patterns from 69 (men, 47; women, 22) surgical colon mucosal specimens were identified by agarose isoelectric focusing. γ-ADH was found to be the predominant form in the mucosa, whereas only β-ADH was detectable in the muscle layer. ALDH1, ALDH2, and ALDH3 were detectable in the mucosa, with cytosolic ALDH1 being the major form. At pH 7.5, the ADH activities in the colon mucosae with the homozygous phenotype (exhibiting γ₁γ₁) and the heterozygous phenotype (exhibiting γ₁γ₁, γ₁γ₂, γ₂γ₂) were determined to be 183 ± 13 and 156 ± 30 nmol/min/g tissue, respectively. The ALDH activities in the ALDH2-active and ALDH2-inactive phenotypes were determined to be 40.2 ± 2.3 and 34.6 ± 2.0 nmol/min/g tissue, respectively. The lack of significant difference in the ALDH activities between these two phenotypic groups can be attributed to the very low expression of the mitochondrial ALDH2 in the colon mucosa. No significant differences in the ADH or the ALDH activities were found between the men and women studied and between the three age groups (20–40, 49–70, and 72–83 years). The ascending, transverse, descending, and sigmoid colons exhibited similar ADH and ALDH activities. The isoenzyme patterns of ADH and ALDH remained unaltered in colon carcinomas, except that a significant reduction of the enzyme activities was found in the cancer tissue as compared with the adjacent normal portions. it is concluded that human colon mucose exhibits significant amounts of ethanol- and acetaldehyde-oxidizing activities.     

Comparison of Salivary and Serum Alkaline Phosphates Level and Lactate Dehydrogenase Levels in Patients with Tobacco Related Oral Lesions with Healthy Subjects - A Step Towards Early Diagnosis

Aim: To evaluate and compare salivary and serum levels of Alkaline Phosphates and Lactate Dehydrogenase in patients without the habit of tobacco, in patients with the habit of tobacco, in patients with benign oral lesions and in patients with oral premalignant lesions and oral malignant lesions. Material and Methodology: This study was comprised of 500 subjects, Group I: 100 healthy individuals without the habit of tobacco usage formed the control group. Group II: 100 patients with the habit of tobacco/ smoking consumption without any oral lesion. Group III: 100 patients with benign oral lesions. Group IV: 100 patients having the history of tobacco consumption and having apparent precancerous lesions like leukoplakia, erythroplakia. Group V:100  patients having frank oral cancer. The grade of dysplasia in these patients was statically correlated with the levels of serum and salivary ALP and LDH. Results: This study revealed that there was high expression of both serum and salivary ALP and LDH  in group IV and Group V as compared with the other groups and mean difference showed a statistically significant p value of less than 0.01. This study revealed that the in group V, the highest level of serum and salivary ALP was found in those patients who were reported with poorly differentiated oral cancer. Conclusion: Both Alkaline phosphates and Lactate dehydrogenase could be considered a sensitive markers for the detection of dysplasia with already existing precancancerous and cancerous lesions.     

六味地黄口服液对冷应激小鼠的保护作用

目的本研究通过冷刺激小鼠模型观察新老两种工艺制备的六味地黄口服液对冷应激损伤的影响及作用比较。方法将80只小鼠随机分成8组（正常组、冷应激组和两种六味地黄口服液不同剂量组），连续灌胃7d，每日于（10±0.5）℃冷水中游泳5min，于末次实验后，取血清、脑、胸腺及脾组织，分别测定血清和脑组织超氧化物歧化酶（SOD）、乳酸脱氢酶（LDH）活性和丙二醛（MDA）含量，同时计算胸腺、脾脏指数。结果与模型组比较，六味地黄口服液可使冷应激小鼠血清中SOD活性升高；MDA含量和LDH活性降低（P〈0.05）；脑组织中MDA含量降低。两种工艺六味地黄口服液中、大剂量给药组可以增大胸腺和脾脏指数。结论六味地黄口服液对冷刺激引起的氧化应激损伤有明显保护作用。     

解酒药改善心肌梗死后心力衰竭大鼠心脏功能的观察研究

目的观察使用解酒药是否能改善心肌梗死后心力衰竭大鼠的心脏功能。方法将60只体重相近的成年雄性SD大鼠采用冠状动脉结扎法建立急性心肌梗死模型后,随机分为4组,灌胃不同药物,分别为阴性对照生理盐水组(0.3ml/(kg.d)),阳性对照依那普利组(10mg/(kg.d)),解酒药组(0.3g/(kg.d))以及解酒药和依那普利联合用药组(0.3g/(kg.d)解酒药加10mg/(kg.d)依那普利)。所有大鼠术后每组每天定时进行药物干预,并每周定时测量体重。术后4周采集超声心动图各参数(室间隔厚度、后壁厚度、左室舒张末内径、左室收缩末内径、左室短轴缩短率、左室射血分数)来评价大鼠的心功能。结果与术前相比,术后4周解酒药组和联合用药组大鼠的体重无明显变化,而依那普利组和生理盐水组大鼠体重上升。术后4周心脏超声结果显示:与生理盐水组比较,依那普利组、解酒药组和联合用药组的左室射血分数、左室短轴缩短率均有升高(P<0.05),但三组间无显著性差异;而室间隔厚度、后壁厚度、左室舒张末内径、左室收缩末内径等参数,四组间无显著性差异。结论解酒药能有效改善心肌梗死后心力衰竭的大鼠心功能。     

冠心康颗粒药效学实验研究

目的:本次实验对冠心康颗粒(简称GG)药效学指标进行了考察。方法:1.用结扎冠状劝脉造成家兔急性心肌缺血模型,考察GG对家兔心肌梗塞面积和血清中乳酸脱氩酶的影响。2.小鼠常压耐缺氧试验.结果:GG高(4.65g/kg),中(3.10g/kg)剂量组明显降低心肌梗塞程度,明显抑制ST段抬高,明显降低乳酸脱氩酶的活性,与生理盐水组有非常显著差异(P值均<0.01)。GG具有明显延长小鼠耐缺氧时间,GG 16.5g/kg和11g/kg组小鼠生存延长分别为37.24%和24.27%,与生理盐水组有非常显著差异(P值均<0.01)。结论:冠心康颗粒具有延长小鼠耐缺氧时间,对实验性心肌缺血家兔具有抗心肌缺血、缩小心肌梗塞范围的作用.     

Ethanol-enhanced cytotoxicity of alkylating agents

BACKGROUND: Although ethanol itself is not genotoxic, chronic alcohol consumption increases the risk of neoplastic disease. The mechanism by which ethanol exerts a cocarcinogenic effect is not well established, and the aim of this study was to determine whether exposure to ethanol increased the cytotoxicity of known carcinogens. METHODS: To assess cell survival, the ability of Chinese hamster A10 cells, which express alcohol dehydrogenase, to form colonies was determined after exposure to ethanol and other substances, including both genotoxicants and non-DNA-reactive cytotoxic agents. RESULTS: 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG) is an alkylating agent that forms covalent bonds with DNA. The cytotoxicity of MNNG at concentrations of 0.17 to 0.68 microM was markedly enhanced when cells were also treated with 50 mM ethanol. When combined with 0.34 microM MNNG, concentrations of ethanol as low as 2 mM exacerbated the toxicity of this alkylating agent. When these experiments were repeated in the presence of 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, pretreatment with ethanol did not affect the toxicity of MNNG. When ethanol treatment was combined with exposure to other carcinogens, as well as agents that do not directly damage DNA, the cytotoxicity of the DNA-reactive agents 4-nitroquinoline-N-oxide, mitomycin C, and 6-chloro-9-(3-[2-chloroethyl]aminopropylamino)-2-methoxyacridine was modestly enhanced, and that of a second alkylating agent, ethyl methanesulfonate, was markedly increased. CONCLUSIONS: The results are consistent with impairment of DNA repair processes, particularly base excision repair, by acetaldehyde, as a mechanism by which ethanol increases the genotoxicity of certain genotoxic agents.     

神经酰胺对小鼠皮层神经元乳酸脱氢酶代谢的影响

目的研究神经酰胺对小鼠皮层神经元乳酸脱氢酶(LDH)代谢的影响。方法在培养的小鼠皮层神经元上清中分别加入50、100、200、500、1000、2000nmol/L的神经酰胺,分别作用0、1、4、8、12、16、24、36h,测定LDH浓度,计算其代谢率和漏出率。结果可显著改变乳酸脱氢酶(LDH)在细胞内外的分布,随神经酰胺作用时间的延长或剂量的增加,细胞外LDH含量显著升高,但神经酰胺对细胞总LDH代谢无影响。结论神经酰胺可增加LDH漏出率,但对细胞总LDH代谢无影响。