Isolation of a bacterium that degrades urethane compounds and characterization of its urethane hydrolase

A bacterium which degrades urethane compounds was isolated and identified as Rhodococcus equi strain TB-60. Strain TB-60 degraded toluene-2,4-dicarbamic acid dibutyl ester (TDCB) and accumulated toluene diamine as the degradation product. The enzyme which cleaves urethane bond in TDCB was strongly induced by acetanilide. The purified enzyme (urethane hydrolase) was found to be homogeneous on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The molecular weight was estimated to be 55 kDa. The optimal temperature and pH were 45°C and 5.5, respectively. The enzyme hydrolyzed aliphatic urethane compound as well as aromatic ones. The activity was inhibited by HgCl₂, p-chrolomercuribenzoic acid, and phenylmethylsulfonyl fluoride, suggesting that cysteine and/or serine residues play an important role in the activity. The enzyme catalyzed the hydrolysis of anilides, amides, and esters as well as TDCB. It was characterized as a novel amidase/esterase, differing in some properties from other known amidases/esterases.     

Thermal salivation in rats, anesthetized with barbiturates, chloralose, urethane and ketamine

1. The relationship between thermal salivation (TS) and thermoregulation was studied in anesthetized rats. 2. Of the 6 anesthetics used, ketamine-anesthetized rats secreted the largest amount of saliva. Salivation, however, was thermal and not induced by ketamine itself. 3. Ketamine-anesthetized rats readily secreted saliva at core temperatures less than 40 degrees C but TS was remarkably enhanced by hyperthermia of 40-42.5 degrees C. 4. The equilibrium phase in the triphasic heat response of core temperature was a consequence of equilibrium between heat gain and heat loss by salivation.     

氨基甲酸乙酯水解酶的家族生物信息学分析

氨基甲酸乙酯(Ethyl carbamate,EC)是酒精饮料生产过程中自然产生的副产物,具有潜在的致癌性和遗传毒性,成为影响人们健康的隐患.利用氨基甲酸乙酯水解酶(Urethane hydrolase,UH)消除酒精饮料中已存在的EC具有直接、高效的作用.为了进一步探索EC水解酶的功能与作用,本实验利用生物信息学的方...     

Clastogenic activity of urethane in mice

Single intraperitoneal (i.p.) treatment of male and female BDF1 (C57B1 × DBA2) mice with urethane (0.5 or 1.0 g/kg) caused a significant increase in micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after 24 h. The clastogenic effect observed was dose-, sex- and age-dependent, the male and younger (6–8 weeks old) animals being more susceptible than the female and older (6 months of age) mice. 3-week oral treatment of female Balb/c mice with urethane (3 g/l added to the drinking water) caused an up to 4-fold increase in the number of micronucleated normochromatic erythrocytes (MNNCE) in mouse peripheral blood. In a month after the carcinogen treatment was stopped, the number of MNNCE dropped to the control values. In addition, a single i.p. treatment of pregnant BDF1 mice on day 17 of gestation with urethane (1.0 g/kg) caused a 514.3% (p < 0.001) elevation of MNPCE in mouse fetal liver after 24 h as well as a 154.4% (p < 0.05) increase in MNPCE frequency in the fetal peripheral blood. At this time point, the clastogenic response in mouse fetal liver erythroblasts was less pronounced than that detected in the maternal bone marrow cells. Urethane is a strong clastogen in mice when administered either intraperitoneally or orally and the micronucleus test applied to adult and fetal erythroblasts is a convenient method of choice for studying the acute and subchronic clastogenicity of this carcinogen, its transplacental effects as well as the influence of modifying factors on these processes.     

Thermal salivation in rats anesthetized with barbiturates,chloralose, urethane and ketamine

1. The relationship between thermal salivation (TS) and thermoregulation was studied in anesthetized rats.2. Of the 6 anesthetics used, ketamine-anesthetized rats secreted the largest amount of saliva. Salivation, however, was thermal and not induced by ketamine itself.3. Ketamine-anesthetized rats readily secreted saliva at core temperatures less than 40°C but TS was remarkably enhanced by hyperthermia of 40–42.5°C.4. The equilibrium phase in the triphasic heat response of core temperature was a consequence of equilibrium between heat gain and heat loss by salivation.     

Effect of pentobarbital,chloralose, and urethane on inhibitory postsynaptic potentials of cortical neurons

The effect of pentobarbital, chloralose, and urethane on IPSPs arising in auditory cortical neurons in response to electrical stimulation of geniculocortical fibers was studied in experiments on cats immobilized with D-tubocurarine. Pentobarbital (60–80 mg/kg body weight, intraperitoneally) sharply reduced the number of neurons responding by spikes to geniculocortical stimulation. Only short-latency responses remained. The number of neurons responding with IPSPs was unchanged. Pentobarbital increased the duration of the IPSPs by 1.5–2 times and shortened their latent periods. Under the influence of chloralose (60 mg/kg, intraperitoneally) the number of responses of EPSP—spike—IPSP type was increased and the duration of the IPSPs also was increased by 3–4 times. The latent period of the primary IPSPs was shortened. Unlike pentobarbital and chloralose, urethane (1000 mg/kg, intravenously) reduced the duration of the IPSPs to 30 msec. About 2% of IPSPs recorded before anesthesia had a latent period of 1.0–1.5 msec. Under the influence of anesthesia the relative number of these IPSPs increased to 5.7%. It it postulated that they are monosynaptic. The mechanism of action of general anesthetics on the cortical inhibitory system is discussed.     

The metabolism of urethane and related compounds

1. Urethane is metabolized in the rat, rabbit and man by a process of N-hydroxylation. This occurs to a smaller extent when methyl, n-propyl and n-butyl carbamates are administered to the rat and rabbit. 2. Other metabolites which have been detected in urine of animals dosed with urethane and N-hydroxyurethane are ethylmercapturic acid, ethylmercapturic acid sulphoxide and N-acetyl-S-carbethoxycysteine. 3. Substances which appear to be S-ethylglutathione and S-ethylglutathione sulphoxide have been detected in the bile of rats dosed with urethane or N-hydroxyurethane. 4. Methyl, ethyl, n-propyl and n-butyl N-hydroxycarbamates are excreted unchanged in the urine of rats dosed with these compounds to extents depending on the dose administered. 5. Animals dosed with methyl, ethyl, n-propyl or n-butyl carbamate or the corresponding N-hydroxycarbamate excrete the corresponding carbamate and N-hydroxycarbamate in the urine. 6. Methyl, n-propyl and n-butyl carbamates and N-hydroxycarbamates are excreted more slowly than are urethane and N-hydroxyurethane. 7. The probable role of N-hydroxyurethane and the processes of alkylation and carbethoxylation, and of hydroxylamine, nitroxyl and hyponitrous acid in carcinogenesis and chemotherapy with urethane, have been discussed.     

Clastogenic activity of urethane in mice.

Single intraperitoneal (i.p.) treatment of male and female BDF1 (C57B1 x DBA2) mice with urethane (0.5 or 1.0 g/kg) caused a significant increase in micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after 24 h. The clastogenic effect observed was dose-, sex- and age-dependent, the male and younger (6-8 weeks old) animals being more susceptible than the female and older (6 months of age) mice. 3-week oral treatment of female Balb/c mice with urethane (3 g/l added to the drinking water) caused an up to 4-fold increase in the number of micronucleated normochromatic erythrocytes (MNNCE) in mouse peripheral blood. In a month after the carcinogen treatment was stopped, the number of MNNCE dropped to the control values. In addition, a single i.p. treatment of pregnant BDF1 mice on day 17 of gestation with urethane (1.0 g/kg) caused a 514.3% (p less than 0.001) elevation of MNPCE in mouse fetal liver after 24 h as well as a 154.4% (p less than 0.05) increase in MNPCE frequency in the fetal peripheral blood. At this time point, the clastogenic response in mouse fetal liver erythroblasts was less pronounced than that detected in the maternal bone marrow cells. Urethane is a strong clastogen in mice when administered either intraperitoneally or orally and the micronucleus test applied to adult and fetal erythroblasts is a convenient method of choice for studying the acute and subchronic clastogenicity of this carcinogen, its transplacental effects as well as the influence of modifying factors on these processes.     

Reaction of urethane with nucleic acids in vivo

1. [1-(14)C]Ethyl carbamate, ethyl [carboxy-(14)C]carbamate, [1-(14)C]ethanol and sodium hydrogen [(14)C]carbonate were injected intraperitoneally into C57 mice, and nucleic acids and proteins were separated from the liver and lungs with phenol as described by Kirby (1956). 2. Chromatographic analysis of the hydrolytic products of the urethane-labelled RNA showed the presence of a single radioactive compound differing in behaviour from the major pyrimidine nucleotides and purines. 3. The products from RNA labelled by [1-(14)C]ethyl carbamate or ethyl [carboxy-(14)C]carbamate appeared chromatographically identical but could not be detected in the RNA of mice given [1-(14)C]ethanol or sodium hydrogen [(14)C]-carbonate. 4. The labelled product appeared to be the ethyl ester of cytosine-5-carboxylic acid formed by the reaction of urethane with RNA in vivo. 5. A direct reaction between labelled urethane or the labelled metabolite of urethane, [1-(3)H]-ethyl N-hydroxycarbamate, and RNA was not detected.