H. Peter

Metabolism of methyl chloride by human erythrocytes

Erythrocyte cytoplasm of rats, mice and humans was incubated in head space vials with methyl chloride and the decline in concentration of the substance monitored as a parameter of metabolism. The production of S-methylglutathione was controlled by tlc. Rats, mice, bovines, pigs, sheep and rhesus monkeys showed no conversion of methyl chloride in erythrocyte cytoplasm. About 60% of the human blood samples showed a significant metabolic elimination of the substance (conjugators), whereas about 40% did not (non-conjugators). The production of S-methylglutathione indicated enzymatic metabolism of the substance by glutathione S-transferases. In literature, a “major” and “minor” form of human erythrocyte glutathione S-transferase has been described. The results indicate that the “minor” form is probably responsible for the unique metabolism of methyl chloride in human erythrocytes.

A comparative investigation of the metabolism of methyl bromide and methyl iodide in human erythrocytes

SummaryHuman erythrocyte cytoplasm was incubated in head space vials with either methyl bromide or methyl iodide. The decline in concentration of the two methyl halides was monitored by gas chromatography. Simultaneously, the production of S-methylglutathione was determined by thin layer chromatography. In parallel experiments, boiled erythrocyte cytoplasm was used in order to determine non-enzymatic conjugation. Furthermore, inhibition experiments with sulfobromophthalein were performed. The results were compared with previous findings on the metabolism of methyl chloride. In contrast to methyl chloride, both methyl bromide and methyl iodide showed a significant non-enzymatic conjugation with glutathione. In addition, an enzymatic conjugation could be observed in the erythrocyte cytoplasm of the majority of the population, whereas a minority lacks this enzymatic activity. This is consistent with findings on methyl chloride. Inhibition experiments show that a minor form of the erythrocyte glutathione transferase may be responsible for the enzymatic conjugation. Of the three monchalogenated methanes, methyl bromide is the substrate with the highest affinity for the conjugating enzyme(s). In the case of methyl iodide, non-enzymatic reaction overweighs the enzymatic process. There are possible implications of the results for occupational health and the toxicity of the substances.

Dissociation of a new glutathione S-transferase activity in human erythrocytes

Human glutathione S-transferases (GST) can be distinguished according to their isoelectric point (pI); a distinction being made between «basic», «neutral», and «acidic» enzymes. These correspond to the three gene families α,μ and π. Isolation of these enzymes is generally performed by affinity chromatography on different sepharose matrices, using 1-chloro-2,4-dinitrobenzene (CDNB) as test substrate [1]

Determination of 7-(2-hydroxyethyl)Guanine with Gas Chromatography/Mass Spectrometry as a Parameter for Genotoxicity of Ethylene Oxide

The formation of epoxides from vinyl compounds is the initial step in genotoxicity of this class of chemicals. In a pharmacokinetic experiment with rats, Bolt and Filser (1987) showed, that exhaled ethylene oxide can be used to estimate the risk of an exposure to ethylene. In Fig. 1 some sites of reaction of ethylene oxide with cellular materials are shown. The alkylation of DNA mainly leads to an addition of a hydroxyethyl group to the N-7 position of guanine resulting in 7-(2-hydroxyethyl)guanine. Although this product does not effect the hydrogen bond to the complementary DNA strand, and therefore, may not be responsible for the genotoxic effects, it seems to be a good indicator for the detection of geno- toxic lesions because it represents about 90% of the alkylated DNA sites. Additionally 7-(2-hydroxyethyl)guanine can easily be released from DNA by heating.

Pharmacokinetics of isoprene in mice and rats

Pharmacokinetic analysis of isoprene inhaled by male Wistar rats and male B6C3F1 mice showed saturation kinetics in both species. Below atmospheric concentrations of 300 ppm in rats and in mice the rate of metabolism is directly proportional to the concentration. The low accumulation of isoprene in the body at low atmospheric concentrations suggests transport limitation of the metabolism. Only small amounts of isoprene taken up are exhaled as unchanged substance (15% in rats and 25% in mice). Its half life in rats is 6.8 min and in mice 4.4 min. At concentrations above 300 ppm the rate of metabolism does not increase further in proportion to the atmospheric concentration. It finally approaches maximal values of 130 mumol/(h X kg) body weight at atmospheric concentrations above 1500 ppm in rats, and 400 mumol/(h X kg) body weight at concentrations above 2000 ppm in mice. This indicates limited production of the two possible mono-epoxides of isoprene at high concentrations. Isoprene is endogenously produced and is systemically available. Its production rate is 1.9 mumol/(h X kg) in rats, and 0.4 mumol/(h X kg) in mice, respectively. Part of the endogenous isoprene is exhaled by the animals but it is metabolized to a greater extent: the rate of metabolism of endogenously produced and systemically available isoprene is 1.6 mumol/(h X kg) (rats) and 0.3 mumol/(h X kg) (mice).

Pharmacokinetics of the neurotoxin n-hexane in rat and man

The pharmacokinetics of inhaled n-hexane in rat and man were compared. In the rat metabolism was saturable. Up to 300 ppm, the metabolic rate was directly proportional to the concentration in the atmosphere, reaching 47 μmol/(h· kg). Only 17% of n-hexane was exhaled unchanged. Above 300 ppm, the amount of n-hexane in the body rose with increasing atmospheric concentrations from 1.6 up to a limiting value of 9.6, which corresponded to the thermodynamic distribution coefficient of n-hexane between the organism and the atmosphere. Up to 3000 ppm, the rate of metabolism increased to 245 μmol/ (h· kg); only a slow further increase was found up to 7000 ppm (285μmol/(h· kg)).In man the steady-state concentrations of n-hexane were about 1 ppm. The metabolic clearance was 1321/h, and n-hexane accumulated to a factor of 2.3 in the organism. The thermodynamic distribution coefficient was calculated to be 12. Twenty per cent of n-hexane in the body was exhaled unchanged.At low concentrations the rate of metabolism of n-hexane is limited in both species by transport to the enzyme system. Under these conditions the rate of metabolism of n-hexane should not be influenced by xenobiotics which induce the n-hexane metabolizing enzyme system.

Formation of DNA adducts in F-344 rats after oral administration or inhalation of [14C]methyl bromide

The genotoxic effects of methyl bromide were investigated in a DNA-binding study. [14C]Methyl bromide was administered to male and female F-344 rats orally, or by inhalation from a closed exposure system. DNA adducts were detected in the liver, lung, stomach and forestomach. [14C]3-Methyladenine, [14C]7-methylguanine and [14C]O6-methylguanine were identified using a combination of three different methods of hydrolysing DNA, followed by HPLC or gas chromatography-mass spectrometry. After both oral and inhalation exposure, the highest levels of methylated guanines, especially those of [14C]O6-methylguanine, were found in the stomach and forestomach of the rats. These results clearly demonstrate a systemic DNA-alkylating potential of methyl bromide.

Pharmacokinetics of Propylene and its Reactive Metabolite Propylene Oxide in Sprague-Dawley Rats

Inhaled propylene gas (PE) is metabolized by the rat to its epoxide propylene oxide (PO) (Svennson and Ostermann-Golkar 1984). The latter was carcinogenic in the rat (Dunkelberg 1982; Lynch et al 1984; Kuper et al 1988). In contrast, its metabolic precursor PE was not carcinogenic in a long-term inhalation study (Quest et al 1984). The aim of the present work was to provide an explanation for these different findings through an evaluation of the pharmacokinetics of PE and PO.

DNA-binding assay of methyl chloride

Fischer-344 rats and B6C3F1 mice of both sexes were exposed in closed chambers to 14C-labeled methyl chloride. Different clearance values from the gas phase of the system indicated that, based on body weight, mice metabolized the test compound much faster than rats. After isolation of DNA and nucleoproteins from liver and kidneys radioactivity was found in all macromolecular samples; this was ascribed to metabolic C1-incorporation. Radioactivity incorporation was particularly high in DNA of mouse kidneys, suggesting a high turnover to active C1 bodies (formaldehyde, formate) in this tissue.Analyses of DNA samples from kidneys of female and male mice showed neither 7-N-methylguanine nor O6-methylguanine. Hence, the formation of tumors in B6C3F1 mice exposed to high concentrations of methyl chloride is not based on methylation of DNA in this tissue.

DNA adducts of halogenated hydrocarbons

SummaryAlthough formation of DNA adducts has been postulated for several halomethanes, no chemical identification of such adducts has been performed so far. There is, however, evidence that methyl chloride does not act biologically as a DNA methylating agent. 1,2-Dichloroethane and 1,2-dibromoethane are activated through conjugation with glutathione. There is some evidence for formation on an N-7 adduct of guanine which carries an ethyl-S-cysteinyl moiety.Extensive work has been published on adducts of vinyl chloride, both in vitro and in vivo. The major DNA adduct is 7-(2-oxoethyl)guanine; a minor adduct appears to be N2,3-ethenoguanine. Other “etheno” adducts, i.e., 1,N6-ethenoadenine and 3,N4-ethenocytosine, are readily formed with DNA, vinyl chloride, and a metabolizing system in vitro and with RNA in vivo, but are usually not detected as DNA adducts in vivo.The data on DNA alkylation by vinyl chloride (and vinyl bromide) metabolites are compared with those of structurally related compounds (acrylonitrile, vinyl acetate, vinyl carbamate).