Quantitative Analysis of Ethyl Carbamate in Distillers Grains Co-products and Bovine Plasma by Gas Chromatography-Mass Spectrometry.

Ethyl carbamate (EC) is a fermentation byproduct in foods and beverages and classified as a Group 2A probable human carcinogen. Each year, greater than 40 million metric tons of fermentation co-products from the U.S. ethanol industry are fed to food animals. A gas chromatography-mass spectrometry assay was developed to quantify EC extracted from various distillers grains co-products with a limit of detection at 0.7 ng/g (on an as-fed basis). EC was detected in all the distillers grains co-products surveyed in this study. Corn condensed distillers solubles contained the highest concentration of EC, ranging from 1618 to 2956 ng/g. Concentrations of EC in other types of distillers grains co-products varied from 17 to 917 ng/g. Cattle fed distillers grains co-products that constituted 19-38% of the total feed (as-fed) were found to contain 2-3 ng/mL of EC in blood plasma. No EC was detected in blood plasma from grass-fed control cattle.

Effect of ethanol on the tumorigenicity of urethane (ethyl carbamate) in B6C3F1 mice.

Urethane is a carcinogen to which there is widespread exposure through the consumption of fermented foods and alcoholic beverages. In this study, we have assessed the carcinogenicity of urethane in combination with ethanol. Male and female B6C3F(1) mice (48 mice per sex per group) were exposed to 0, 10, 30, or 90 ppm urethane in the presence of 0%, 2.5%, or 5% ethanol in drinking water ad libitum for two years, at which time the extent of tumorigenesis was assessed. Additional mice (four per sex per group) received the same doses for four weeks to assess serum levels of urethane and ethanol, DNA adduct formation, and the induction of microsomal cytochromes P450, cell proliferation, and apoptosis. Urethane decreased cell replication in the livers of female, but not male, mice, decreased cell replication in the lungs of both sexes, and induced cytochrome P450 2E1 in the livers of female mice. Hepatic levels of the DNA adduct 1,N(6)-ethenodeoxyadenosine were increased by exposure to urethane and decreased by treatment with ethanol. Animal weights and survival were not affected by ethanol; in contrast, urethane administration decreased body weights and survival. Urethane caused dose-dependent increases in liver, lung, and harderian gland adenoma or carcinoma and hemangiosarcoma of the liver and heart in both sexes, mammary gland and ovarian tumors in females, and squamous cell papilloma or carcinoma of the skin and forestomach in males. The increase in hepatocellular tumors occurred in a relatively linear manner and was attributed to the formation of 1,N(6)-ethenodeoxyadenosine in hepatic DNA coupled with an increase in cell replication. Hemangiosarcomas were observed only at the 90 ppm urethane dose and were probably a result of high-dose urethane-induced toxicity. Lung alveolar/bronchiolar and harderian gland adenoma or carcinoma increased in a relatively linear manner, suggestive of a genotoxic mechanism for tumor induction. Ethanol induced a dose-dependent trend in hepatocellular adenoma or carcinoma in male mice, with the incidence being marginally increased at the highest dose. In female mice administered 10 ppm and 90 ppm urethane, ethanol caused dose-related increases in alveolar/bronchiolar adenoma or carcinoma and hemangiosarcoma of the heart, respectively. This may be due to ethanol decreasing the first-pass clearance of urethane, thus, increasing systemic distribution. In male mice a different relationship was observed: ethanol caused a dose-related decrease in alveolar/bronchiolar and harderian gland adenoma or carcinoma in mice administered 30 ppm urethane.

Urethane suppresses renal sympathetic nerve activity in Wistar rats.

AIM: To investigate the effects of urethane on renal sympathetic nerve activity (RSNA) in Wistar rats. MATERIALSAND METHODS: 46 rats were randomly allocated in two groups: group A in which rats were injected with urethane; group B in which barbital sodium was used as a control. The changes of RSNA, blood pressure (BP) and heart rate (HR) of each group were evaluated and analyzed. RESULTS: Compared to the control group, the value of RSNA, BP and HR were all significant decreased in rats of group A after urethane injection (p < 0.05). CONCLUSIONS: These results suggest that urethane could affect RSNA through somatosensory system.

The kinetics of urethane elimination in the mouse.

Data from several investigators suggest that the prevalence of urethane-induced lung adenomas in the mouse is more nearly linearly related to the square of the urethane dose than to the dose itself. However, the relationship between urethane dose and integrated internal exposure to urethane has not been established. Outbred male Swiss mice between 41 and 45 days old were injected ip with one of seven doses of urethane ranging from 0.4 to 1.8 mg/g. The rate of elimination of urethane from the blood was followed by assaying the ethanol liberated from urethane by alkaline hydrolysis. The results indicated that urethane elimination is saturable, and saturated at all doses used, with a Vmax of 0.087 mg/ml/hr. Thus, internal exposure to urethane, measured as the area under the blood concentration, time curve, is not linearly related to urethane dose. In the range of doses used in this study, the area under the curve is C0(2)/2Vmax or (D/VD)2 (1/2Vmax), where C0 is the initial concentration; D, the dose; and VD, the volume of distribution. This relationship can be used to predict internal exposure to urethane as a function of dose.

Measurement of ethyl carbamate in blood by capillary gas chromatography/mass spectrometry using selected ion monitoring.

Methodology is presented for convenient, reproducible and direct measurement of blood concentrations of ethyl carbamate, an experimental animal carcinogen. Extraction techniques requiring 20 microliters of blood and selected ion monitoring using ethyl (13C, 15N)carbamate as internal standard enabled quantification of ethyl carbamate concentrations ranging from 50 ng ml-1 to 100 micrograms ml-1. Coefficients of variation at several representative concentrations averaged less than 4%. The method was used to determine the time course of elimination of ethyl carbamate from mice receiving doses of 125 mumol kg-1.

Flow measurement of blood radioactivity in animals.

In order to permit flow measurement of blood radioactivity in an animal after establishing extracorporeal circulation, counting cells were constructed of various plastic scintillator materials. The cell was placed in a liquid scintillation spectrometer replacing the sample vial. Stable and reproducible counting rates were obtained only with cells made of blue Altustipe plastic. The counting efficiency was 1.8%. Using this detection system, continuous records were produced of blood radioactivity in miniature pigs after administration of 14C-urethane and [14C]methyl-piperazinyl pyrazino benzoxazepine maleate.

Consideration of the mechanism of pulmonary adenogenesis in urethane-treated Swiss mice.

A number of investigators have observed a quadratic relationship between acute urethane dose and cumulative pulmonary adenoma incidence in mice. The hypothesis was tested that this dose-effect relationship may be explained by consideration of the elimination kinetics of urethane. Single doses of 0.4 to 1.8 mg urethane per g body weight were given ip to 6-week-old Swiss-Cox mice. The measure of internal exposure to the intact urethane molecule for a given external urethane dose was taken to be the area under the curve (AUC alpha) of a blood urethane concentration versus time plot. AUC alpha was linearly related to tumor prevalence. When urethane elimination was induced by pretreatment of the mice with p,p'-DDT, the linear relationship of AUC alpha to tumor prevalence was shifted. Reduction of tumor prevalence by p,p'-DDT pretreatment was more marked than that predicted on the basis of exposure to urethane. Thus, the kinetic evidence is consistent with biochemical evidence from other investigators supporting the premise that activation of urethane to a reactive metabolite is required for adenoma formation in the mouse lung. While exposure to the adenogenic moiety is evidently closely proportional to internal exposure to urethane in both pretreated and non-pretreated mice, p,p'-DDT pretreatment causes a shift in this proportionality.

Metabolic changes induced by urethane-anesthesia in rats.

1. Fasting hyperglycemia was observed in urethane-anesthetized rats. No significant changes had been observed in fed animals. The effect is dose-dependent, being ineffective doses lesser than 1.4 g/kg of body weight. 2. Urethane originates a rise in glycemia during the first 10 min of anesthesia followed by control values at 30 min, and a latter hyperglycemic phase for more than 60 min that remain at 2 hr. 3. The negative correlationship between plasma glucose, lactate and amino acid levels suggest that gluconeogenesis may be the main responsibility of the observed hyperglycemia during the first phase, but it is possible that during the second phase a decrease in the consumption of glucose may take place as a consequence of the competitive effects of ketone bodies increased during the first 30 min of anesthesia. 4. We postulate that the mechanism of the hyperglycemic response to urethane is a sympathetic response with release of catecholamines both in the liver and in the adrenal gland which enhances gluconeogenesis and lipolysis.

Suitability of urethane anesthesia for physiopharmacological investigations in various systems. Part 1: General considerations.

The suitability of urethane anesthesia for physiopharmacological investigations is reviewed. Total dose administered and route of administration are recognized as factors having a great influence on both resting parameters and biological responses to drugs. A peculiar characteristic of urethane is represented by its ability to induce a surgical plane of anesthesia without affecting neurotransmission in various subcortical areas and the peripheral nervous system. This makes urethane a suitable general anesthetic for studying neural function in both central and peripheral nervous systems and accounts for the preservation of a number of reflex responses in urethane-anesthetized animals.