A physiologically-based pharmacokinetic(PB-PK) model for ethylene dibromide: relevance of extrahepatic metabolism.

A physiologically-based pharmacokinetic (PB-PK) model was developed for ethylene dibromide (1,2-dibromoethane, EDB) for rats and humans, partly based on previously published in vitro data (Ploemen et al., 1997). In the present study, this PB-PK model has been validated for the rat. In addition, new data were used for the human class ThetaGST T1-1. Validation experiments are described in order to test the predictive value of kinetics to describe "whole-body" metabolism. For the validation experiments, groups of cannulated rats were dosed orally or intravenously with different doses of EDB. Obtained blood concentration-time curves of EDB for all dosing groups were compared to model predictions. It appeared that metabolism, which previously was assumed to be restricted to the liver, was underestimated. Therefore, we extended the PB-PK model to include all the extrahepatic organs, in which the enzymes involved in EDB metabolism have been detected and quantified. With this extended model, the blood concentrations were much more accurately described compared to the predictions of the "liver-model". Therefore, extrahepatic metabolism was also included in the human model. The present study illustrates the potential application of in vitro metabolic parameters in risk assessment, as well as the use of PB-PK modelling as a tool to understand and predict in vivo data.

Liver AP-1 activation due to carbon tetrachloride is potentiated by 1,2-dibromoethane but is inhibited by alpha-tocopherol or gadolinium chloride.

Experimental acute intoxication by prooxidant haloalkanes produces marked stimulation of hepatic lipid peroxidation and cytolysis, which is followed by tissue regeneration. Our aim was to clarify the role of oxidative imbalance in the activation of the redox-sensitive transcription factor, activator protein-1 (AP-1), which is involved in tissue repair. Rats were poisoned with a very low concentration of carbon tetrachloride, given alone or in combination with another hepatotoxin, 1,2-dibromoethane, to provide varying extents of oxidative damage. The level of AP-1-DNA binding was analyzed by electrophoretic mobility shift assay on liver extracts, obtained from rats killed 6 h after poisoning. Stimulation of lipid peroxidation and AP-1 upregulation were already established when the hepatic damage due to carbon tetrachloride +/-1,2-dibromoethane was beginning to appear. Rat supplementation with the antioxidant vitamin E completely inhibited AP-1 upregulation, thus supporting a causative role of membrane lipid oxidation in the observed modulation of the transcription factor. Moreover, activation of Kupffer cells appears to be a crucial step in the increased AP-1 binding to DNA, the latter being largely prevented by gadolinium chloride, a macrophage-specific inhibitor.

Role of kidney S9 in the mutagenic properties of 1,2-dibromoethane.

The mutagenic properties of 1,2-dibromoethane (DBE) were studied in the Ames Salmonella typhimurium assay using the strains TA 1535 and TA 100. Kidney S9 fraction alone did not modify the direct mutagenic activity of DBE; but an addition of kidney S9 to liver S9 fraction yielded a higher mutagenic activity of DBE than with liver S9 fraction alone. Moreover, the addition of glutathione (GSH) to kidney S9 increased the mutagenic activity of DBE. Methimazole, a competitive inhibitor of the flavin-containing monooxygenase, reduced mutagenic activity suggesting that this enzyme may contribute to renal damage from DBE. No mutagens could be detected in the urine of rats treated with DBE.

Neoplastic expression in murine cells induced by halogenated hydrocarbons.

The neoplastic expression in mouse embryo fibroblasts exposed to 1,2-dibromoethane and its chloroanalogue, 1,2-dichloroethane in vitro, was examined. Both substances are widely used as fumigants for carpet and upholstery, as gasoline additives, and as organic solvents. Both are known to be highly toxic, mutagenic, and carcinogenic agents. C3H10T1/2 cells treated with these haloalkanes exhibited altered morphology and were selected further by cloning in soft agar. Soft agar clones were found to induce a 100% multitumor occurrence in the nude mouse model. These results suggest that this pair of mutagens have altered the normal phenotype of mouse embryo cells, and these cells have become neoplastic. These neoplastic cell lines will be useful as an in vitro model to study the role of genetic changes in the transformation processes induced by halogenated hydrocarbons.

The spermicidal effect of ethylene dibromide in bulls and rams.

The spermicidal effect of ethylene dibromide (EDB) in bulls and rams is reviewed. Following oral or parenteral administration EDB was found to cause, by its alkylating effect in the testes of treated bulls and rams, lysis of the chromatin of the elongating spermatids during the time of somatin histones replacement by the sperm protamines. However, while in the bulls the abnormal spermatozoa issued from the affected spermatids were also collected in the ejaculates, this was not the case with treated rams. In the latter animals the abnormal spermatids seem to be phagocytized in the epididymis before their arrival in the ejaculate. In addition, whereas the alkylating effect of EDB occurred also in the upper parts of the epididymis of the bulls, causing tail and acrosome defects to the spermatozoa, in the rams such an effect seems to occur all along the epididymal duct. These differences between bulls and rams in the sites of the genital tract where the drug takes effect, and in the mechanism of this effect, reveal probable differences in the physiology of the reproductive tract between these species.

In vivo studies on halogen compound interactions. I. Effects of carbon tetrachloride plus 1,2-dibromoethane on liver necrosis.

Investigation of the hepatotoxicity of a mixture of two halogen compounds--carbon tetrachloride (CT) and 1,2-dibromoethane (DBE) used in association, especially in agriculture--is reported. Their simultaneous administration in the rat potentiates the necrosis provoked by exposure to CT alone. This damage can be totally prevented by prior treatment with vitamin E, which raises the liver antioxidant level. Determination of the TBA-reacting substances released from the liver homogenates of animals treated with one or both substances also corroborates the importance of lipid peroxidation in the pathogenesis of this potentiation. Furthermore, no significant difference between poisoning by CT and CT + DBE is noted when liver GSH values are measured under the same experimental conditions. This finding rules out the possibility that potentiation is only the simply sum of the damage caused by the two compounds (lipid peroxidation due to a radical initiator for CT, depletion of GSH for DBE). It is thus something more than a mere combination of two mechanisms of action and further investigation is required to unravel its more complex pathogenesis.

Forestomach lesions induced by butylated hydroxyanisole and ethylene dibromide: a scientific and regulatory perspective.

Selected pathology lesions from 9 studies, 5 with butylated hydroxyanisole (BHA) and 4 with ethylene dibromide (EDB) are reviewed and their relative importance in regulatory evaluation is discussed. When Fischer 344 (F344) rats were fed BHA at 0.5% and 2.0% of the diet for 2 years, an increased number of rats of both sexes had epithelial hyperplasia of the forestomach at both treatment levels, compared to controls. At the 2.0% level, an increased number of rats had forestomach papilloma or forestomach squamous cell carcinoma. In a second study, in which F344 rats were fed BHA at 1.0% and 2.0% of the diet for 2 years, increased numbers of rats in both treatment groups were reported to have hyperplasia or papilloma of the forestomach. At the 2.0% level, increased numbers of rats developed squamous cell carcinoma of the forestomach. More Syrian golden hamsters fed BHA at 1.0% and 2.0% of the diet for 2 years reportedly had hyperplasia, papilloma or squamous cell carcinoma of the forestomach than did nontreated animals. Ingestion of BHA at 0.5% and 1.0% of the diet by B6C3F1 mice for 2 years was reported to produce an increase of animals with hyperplasia or papilloma of the forestomach at both dosage levels, compared to nontreated mice. When beagle dogs were fed BHA at 1.0% and 1.3% of the diet for 180 days, no lesions/tumors of the distal esophagus or stomach were identified at gross necropsy or by light or electron microscopy. When EDB was administered by gavage to Osborne-Mendel rats and B6C3F1 mice under conditions of the National Toxicology Bioassay Program, more rats and mice, both male and female, developed squamous cell carcinoma of the forestomach than did nontreated groups. EDB administered via inhalation to F344 rats and B6C3F1 mice did not cause squamous cell carcinoma of the forestomach; however, other neoplasms occurred which were considered to be treatment-related. Information gleaned from the BHA and EDB studies with multiple animal species facilitated regulatory decision-making regarding the potential toxicity/carcinogenicity of these compounds to man.

Effects of time-variant exposure on toxic substance response.

Sources of time-variant exposure to toxic substances are identified and examined for their effects on the estimation of response. It is shown that only time-averaged target tissue concentrations are required to obtain rigorous risk estimates from the one-hit and multihit models. In contrast, detailed concentration histories need to be retained throughout analyses involving two-event models with intermediate-stage clonal growth advantage (clonal two-stage) and multistage models. Cumulative incidence ratios, based on the exact to time-averaged treatment of concentration time dependencies, are evaluated for substances whose toxic responses exhibit moderate (arsenic) and strong (ethylene dibromide) dependence on time of actual exposure. These ratios reveal that time-averaged dose approximations may lead to several orders of magnitude error in both the multistage and clonal two-stage models if exposure periods are short, and that 3.4-fold (arsenic) and 8-fold (ethylene dibromide) errors still exist even when an actual two-thirds lifetime exposure is averaged over a full lifetime. Finally, the effects of time-variant exposure on risk estimation due to migration and birth-death in an epidemiological setting are examined. A residence time distribution calculation shows that, if these effects are ignored for a population orally exposed to arsenic and characterized by an out-migration rate in excess of 5%/yr, response errors will exceed an order of magnitude.

In vivo cytogenetic studies on mice exposed to ethylene dibromide.

The pesticide, ethylene dibromide (EDB), was evaluated with in vivo cytogenetic assays to determine its genotoxicity. CD1 male mice were exposed to EDB through intraperitoneal injections. Bone marrow cells isolated from femora were analyzed for sister-chromatid exchange (SCE), chromosome aberration and micronucleus formation. The results showed that only certain concentrations of EDB tested caused a slight but significant increase in SCEs and chromosome aberrations. However, these increases were not dose-related. No increase in the polychromatic erythrocytes with micronuclei was observed following EDB exposure. Also, EDB did not cause cell-cycle delay in comparison with controls. Thus, it appears that EDB is not an effective genotoxic agent in vivo in mice.

Modulation of the mitotic action of ethylene dibromide.

Refeeding rats treated with a single high dose of ethylene dibromide (1,2-dibromoethane, EDB) induced liver DNA synthesis. The peak of DNA synthesis, as measured by [methyl-3H]thymidine incorporation was attained after 24 h in refed rats and at 48 h in fasted ones. Fasting enhances the EDB action leading to liver cell necrosis, as shown by elevation of serum enzymes' activities, glutamic pyruvic transaminase (GPT) and sorbital dehydrogenase (SDH). A low dose of EDB administered during 2 and 3 weeks slightly enhanced the liver DNA synthesis and elevated the activity of serum enzymes. Phenobarbitone (PB) treatment of rats together with low dose of EDB during 2 weeks prevented the enzyme activity elevation and attenuated the DNA synthesis. Diethyldithiocarbamate (DDC) pretreatment potentiated the DNA synthesis in fed rats after both a small dose of EDB for 2 weeks and after a single high-dose treatment. In DDC pretreated rats, the high single dose of EDB caused biochemical perturbations in serum and liver representative of liver cell necrosis; changes in serum enzymes' activities also were noticed as early as 2 h after EDB toxication. The possible function of modulators on the mitogenic or the necrogenic action of EDB is discussed.