Studies with alkylating esters—II: A chemical interpretation through metabolic studies of the antifertility effects of ethylene dimethanesulphonate and ethylene dibromide
Abstract
The metabolism of 1,2-14C-ethylene dimethanesulphonate (EDS) has been followed in the rat and mouse and compared with that of 1,2-14C-ethylene dibromide (EDB). EDS is excreted unchanged in urine together with S-(2-hydroxyethyl)-cysteineN-acetate and S-(2-hydroxyethyl)-cysteine-N-acetate-S-oxide. EDB is not excreted unchanged but is metabolised mainly to S-(2-hydroxyethyl)-cysteine and its N-acetate.
The distribution of radioactive label in mouse tissues is different for both compounds which may indicate a difference in the origin of cysteinal units for alkylation. Both ethylene dimethanesulphonate and ethylene dibromide react efficiently in vitro with SH containing compounds by a reaction analogous to the “sulphur stripping” action of Myleran, although the metabolism of EDS and EDB would not indicate such a reaction takes place in vivo. The antispermatogenic actions of EDS differ from those of its homologues, such as Myleran, but show some resemblance to those of EDB. These effects of EDS and EDB may indicate that both compounds have latent “mustard-like” activity, and this possibility is discussed in terms of the pharmacological effects produced by EDS and its chemical reactions with nucleophiles.
References (14)
- K. Edwards et al.
Biochem. Pharmac.
(1969) - E. Boyland
Biochem. Pharmac.
(1961) - J.J. Roberts et al.
Biochem. Pharmac.
(1961) - R.F. Hudson et al.
Biochem. Pharmac.
(1958)G.M. TimmisAnn. N. Y. Acad. Sci.
(1958) - T.A. Connors et al.
Biochem. Pharmac.
(1958) - H. Jackson
Br. Med. Bull.
(1964) - A.R. Jones et al.
Experientia
(1968)
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Activation of α<inf>1</inf>-adrenergic receptors potentiates the nephrotoxicity of ethylene dibromide
2003, ToxicologyEthylene dibromide (EDB) has been used as a model compound for eliciting hepato- and nephrotoxicity. Conjugation with glutathione (GSH) has been shown to play a role in the bioactivation of EDB. The aim of this study was to determine whether activation of α1-adrenergic receptors, which causes a decrease in cellular GSH levels, could modulate the nephrotoxicity of EDB. For this purpose, male ICR mice were treated with EDB and/or the α-adrenergic agonist, phenylephrine (Pe), or the α-adrenergic antagonist, phentolamine (Phe). Animals treated with EDB (40 mg/kg, i.p.) had a 9.3-fold increase in urinary γ-glutamyltranspeptidase (GGTP: EC 2.3.2.2) activity and a 38% decrease in renal non-protein bound sulfhydryl (NPSH) levels; however, animals co-treated with EDB and Pe (50 mg/kg, i.p.) exhibited a 27.8-fold increase in urinary GGTP activity and a 60% decrease in NPSH levels. The enhanced presence of urinary GGTP and decrease in cellular levels of NPSH was nearly blocked by treating animals concomitantly with EDB and Phe (10 mg/kg, i.p.) or EDB, Pe, and Phe. Histopathological examination revealed the enhanced degree of tissue damage and necrosis following treatment with EDB and Pe, and the protective effect of Phe at ameliorating EDB toxicity. These results indicate that factors that can influence α-adrenergic receptors may be critical in assessing dose–response data used in the risk assessment process.
The use of human in vitro metabolic parameters to explore the risk assessment of hazardous compounds: The case of ethylene dibromide
1997, Toxicology and Applied PharmacologyEthylene dibromide (1,2-dibromoethane, EDB) is metabolized by two routes: a conjugative route catalyzed by glutathioneS-transferases (GST) and an oxidative route catalyzed by cytochrome P450 (P450). The GST route is associated with carcinogenicity. An approach is presented to use human purified GST and P450 enzymes to explore the importance of these metabolic pathways for manin vivo.This strategy basically consists of four steps: (i) identification of the most important isoenzymesin vitro,(ii) scaling to rate per milligram cytosolic and microsomal protein, (iii) scaling to rate per gram liver, and (iv) incorporation of data in a physiologically based pharmacokinetic (PBPK) model. In the first step, several GST isoenzymes were shown to be active toward EDB and displayed pseudo-first-order kinetics, while the EDB oxidation was catalyzed by CYP2E1, 2A6, and 2B6, which all displayed saturable kinetics. In the second step, the predictions were in agreement with the measured activity in a batch of 21 human liver samples. In the third step, rat liver P450 and GST metabolism of EDB was predicted to be in the same range as human metabolism (expressed per gram). Interindividual differences in GST activity were modeled to determine “extreme cases.” For the most active person, an approximately 1.5-fold increase of the amount of conjugative metabolites was predicted. Lastly, it was shown that the GST route, even at low concentrations, will always contribute significantly to total metabolism. In the fourth step, a PBPK model describing liver metabolism after inhalatory exposure to EDB was used. The saturation of the P450 route was predicted to occur faster in the rat than in man. The rat was predicted to have a higher turnover of EDB from both routes. Nevertheless, when all data are combined, it is crucial to recognize that the GST remains significantly active even at low EDB concentrations. The limitations and advantages of the presented strategy are discussed.
Potential role of the flavin-containing monooxygenases in the metabolism of endogenous compounds
1995, Chemico-Biological InteractionsSeveral xenobiotics and their corresponding cysteine S-conjugates are metabolized in vivo to cysteine S-conjugate sulfoxides and/or N-acetylcysteine S-conjugate sulfoxides. Homocysteine S-conjugates, such as methionine and ethionine, are also metabolized in vivo to sulfoxides. The enzymatic basis for these metabolic reactions is not known. Recently, the rat liver and kidney activities were found to be associated with flavin-containing monooxygenases that are structurally and immunochemically related to known FMO1 isoforms. Further evidence for FMO1 being the major FMO isoform involved in sulfoxidation was obtained from kinetic studies with cDNA-expressed rabbit FMOs. Endogenous cysteine S-conjugates, e.g. cysteinylcatecholamines, cysteinylleukotrienes, lanthionine and djenkolic acid may also be substrates for FMOs, since can be considered a model for these compounds. Methionine, an endogenous homocysteine S-conjugate, was shown to be a substrate for cDNA-expressed rabbit FMO1, FMO2, and FMO3, however, the methionine sulfoxidation reaction was preferentially catalyzed by FMO3. These results suggest that FMOs may also play a role in the in vivo metabolism of endogenous homocysteine S-conjugates.
Hamster leydig cells are less sensitive to ethane dimethanesulfonate when compared to rat leydig cells both in vivo and in vitro
1995, Toxicology and Applied PharmacologyIt has been reported that ethane dimethanesulfonate (EDS) is a Leydig cell toxicant that affects rats and hamsters (Kerr et al., 1987), while, in contrast, the Leydig cells of mice are relatively insensitive to the toxicant. In the rat, there is a rapid decline in levels of testosterone (T) within hours after EDS administration. However, T production, spermiogenesis, and fertility are restored within a few weeks as new Leydig cells are formed from undifferentiated cells in the interstitium of the testis. In an earlier study, we found, as expected, that ejaculated sperm counts (ESCs) reached a nadir 10 days after adult rats were dosed with EDS at 65 mg/kg ip along with serum and testicular T, testis and seminal vesicle weights, and in vitro T production, while, in contrast, EDS at 65 mg/kg had no effect on these endpoints in the Syrian hamster (Gray et al., 1992). In the current study, when EDS was administered to 6, 12, and 18 month old hamsters at 100 mg/kg, it produced subtle effects on serum T and sex accessory gland weights, while dramatic effects were seen in similarly exposed rats. In addition, when testes were examined by light microscopy all treated rats displayed severely reduced Leydig cell numbers, while, in contrast, only one-third of the EDS-treated hamsters were affected, having moderately reduced Leydig cell numbers. In support of the histological data, 3 β-HSD enzyme activity was reduced by 99% of control in EDS-treated rats, but it was reduced by only 35% of control in EDS-treated hamsters. An in vitro analysis of the effects of EDS on LH-stimulated T production by quartered testes demonstrated that the hamster testis was less sensitive to the direct effects of EDS than the rat testis. The IC50 after 3 hr in culture was greater than 1800 μg EDS/ml for the hamster quarter testes, while the IC50 for the rat quarter testes was 320 μg EDS/ml. In summary, these results demonstrate in vivo and in vitro that Leydig cells of hamsters are less sensitive to EDS than those of the adult rat.
Mechanism by which ethane dimethanesulfonate kills adult rat leydig cells: Involvement of intracellular glutathione
1993, Toxicology and Applied PharmacologyWe have previously demonstrated that ethane-1,2-dimethane-sulfonate (EDS) kills adult, but not immature, rat Leydig cells in vivo and in vitro. The mechanism responsible for this selective toxicity is not known. Here we report that the cytotoxic effects of EDS on adult rat Leydig cells were not dependent upon new protein synthesis or cytochrome P450 enzyme activity. To determine whether inhibition of glutathione synthesis protects Leydig cells from the cytotoxic effects of EDS, adult rat Leydig cells were cultured in the presence or absence of 4 mM buthionine sulfoximine (BSO; 2 hr), a specific inhibitor of glutathione synthesis, and subsequently with increasing doses of EDS (3 hr). Following EDS addition, the ability of the cells to produce testosterone in response to LH stimulation, and to synthesize protein ([35S]methionine incorporation) were evaluated. In both cases, Leydig cells cultured in the presence of BSO were far less sensitive than Leydig cells cultured in medium alone (control) to EDS effects on testosterone production (control: EC50 60 μg EDS/ml; BSO: EC50 > 1500 μg EDS/ml) and [35S]methionine incorporation (control: EC50 = 95 μg EDS/ml; BSO: EC50 = 1560 μg EDS/ml). This protective effect of BSO was abolished by restoring intracellular glutathione levels with glutathione ethyl ester (8 mM; GSHEE). Interestingly, none of these treatments altered the viability (i.e., [35S]methionine incorporation) of immature rat Leydig cells (EC50 = 420 μg EDS/ml for control, GSHEE, and BSO groups). Taken together, these data suggest that the mechanism by which EDS kills adult rat Leydig cells may involve Leydig cell glutathione.
Covalent binding of ethylene dibromide and its metabolites to albumin
1992, Toxicology LettersThe present study was undertaken to determine covalent binding of [1,2-14C]ethylene dibromide (EDB) to albumin under in vivo and in vitro conditions. For the in vivo covalent binding, 25 body weight of [1,2-14C]EDB was given daily to male rats for 12 consecutive days and the animals were sacrificed at 24 h following the last dose. Blood was withdrawn from inferior vena cava in heparinized tubes and plasma was separated, dialyzed against ice-cold 10 mM phosphate buffer (pH 7.4) and then subjected to size-exclusion high-performance liquid chromatography (SE-HPLC). A major radioactive peak eluted at an elution volume corresponding to 65 000 dalton molecular mass was found to be associated to albumin at a level of 0.14 nmol equivalent protein. For the in vitro covalent binding, human plasma or purified albumin was incubated with [1,2-14C]EDB in the presence of phenobarbital-treated rat liver microsomes and NADPH-generating system for 2 h at 37°C. The 100 000 × g supernatant of the incubation mixture was dialyzed extensively and analyzed as described for the in vivo studies. Approximately 0.28 nmol equivalent protein was found to be associated to albumin (about 2-fold higher than the in vivo binding). Binding of 14C-label to albumin under in vivo and in vitro conditions was further supported by the affinity chromatography of albumin fraction isolated by SE-HPLC. Reversed-phase HPLC analysis of pronase digest of the albumin obtained from in vitro studies indicated formation of several amino acid adducts of EDB and/or its metabolites. Structure elucidation of such amino acid adducts will be helpful in developing a relatively non-invasive method of measuring the EDB exposure.