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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.

Rats were exposed, in a closed desiccator jar chamber, to concentrations of various halogenated C1 and C2 compounds at which the metabolizing capacities were saturated (Vmax conditions). Within the exposure period of 50 h concentrations of the xenobiotic and of exhaled acetone were monitored in the gas phase of the system. The quantitative extent of acetone exhalation was dependent on the individual compound examined. Acetone exhalation was stimulated in presence of vinyl chloride, vinyl bromide, vinyl fluoride, vinylidene fluoride, cis- and trans-1,2-dichloroethylene, trichloroethylene, perchloroethylene, methylene chloride, chloroform, carbon tetrachloride and 1,1,2-trichloroethane. No stimulation of acetone exhalation occurred with 1,1,1-trichloroethane and with the reference hydrocarbon n-hexane. Also, acetone exhalation was evoked by infusions of either fluoroacetate or chloroacetate, two anticipated or proven metabolites of some haloethylenes; the infusion rates of which were based on calculations of the metabolic rates of vinylidene fluoride and of vinyl chloride, respectively.

When rats were dosed with 14 microgram/kg 3H-isoproterenol, 3H-radioactivity was measurable in the liver until 48 h. This amount was not different in livers of animals which have been pretreated with diethyl maleate. After exhaustive extraction, a significant amount of 3H-radioactivity from isoproterenol could be detected in the proteins of total liver (homogenate), of cytosol and of microsomes. In the cytosol fraction of diethyl maleate pretreated animals twice the amount of isoproterenol-radioactivity was found in the extracted proteins compared to controls. In the microsomal fraction there was no difference between diethyl maleate pretreated and control animals in the amount of radioactivity incorporated into proteins. In all fractions the radioactivity measurable in the extracted proteins declined with a half life time of about 24 h. The in vivo results on covalent binding of isoproterenol are compared to the irreversible protein binding of ethinyloestradiol in vivo. Quantitatively, these in vivo data are compared to the results on irreversible protein binding obtained during incubations of isoproterenol or ethinyloestradiol with rat liver microsomes.

Halogenated ethylenes are metabolized to reactive intermediates which covalently bind to different cellular targets. Vinyl chloride and vinyl bromide metabolites bind to DNA, preferably to N-7 of deoxyguanosine. With RNA, 1,N6-ethenoadenosine and, 3,N4-ethenocytidine moieties are formed. All the haloethylenes in which this effect has been studied form metabolites capable of alkylating proteins, preferably at free sulfhydryl groups. Also, there is alkylation by haloethylene metabolites of cellular coenzymes. An observed increased exhalation of acetone by rats exposed to different haloethylenes can possibly be explained by alkylation of cytosolic coenzyme A. Such metabolic effects may serve as an indicator for reactive metabolite formation in vivo and should be more investigated.

Different kinds of experimental liver damage in rats are evaluated as to associated changes in breakdown of natural estrogens. Acute or chronic treatment of rats with ethanol does not influence aromatic hydroxylation of estradiol, as indicated by liver microsomal replacement of tritium from 2,4,6,7-3H-estradiol. More severe liver damage by CCl4 or thioacetamide, which lowers hepatic cytochrome P-450, causes impairment of estrogen degradation: CCl4 dosage leads to a marked decrease in aromatic hydroxylation of estradiol. Whereas thioacetamide in a chronic application schedule has been previously reported to a decrease microsomal aromatic estrogen hydroxylation, a single dose of 300 mg/kg thioacetamide in rats causes increased microsomal formation of estrone from estradiol, which is regarded to be a better (i.e., more lipophilic) substrate for the microsomal estrogen 2-hydroxylase than is estradiol. The data show that hepatotoxic agents may act differentially on hepatic metabolism of endogenous steroids.

Male Wistar rats were exposed to n-hexane concentrations between 50 and 3000 ppm for 8 h, and urinary excretion kinetics of the n-hexane metabolites 1-hexanol, 2-hexanol, 3-hexanol, 2-hexanone, 2,5-hexanedione, and 4,5-dihydroxy-2-hexanone were assessed. The amounts of metabolites excreted were linearly dependent on the n-hexane exposure concentration, up to an exposure of about 300 ppm. Above 300 ppm exposure the metabolite excretion indicated saturation kinetics in the metabolism of n-hexane. In its quantity, the newly described 4,5-dihydroxy-2-hexanone was the second metabolite, its amount in the urine being about ten times higher than that of excreted 2,5-hexanedione. Using gas chromatography-mass spectrometry the occurrence of 4,5-dihydroxy-2-hexanone as an n-hexane metabolite in urine of man was confirmed after exposure of a male volunteer to a mean of 217 ppm n-hexane for 4 h (laboratory exposure). Twenty-six hours after starting this exposure the excretion of 4,5-dihydroxy-2-hexanone (as a result of the n-hexane exposure) reached a level which was four times higher than the excretion of 2,5-hexanedione. The results in both rat and man indicate the relevance of 4,5-dihydroxy-2-hexanone as a metabolite of n-hexane metabolism. Formation of this metabolite may be viewed as a route of detoxification.

Male Wistar rats were exposed to 1000 ppm n-hexane, and the excreted urinary metabolites were analyzed by capillary gas chromatography-mass spectrometry (GC-MS). 1-Hexanol, 2-hexanol, 3-hexanol, 2-hexanone, 2,5-hexanedione, 2,5-dimethyltetrahydrofuran, 2,5-dimethyl-2,3-dihydrofuran and gamma-valerolactone were identified by their retention times and their mass spectra. Quantitative gas chromatographic analyses were performed using an FID. Experiments on the hydrolysis of conjugated n-hexane metabolites revealed that enzymatic hydrolysis (in addition to acid hydrolysis) was not required, as treatment with HCl hydrolyzed conjugates sensitive to acid as well as conjugates sensitive to beta-glucuronidase. By incorporating acid hydrolysis only and by using C18-cartridges for sample extraction, a method was developed that allowed the determination of n-hexane metabolites with a sample preparation time of only 45 min. Assay precision was assessed by repeated analyses of the same urine sample. Coefficients of variation for the individual metabolites ranged from between 1.8 and 3.3.

The state of the literature led to a re-investigation of the alkylation products caused by vinyl chloride metabolites in DNA. When rat liver microsomes, an NADPH-regenerating system, DNA and [14C]vinyl chloride were incubated and, when the DNA was subsequently re-isolated and (enzymatically) hydrolyzed, chromatograms (on Aminex A-6) showed the presence of 1,N6-ethenodeoxyadenosine, 3,N4-ethenodeoxycytidine and 7-N-(2-oxoethyl)guanine (the product of hydrolysis of 7-N-(2-oxoethyl)-deoxyguanosine). By contrast, when rats were exposed to [1,2-14C]vinyl chloride and when the liver DNA of these rats was subjected to similar procedures, no radioactive 'etheno' derivatives could be detected, but a radioactive peak was eluted with 7-N-(2-oxoethyl)guanine. This peak could be transformed into 7-N-(2-hydroxyethyl)guanine; the chromatographic behaviour of which was identical to the reference compound used by Ostermann-Golkar et al. (Biochem. biophys. Res. Commun., 76 (1977) 259). Thus, it is concluded that the compound described by these authors, 7-N-(2-oxoethyl)guanine is in fact the major product of base alkylation in DNA after exposure to vinyl chloride.

The roles of chloroethylene oxide (CEO) and chloroacetaldehyde (CAA) in carcinogenicity of vinyl chloride (VC) have been studied by comparing biological effects of VC exposure with those of 2,2'-dichlorodiethylether (bis(chloroethyl)ether, BCEE) as a metabolic precursor of CAA. Biological end-points investigated were covalent protein binding, nucleic acid (RNA and DNA) alkylation and the potency of the two chemicals to induce preneoplastic ATPase-deficient foci in rat liver. After exposure of rats to [1-14C]BCEE, BCEE derived radioactivity was bound to liver proteins. Analysis of hydrolysates of liver RNA and DNA gave no indication for the formation of either 7-N-(2-oxoethyl)guanine, 1,N6-ethenoadenine or 3,N4-ethenocytosine residues within the nucleic acids. After application of VC, BCEE or chloroethanol [CE), also a precursor of CAA) to young rats, only animals exposed to VC developed preneoplastic hepatocellular ATPase-deficient foci. From these investigations it is concluded, that CEO (which is not formed during metabolism of BCEE and CE), not CAA, is the ultimate carcinogenic principle in VC carcinogenicity.

Acute acrylonitrile intoxications are followed by loss of consciousness, convulsions, respiratory arrest, and may end fatally. Common cyanide antidotes have not proved to be effective. In previous animal experiments we could demonstrate that the cyanide antidotes show some protective effect only after oral acrylonitrile application. Only a minimal amount of inhaled acrylonitrile will be transformed into cyanide, in contrast to pharmacokinetics after oral intake. After acrylonitrile inhalation the toxic effect of the whole molecule ( cyanethylation ) is important, and sulfhydryl compounds show antidotal effects. Further animal experiments demonstrate the superior antidotal effects of N-acetyl-cysteine after acrylonitrile inhalation. Intravenous injection of N-acetyl-cysteine in high doses is recommended according to the treatment of paracetamol poisoning.

Metabolism of 1,3-butadiene to 1,2-epoxybutene-3 in rats follows saturation kinetics. Comparative investigation of inhalation pharmacokinetics in mice also revealed a saturation pattern. For both species "linear" pharmacokinetics apply at exposure concentrations below 1000 ppm 1,3-butadiene; saturation of butadiene metabolism is observed at atmospheric concentrations of about 2000 ppm. For mice metabolic clearance per kg body weight in the lower concentration range where first order metabolism applies was 7300 ml X h-1 (rat: 4500 ml X h-1. Maximal metabolic elimination rate (Vmax) was 400 mumol X h-1 X kg-1 (rat: 220 mumol X h-1 X kg-1. This shows that 1,3-butadiene is metabolized by mice at higher rates compared to rats. Based on these investigations, the metabolic elimination rates of butadiene in both species were calculated for the exposure concentrations applied in two inhalation bioassays with rats and with mice. The results show that the higher rate of butadiene metabolism in mice when compared to rats may only in part be responsible for the considerable difference in the susceptibility of both species to butadiene-induced carcinogenesis.

The time course of free oestradiol and oestrone as well as of total (free and conjugated) oestrone was determined in plasma of women after oral ingestion of 3.75 mg conjugated equine oestrogens, or 9.74 micromol of oestrone sulfate, oestrone, and oestradiol, respectively. All these oestrogen preparations led to transiently increased plasma oestrogen levels which fell to normal values after 48 h. The main difference observed between administration of free oestrone or oestradiol and of conjugated oestrogens (oestrone sulfate or equine oestrogens) was a much more protracted influx of oestrogens from the intestine into the plasma compartment, with a tendency to more sustained plasma levels, if conjugated oestrogens were administered. There was a consistent discontinuity in plasma oestrogen levels 10--12 h after oral ingestion of all the preparations examined indicating enterohepatic circulation. Comparison of "areas under the curve" obtained with the present preparations to similar previous studies on ethinyl oestradiol indicated that the bioavailability of the non-ethinyl oestrogens is by more than one order of magnitude less than that of ethinyl oestradiol after oral administration. The ratio of oestrone/oestradiol was the highest with free oestrone and similar among the other three preparations indicating an increased metabolism of sulfoconjugated oestrone to oestradiol after oral application when compared with free oestrone.

14,15-3H-Norethisterone-4 beta, 5 beta-epoxide, a metabolite of norethisterone, was incubated with several proteins and nucleic acids. After 30 min incubation 0.19 nmol of the epoxide were irreversibly bound per mg albumin which contains free sulfhydryl groups; proteins without SH-groups, such as concanavalin A, gamma-globulin, DNA and RNA, did not irreversibly bind norethisterone epoxide. A superoxide (O2) generating enzyme system comprised of xanthine oxidase and hypoxanthine was capable of catalyzing the irreversible binding of the parent compound, norethisterone, to albumin, indicating that an oxidation product was formed which reacted with the protein. When norethisterone epoxide was incubated for 60 min with hepatic microsomes of rats in absence of NADPH, about 2.0 nmol of the epoxide were irreversibly incorporated per mg microsomal protein. This binding was increased to 5.2 nmol by addition of a NADPH regenerating system. Addition of glutathione and cytosol decreased only the NADPH-dependent protein binding; phenobarbital pretreatment of rats induced this NADPH-dependent binding of norethisterone epoxide to microsomal protein by a factor of 2. In presence of NADPH, binding of the epoxide to microsomal protein depended on substrate concentration used. The results indicate that norethisterone epoxide is able to chemically react with proteins. In addition, hepatic microsomal enzymes convert the epoxide to another metabolite which also can react with proteins.

Rats have been exposed in a closed system to the chlorinated ethylenes vinyl chloride and trichloroethylene and to carbon tetrachloride as a reference compound. Data of uptake of the compounds, of urinary excretion of metabolites, and of exhalation after exposure show that the chlorinated ethylenes are metabolized much faster than carbon tetrachloride, probably due to their common ethylene structure. To eliminate differences in uptake, calculation of metabolites of the three compounds in tissues was based on the amount actually taken up by the animals. Vinyl chloride, trichloroethylene, and carbon tetrachloride showed irreversible binding of metabolites to tissue proteins, mainly of the liver. Irreversible protein binding of either of these compounds ranged within the same order of magnitude, if related to the amount of compound which had been taken up. Also, no differences in the relative portion of irreversibly bound metabolites were found after exposure to different atmospheric concentrations of the three compounds. As already shown for vinyl chloride, trichloroethylene is metabolized in vitro by rat liver microsomes in presence of NADPH-regenerating system to intermediates that irreversibly bind to proteins. Albumin (bovine and rabbit) was a preferred target for binding. In contrast to vinyl chloride, significant irreversible binding of trichloroethylene metabolites also occurred to non-SH-proteins (gamma-globulin, concanavalin A) and to polylysine. Hence it should be inferred that, unlike vinyl chloride, trichloroethylene metabolites not only bind to sulfhydryl groups but also, to a lesser extent, to free amino groups of proteins.

1. Oxidation of estradiol and ethynylestradiol at ring A and ring B by rat liver microsomes and NADPH-regenerating system in vitro is inhibited by the two arylimidazole insecticide synergists, 3-bromophenyl-4(5)-imidazole and 1-naphthyl-4(5)-imidazole, but not by the benzothiadiazole insectide synergists 6-nitro-1,2,3-benzothiadiazole and 5,6-dimethyl-1,2,3-benzothiadiazole. The Ki of the most potent inhibitor, 1-naphthyl-4(5)-imidazole, was 3 X 10(-6) M. 2. 6-Nitro-1,2,3-benzothiadiazole (10(-6) M), which did not inhibit hydroxylation of the estrogens, inhibited oxidation of aniline and demethylation of ethylmorphine, p-nitroanisole, and aminopyrine by 30-70%. 5,6-Dimethyl-1,2,3-benzothiadiazole inhibited only demethylation of p-nitroanisole and aminopyrine. From these results the presence of different hepatic microsomal mixed function oxidases may be inferred. 3. 1-Naphthyl-4(5)-imidazole, the most potent inhibitor of hydroxylation of drugs and estrogen rings A and B, also inhibited microsomal estrogen-16alpha-hydroxylation. 4. These data show that insecticide synergists may effect the breakdown of estrogenic hormones in the organism.

The physiological effect of estrogens on the estrogen-dependent target organs is mediated by estrogen receptors which are present in the cytoplasm of estrogen-sensitive cells. By determining the estrogen receptors in mammary carcinoma tissue, information may be obtained on the susceptibility of the tumor concerned to hormones. As a novel pharmacological principle, the anti-estrogens are able to blockade the hormonal effect of endogenous estrogens on the tissue of the mammary carcinoma. Possible relationships between prolactin and mammary carcinoma become apparent. From the pharmacologist's point of view, these questions are of particular importance in relation to the possibility of predisposition to mammary carcinoma through treatment with reserpine, which is at present under discussion.

Fast transport kinetics of 51Cr (VI) into red blood cells (RBCs) in vitro were studied. No significant species differences were found between RBCs of man and rat. The uptake of 51Cr (VI) by RBCs in whole blood was composed of two different first order processes of different velocities (apparent t 1/2 of 22.7 s and 10.4 min for man and 6.9 s and 10.1 min for rat, respectively). However, even after longer time periods a fixed portion of approximately 15% of the administered dose remained in the plasma and did not penetrate into RBCs Over the entire concentration range studied (10 microM-50 mM), the fast initial uptake followed Michaelis-Menten kinetics. The maximal capacity of this Cr(VI) transport into RBCs of man and rat was 3.1 X 10(8) CrO4(2-) ions X cell-1 X min-1 and 2.5 X 10(8) CrO4(-2) ions X cell-1 X min-1, respectively. It is likely that Cr(VI) is transported into RBCs via a physiological anion carrier ("band-3-protein").

Uptake of 20 micron 51Cr(VI) by resuspended red blood cells (RBC) (0.9% saline) was studied. Inhibition of the specific anion carrier system "band-3-protein" of RBC membrane with "SITS" (4-acetamido-4'-cyanatostilbene-2,2'-disulphonic acid) resulted in a non-competitive type of inhibition for chromium uptake. It is concluded that Cr(VI) is transported by participation with this system. Intracellularly, chromium is almost quantitatively bound to hemoglobin and trapped for the cell's lifetime. Glutathione (GSH) is possibly involved in this process as indicated by incubation experiments of RBC with 10 mM Na2(51)CrO4. In these experiments, a rapid decrease of GSH content of the cells was observed.