Ninzo Murayama

Three forms of trichloroethylene-metabolizing enzymes in rat liver induced by ethanol, phenobarbital, and 3-methylcholanthrene

In vitro metabolism of trichloroethylene (TRI) and trichloroethanol (TCE) was investigated using liver microsomes from control and ethanol-, phenobarbital (PB)-, and 3-methylcholanthrene (MC)-treated rats. At least three forms of enzymes were involved in TRI metabolism. One was a low-Km type normally existing in microsomes from control rats. The ethanol-inducible enzyme was found to be catalytically identical to this low-Km isozyme. Another was a high-Km type which was induced exclusively by PB, and a third was an MC-inducible isozyme with a Km value between those of ethanol- and PB-inducible isozymes. Although MC treatment did not affect the rate of TRI metabolism in vitro, both ethanol and PB treatment markedly enhanced the metabolism. Ethanol-induced enhancement was different from PB-induced enhancement in that ethanol enhanced the metabolism predominantly at low substrate concentrations, whereas PB did so at high concentrations. In addition, TRI metabolism with enzymes from ethanol-treated rats was inhibited by the substrate itself at high concentrations. MC treatment of rats had little or no influence on the rate of TCE metabolism in vitro, whereas both ethanol and PB enhanced the microsomal conversion of TCE to chloral hydrate. As in the case of TRI metabolism, ethanol induced a microsomal TCE-metabolizing enzyme of low Km, whereas PB preferentially induced an enzyme of high Km.

A pharmacokinetic model to study the excretion of trichloroethylene and its metabolites after an inhalation exposure.

For a better understanding of absorption, distribution, excretion, and metabolism of trichloroethylene the time-course of blood concentration of the vapour and urinary excretion of its metabolites was examined using a pharmacokinetic model. After a single experimental exposure in which four men inhaled 100 parts per million (ppm) of trichloroethylene for four hours an elimination curve showed three exponential components, that is, X=1-0005e(-16.71t)+0-449e(-1.710t)+0-255e(-0.2027t), where X is that blood concentration in mg/l and t the time in hours from 0 to 10. The overall rate constant for the disappearance of trichloroethylene was found to a agree with the theoretical one, estimated by means of a mathematical model for the blood concentration data. A D8- XD plot, developed from a mathematical model for urinary excretion, could also give a good estimate of rate constant for the transfer of trichloroethylene in the body. The rate constant thus estimated from urinary excretion was consistent with data on the blood concentration.

Kinetic studies on sex difference in susceptibility to chronic benzene intoxication--with special reference to body fat content.

The sex difference in the susceptibility to haematopoietic disorders induced by benzene was studied kinetically with a special reference to its relation with the body fat content. In rats of both sexes with a large body fat content, benzene was eliminated more slowly and remained in the body for a longer time than in rats with a small body fat content. In accord with this finding, the decrease in white blood cell numbers during a chronic benzene exposure was observed only in the groups of rats which had a large volume of fat tissue. In an experimental human exposure, the elimination of benzene was slower in the females than in the males. The kinetic study revealed that the slower elimination in the females is due primarily to the bulky distribution of body fat tissue in that sex. From these results obtained from the experimental exposure of men and rats to benzene, it was concluded that the human female, with her massive body fat tissue, shows an inherent disposition to be susceptible to a chemical such as benzene which has a high affinity with fat tissue.

Monoclonal antibody-directed assessment of toluene induction of rat hepatic cytochrome P450 isozymes

Cytochrome P450 isozymes induced in rat liver by a range of concentrations of toluene were studied with monoclonal antibodies (MAbs) to specific P450 isozymes and by enzyme assays. Nitrosodimethylamine demethylase activity was significantly increased in microsomes from rats exposed to more than 1000 ppm of toluene, an increase that was dose-dependent. Anti-CYP2E1 significantly inhibited the metabolism of toluene to benzyl alcohol (BA) by about 50%, in microsomes from 1000 to 4000 ppm toluene-exposed rats, at low substrate concentration (0.2 mM). With anti-CYP2B1/2, the rate of BA formation was decreased by 15-17% in microsomes from rats of 2000 and 4000 ppm toluene exposures at high substrate concentration (5.0 mM). On the other hand, anti-CYP2C11/6 inhibited the rate of formation of BA in all of the microsomes, but the extent of inhibition was progressively decreased from 55% in control to 33% in 4000 ppm exposure. Immunoblot analysis with anti-CYP2E1 and anti-CYP2B1/2 revealed stronger immunoreactive bands in microsomes from rats exposed to more than 1000 and 2000 ppm of toluene, respectively. Stronger bands were also observed in microsomes from rats of 2000-4000 ppm toluene exposures with anti-CYP3A1/2, but no immunoreactivity appeared with anti-CYP1A1/2. These results suggest that toluene induces CYP2E1, CYP2B1/2 and CYP3A1/2, but reduces CYP2C11/6, and has no effect on CYP1A1/2.

Immunochemical assessment of the influence of nutritional, physiological and environmental factors on the metabolism of toluene

SummaryFactors influencing the metabolism of toluene were investigated in rats using monoclonal antibody (MAb) to cytochrome P450 (P450). At low toluene concentrations, P450 IIE1 was primarily involved in the metabolism of toluene, whereas P450 IIC11/6 was involved at high concentrations. A low-carbohydrate diet induced P450 IIE1 and resulted in an increase in toluene metabolism. The intake of fat did not influence the metabolism. A lowered protein intake decreased not only the total content of P450 but also the P450 IIC11/6. Fasting and ethanol consumption also enhanced toluene metabolism via the induction of P450 IIE1. The metabolic rate of toluene in adult male rats was 4-fold higher than in immature males and adult females at a high substrate concentration because of the high level of P450 IIC11/6 in adult males, whereas no difference was noted between adult and immature females. Although development did not influence toluene metabolism in males at a low substrate concentration, the metabolic rate in adult female rats was significantly lower than that of immature females and males; this may be due to the decrease in P450 IE1 with development. Diabetic status influenced toluene metabolism in rats by affecting several kinds of P450 isozymes. Toluene exposure also affected its own metabolism by increasing P450 IIE1 and P450 IIB1/2, and decreasing P450 IIC11/6. A significant difference in toluene metabolism was observed among rat, mouse and human liver microsomes. Thus, when considering the factors affecting toluene metabolism, it is important to elucidate the change in specific P450 isozyme composition related to the modifications, and their affinities to toluene.

Simultaneous gas-chromatographic analysis of lower fatty acids, phenols and indoles in faeces and saliva using a fused silica glass capillary column

The simultaneous gas-chromatographic separation of a mixture of 14 lower fatty acids, 11 phenols and 7 indoles was performed by using a fused silica glass capillary column of Carbowax 20M (50 m × 0.2 mm i.d., Carbowax 20M deactivated). Complete separation of the mixture was obtained, except for the peaks of phenols and o-cresol, o-ethylphenol and 3,5-xylenol and pelargonic acid, 2,3-xylenol and 1,2-dimethylindole, whose peaks overlapped, and 2- and 3-methylindoles, which were poorly separated. The optimum conditions are as follows: column temperature, held for 1 min at 100 °C; column oven, heated at 4 °C min–1 from 100 to 220 °C, maintained at 220 °C for 9 min for standard compounds or 29 min for sample specimens, then cooled to 100 °C; and carrier gas (nitrogen) flow-rate, 0.97 ml min–1. The method was applied to the analysis of the lower fatty acids, phenols and indoles in cat and human faeces and non-smoker saliva.

Health care of workers engaged in waste water treatment. 1) The exposure conditions to organic solvents in workers engaged in waste water treatment

In the investigation of the exposure conditions to organic solvents of workers in 35 waste water treatment facilities, it was found that these workers treated various kinds of solvents. Almost all of the facilities treated chloroform, acetone, n-hexane, methanol and carbon tetrachloride. The average exposure concentration of the workers was, however, low. Exposure concentration to organic solvents (chloroform, xylene and carbon tetrachloride) was evaluated in one of the facilities. The average concentration of the respective solvents in the workers did not exceed the threshold limit value of Japan, but the concentration calculated on the basis of combined exposure exceeded the threshold limit value. On the other hand, high concentrations of organic solvents were detected in the room where the solvents were introduced but not in the working rooms, indicating that the workers engaged in waste water treatment were mainly exposed to organic solvents in the former room where they sorted and mixed the solvents and placed them in the incinerator. In conclusion, improvement in working conditions and health care for workers engaged in waste water treatment facilities should be made in order to prevent workers from becoming intoxicated by organic solvents.