Physiologically based biokinetic (PBBK) model for safrole bioactivation and detoxification in rats.

A physiologically based biokinetic (PBBK) model for alkenylbenzene safrole in rats was developed using in vitro metabolic parameters determined using relevant tissue fractions. The performance of the model was evaluated by comparison of the predicted levels of 1,2-dihydroxy-4-allylbenzene and 1'-hydroxysafrole glucuronide to levels of these metabolites reported in the literature to be excreted in the urine of rats exposed to safrole and by comparison of the predicted amount of total urinary safrole metabolites to the reported levels of safrole metabolites in the urine of safrole exposed rats. These comparisons revealed that the predictions adequately match observed experimental values. Next, the model was used to predict the relative extent of bioactivation and detoxification of safrole at different oral doses. At low as well as high doses, P450 mediated oxidation of safrole mainly occurs in the liver in which 1,2-dihydroxy-4-allylbenzene was predicted to be the major P450 metabolite of safrole. A dose dependent shift in P450 mediated oxidation leading to a relative increase in bioactivation at high doses was not observed. Comparison of the results obtained for safrole with the results previously obtained with PBBK models for the related alkenylbenzenes estragole and methyleugenol revealed that the overall differences in bioactivation of the three alkenylbenzenes to their ultimate carcinogenic 1'-sulfooxy metabolites are limited. This is in line with the generally less than 4-fold difference in their level of DNA binding in in vitro and in vivo studies and their almost similar BMDL(10) values (lower confidence limit of the benchmark dose that gives 10% increase in tumor incidence over background level) obtained in in vivo carcinogenicity studies. It is concluded that in spite of differences in the rates of specific metabolic conversions, overall the levels of bioactivation of the three alkenylbenzenes are comparable which is in line with their comparable carcinogenic potential.

Identification of nevadensin as an important herb-based constituent inhibiting estragole bioactivation and physiology-based biokinetic modeling of its possible in vivo effect.

Estragole is a natural constituent of several herbs and spices including sweet basil. In rodent bioassays, estragole induces hepatomas, an effect ascribed to estragole bioactivation to 1'-sulfooxyestragole resulting in DNA adduct formation. The present paper identifies nevadensin as a basil constituent able to inhibit DNA adduct formation in rat hepatocytes exposed to the proximate carcinogen 1'-hydroxyestragole and nevadensin. This inhibition occurs at the level of sulfotransferase (SULT)-mediated bioactivation of 1'-hydroxyestragole. The Ki for SULT inhibition by nevadensin was 4 nM in male rat and human liver fractions. Furthermore, nevadensin up to 20 microM did not inhibit 1'-hydroxyestragole detoxification by glucuronidation and oxidation. The inhibition of SULT by nevadensin was incorporated into the recently developed physiologically based biokinetic (PBBK) rat and human models for estragole bioactivation and detoxification. The results predict that co-administration of estragole at a level inducing hepatic tumors in vivo (50mg/kg bw) with nevadensin at a molar ratio of 0.06, representing the ratio of their occurrence in basil, results in almost 100% inhibition of the ultimate carcinogen 1'-sulfooxyestragole when assuming 100% uptake of nevadensin. Assuming 1% uptake, inhibition would still amount to more than 83%. Altogether these data point at a nevadensin-mediated inhibition of the formation of the ultimate carcinogenic metabolite of estragole, without reducing the capacity to detoxify 1'-hydroxyestragole via glucuronidation or oxidation. These data also point at a potential reduction of the cancer risk when estragole exposure occurs within a food matrix containing SULT inhibitors compared to what is observed upon exposure to pure estragole.

Prediction of isoprene diepoxide levels in vivo in mouse, rat and man using enzyme kinetic data in vitro and physiologically-based pharmacokinetic modelling.

The present study was designed to explain the differences in isoprene toxicity between mouse and rat based on the liver concentrations of the assumed toxic metabolite isoprene diepoxide. In addition, extrapolation to the human situation was attempted. For this purpose, enzyme kinetic parameters K(m) and V(max) were determined in vitro in mouse, rat and human liver microsomes/cytosol for the cytochrome P450-mediated formation of isoprene mono- and diepoxides, epoxide hydrolase mediated hydrolysis of isoprene mono- and diepoxides, and the glutathione S-transferases mediated conjugation of isoprene monoepoxides. Subsequently, the kinetic parameters were incorporated into a physiologically-based pharmacokinetic model, and species differences regarding isoprene diepoxide levels were forecasted. Almost similar isoprene diepoxide liver and lung concentrations were predicted in mouse and rat, while predicted levels in humans were about 20-fold lower. However, when interindividual variation in enzyme activity was introduced in the human model, the levels of isoprene diepoxide changed considerably. It was forecasted that in individuals having both an extensive oxidation by cytochrome P450 and a low detoxification by epoxide hydrolase, isoprene diepoxide concentrations in the liver increased to similar concentrations as predicted for the mouse. However, the interpretation of the latter finding for human risk assessment is ambiguous since species differences between mouse and rat regarding isoprene toxicity could not be explained by the predicted isoprene diepoxide concentrations. We assume that other metabolites than isoprene diepoxide or different carcinogenic response might play a key role in determining the extent of isoprene toxicity. In order to confirm this, in vivo experiments are required in which isoprene epoxide concentrations will be established in rats and mice.

Structure-activity study on the quinone/quinone methide chemistry of flavonoids.

A structure-activity study on the quinone/quinone methide chemistry of a series of 3',4'-dihydroxyflavonoids was performed. Using the glutathione trapping method followed by HPLC, (1)H NMR, MALDI-TOF, and LC/MS analysis to identify the glutathionyl adducts, the chemical behavior of the quinones/quinone methides of the different flavonoids could be deduced. The nature and type of mono- and diglutathionyl adducts formed from quercetin, taxifolin, luteolin, fisetin, and 3,3',4'-trihydroxyflavone show how several structural elements influence the quinone/quinone methide chemistry of flavonoids. In line with previous findings, glutathionyl adduct formation for quercetin occurs at positions C6 and C8 of the A ring, due to the involvement of quinone methide-type intermediates. Elimination of the possibilities for efficient quinone methide formation by (i) the absence of the C3-OH group (luteolin), (ii) the absence of the C2=C3 double bond (taxifolin), or (iii) the absence of the C5-OH group (3,3',4'-trihydroxyflavone) results in glutathionyl adduct formation at the B ring due to involvement of the o-quinone isomer of the oxidized flavonoid. The extent of di- versus monoglutathionyl adduct formation was shown to depend on the ease of oxidation of the monoadduct as compared to the parent flavonoid. Finally, unexpected results obtained with fisetin provide new insight into the quinone/quinone methide chemistry of flavonoids. The regioselectivity and nature of the quinone adducts that formed appear to be dependent on pH. At pH values above the pK(a) for quinone protonation, glutathionyl adduct formation proceeds at the A or B ring following expected quinone/quinone methide isomerization patterns. However, decreasing the pH below this pK(a) results in a competing pathway in which glutathionyl adduct formation occurs in the C ring of the flavonoid, which is preceded by protonation of the quinone and accompanied by H(2)O adduct formation, also in the C ring of the flavonoid. All together, the data presented in this study confirm that quinone/quinone methide chemistry can be far from straightforward, but the study provides significant new data revealing an important pH dependence for the chemical behavior of this important class of electrophiles.

The interplay of glutathione-related processes in antioxidant defense.

This review summarizes current knowledge on glutathione (GSH) associated cellular processes that play a central role in defense against oxidative stress. GSH itself is a critical factor in maintaining the cellular redox balance and has been demonstrated to be involved in regulation of cell signalling and repair pathways. Enhanced expression of various enzymes involved in GSH metabolism, including glutathione peroxidases, γ-glutamyl cysteinyl synthetase (γ-GCS), glutathione S-transferases (GST) and membrane proteins belonging to the ATP-binding cassette family, such as the multidrug resistance associated protein, have all been demonstrated to play a prominent role in cellular resistance towards oxidative stress. This review stresses the fact that aco-ordinateinterplay between these systems is essential for efficient protection against oxidative stress.

Prediction of interindividual variation in drug plasma levels in vivo from individual enzyme kinetic data and physiologically based pharmacokinetic modeling.

A strategy is presented to predict interindividual variation in drug plasma levels in vivo by the use of physiologically based pharmacokinetic modeling and human in vitro metabolic parameters, obtained through the combined use of microsomes containing single cytochrome P450 enzymes and a human liver microsome bank. The strategy, applied to the pharmaceutical compound (N-[2-(7-methoxy-1-naphtyl)-ethyl]acetamide), consists of the following steps: (1) estimation of enzyme kinetic parameters K(m) and V(max) for the key cytochrome P450 enzymes using microsomes containing individual P450 enzymes; (2) scaling-up of the V(max) values for each individual cytochrome P450 involved using the ratio between marker substrate activities obtained from the same microsomes containing single P450 enzymes and a human liver microsome bank; (3) incorporation into a physiologically based pharmacokinetic model. For validation, predicted blood plasma levels and pharmacokinetic parameters were compared to those found in human volunteers: both the absolute plasma levels as well as the range in plasma levels were well predicted. Therefore, the presented strategy appears to be promising with respect to the integration of interindividual differences in metabolism and prediction of the possible impact on plasma and tissue concentrations of drugs in humans.

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.

Glutathione conjugation as a bioactivation reaction.

In general, glutathione conjugation is regarded as a detoxication reaction. However, depending on the properties of the substrate, bioactivation is also possible. Four types of activation reaction have been recognized: direct-acting compounds, conjugates that are activated through cysteine conjugate beta-lyase, conjugates that are activated through redox cycling and lastly conjugates that release the original reactive parent compound. The glutathione S-transferases have three connections with the formation of biactivated conjugates: they catalyze their formation in a number of cases, they are the earliest available target for covalent binding by these conjugates and lastly, the parent alkylating agents are regularly involved in the induction of the enzymes. Individual susceptibility for each of these agents is determined by individual transferase subunit composition and methods are becoming available to assess this susceptibility.

Determining the best animal model for human cytochrome P450 activities: a comparison of mouse, rat, rabbit, dog, micropig, monkey and man.

1. In the present study, nine cytochrome P450 enzyme activities in seven species were characterized to allow a practical means of comparing this important metabolic step between various test animals and man. 2. Enzyme activities and kinetic parameters were first determined towards marker substrates for human cytochrome P450 enzymes. Inhibition profiles were then determined with both antibodies directed against various cytochrome P450 enzymes and with chemical inhibitors. 3. Both the enzyme kinetic parameters/enzyme activities, and the inhibition profiles obtained for the animal species were compared with those obtained for human liver microsomes in order to postulate the animal species most similar to man with regard to each individual cytochrome P450 enzyme activity. 4. It was found that, as expected, none of the tested species was similar to man for all the measured P450 enzyme activities, but that in each species only some of the P450 enzyme activities could be considered as similar to man. 5. When it is known which human cytochrome P450 enzymes are involved in the metabolism of a compound, the comparative data presented here can be used for selecting the most suitable species for in vitro and in it no experiments.

In vitro and in vivo inhibition of human flavin-containing monooxygenase form 3 (FMO3) in the presence of dietary indoles.

The effect of consumption of glucosinolate-containing Brussels sprouts on flavin-containing monooxygenase functional activity in humans was investigated in 10 healthy, male, non-smoking volunteers. After a 3-week run-in period, 5 volunteers continued on a glucosinolate-free diet for 3 weeks (control group), and 5 others consumed 300 g of cooked Brussels sprouts per day (sprouts group). Human flavin-containing monooxygenase activity was measured by determining the levels of urinary trimethylamine and trimethylamine N-oxide. In the control group similar trimethylamine to trimethylamine N-oxide ratios were observed, while in the sprouts group the trimethylamine to trimethylamine N-oxide ratios were increased 2.6- to 3.2-fold, and thus flavin-containing monooxygenase functional activity was decreased significantly. To investigate the molecular basis for the in vivo inhibition of functional human flavin-containing monooxygenase activity, in vitro studies were carried out examining the effect of acid condensation products of indole-3-carbinol, anticipated to be formed after transit of Brussels sprouts through the gastrointestinal system, on the prominent cDNA-expressed human flavin-containing monooxygenase form 3 enzymes. Two indole-containing materials were observed to be potent inhibitors of human flavin-containing monooxygenases, having Ki values in the low micromolar range. The results suggested that acid condensation products expected to be formed upon transit of Brussels sprouts materials through the gastrointestinal system were potent competitive inhibitors of human flavin-containing monooxygenase form 3 enzymes. The findings indicate that daily intake of Brussels sprouts may lead to a decrease in human flavin-containing monooxygenase activity, and this may have consequences for metabolism of other xenobiotics or dietary constituents.

The role of biotransformation in dietary (anti)carcinogenesis.

The fact that dietary compounds influence the susceptibility of human beings to cancer, is widely accepted. One of the possible mechanisms that is responsible for these (anti)carcinogenic effects is that dietary constituents may modulate biotransformation enzymes, thereby affecting the (anti)carcinogenic potential of other compounds. This ambiguous theme is the basis for the present paper. The possible effects of enzymatic bioactivation and detoxification of dietary constituents are discussed using two representative examples of phase I and phase II biotransformation enzymes i.e., cytochrome P450 and glutathione S-transferase. Furthermore, the impact of genetic polymorphisms of these two enzyme systems is considered. Although it is very difficult on the basis of the enzyme inducing or inhibiting properties of dietary compounds, especially to characterize them as anticarcinogenic, for certain constituents it is acknowledged that they have anticarcinogenic properties. As such, this provides for an important mechanistic substantiation of the established cancer chemopreventive effect of a diet rich in fruits and vegetables.

Interactions of prostaglandin A2 with the glutathione-mediated biotransformation system.

The cyclopentenone prostaglandin A2 (PGA2) is known to inhibit cell proliferation, and metabolism of this compound thus might be important in controlling its ultimate function. The glutathione-related metabolism of PGA2 was therefore investigated both with purified glutathione S-transferase P1-1 (GSTP1-1) and with IGR-39 human melanoma cells. Firstly, the irreversible inhibition of human GSTP1-1 and its mutants C47S, C101S, and C47S/C101S was studied. PGA2 appeared to inhibit GSTP1-1 mainly by binding to the cysteine 47 moiety of the enzyme. This binding was reversed by a molar excess of GSH, indicating that retro-Michael cleavage occurs. Secondly, after exposing IGR-39 human melanoma cells to PGA2, both diastereoisomers of the PGA2-glutathione conjugate are excreted into the medium, although with a clear excess of the S-form, due to its preferential formation by the GSTP1-1 present in the cells. Thirdly, the effect of PGA2 on intracellular GST activity was determined by quantification of the excreted glutathione conjugate S-(2,4-dinitrophenyl)glutathione (DNPSG) after exposure to 1-chloro-2,4-dinitrobenzene. DNPSG excretion was inhibited after incubation with 10 or 20 microM PGA2 for 1 or 4 hr, as a result of glutathione depletion, reversible GST inhibition, and covalent modification of intracellular GST. Furthermore, PGA2 also inhibited transport of DNPSG by the multidrug resistance-associated protein, an effect that was reversible and competitive. In conclusion, PGA2 modulates all three aspects of the glutathione-mediated biotransformation system, i.e. GSH levels, GSTP1-1 activity, and transport of GSH conjugates. A role for GSTP1-1 as a specific transport protein inside the cell is indicated.

Conjugation of isoprene monoepoxides with glutathione, catalyzed by alpha, mu, pi and theta-class glutathione S-transferases of rat and man.

In the present study, the enzymatic conjugation of the isoprene monoepoxides 3,4 epoxy-3-methyl-1-butene (EPOX-I) and 3,4-epoxy-2-methyl-1-butene (EPOX-II) with glutathione was investigated, using purified glutathione S-transferases (GSTs) of the alpha, mu, pi and theta-class of rat and man. HPLC analysis of incubations of EPOX-I and EPOX-II with [35S]glutathione (GSH) showed the formation of two radioactive fractions for each isoprene monoepoxide. The structures of the EPOX-I and EPOX-II GSH conjugates were elucidated with 1H-NMR analysis. As expected, two sites of conjugation were found for both isoprene epoxides. EPOX-II was conjugated more efficiently than EPOX-I. In addition, the mu and theta class glutathione S-transferases were much more efficient than the alpha and pi class glutathione S-transferases, both for rat and man. Because the mu- and theta-class glutathione S-transferases are expressed in about 50 and 40-90% of the human population, respectively, this may have significant consequences for the detoxification of isoprene monoepoxides in individuals who lack these enzymes. Rat glutathione S-transferases were more efficient than human glu tathione S-transferases: rat GST T1-1 showed about 2.1-6.5-fold higher activities than human GST T1-1 for the conjugation of both EPOX-I and EPOX-II, while rat GST M1-1 and GST M2-2 showed about 5.2-14-fold higher activities than human GST M1a-1a. Most of the glutathione S-transferases showed first order kinetics at the concentration range used (50-2000 microM). In addition to differences in activities between GST-classes, differences between sites of conjugation were found. EPOX-I was almost exclusively conjugated with glutathione at the C4-position by all glutathione S-transferases, with exception of rat GST M1-1, which also showed significant conjugation at the C3-position. This selectivity was not observed for the conjugation of EPOX-II. Incubations with EPOX-I and EPOX-II and hepatic S9 fractions of mouse, rat and man, showed similar rates of GSH conjugation for mouse and rat. Compared to mouse and rat, human liver S9 showed a 25-50-fold lower rate of GSH conjugation.

Conversion of pentahalogenated phenols by microperoxidase-8/H2O2 to benzoquinone-type products.

This study reports the microperoxidase-8 (MP8)/H2O2-catalyzed dehalogenation of pentafluorophenol and pentachlorophenol, compounds whose toxic effects and persistence in the environment are well documented. The primary products of this dehalogenation reaction appear to be the corresponding tetrahalo-p-benzoquinones. Under the conditions used, the fluorinated phenol and its intermediate products are more susceptible to degradation than the corresponding chlorinated analogue and its products. The main degradation products of tetrachloro-p-benzoquinone and tetrafluoro-p-benzoquinone were identified as trichlorohydroxy-p-benzoquinone and trifluorohydroxy-p-benzoquinone, respectively. This secondary conversion of tetrafluoro-p-benzoquinone and tetrachloro-p-benzoquinone was not mediated by MP8, but was driven by H2O2. Evidence is presented for a mechanism where H2O2 molecules and not hydroxide anions are the reactive nucleophilic species attacking the tetrahalo-p-benzoquinones. In addition to the formation of the trihalohydroxy-p-benzoquinones, the formation of adducts of the tetrahalo-p-benzoquinone products with ethanol, present in the incubation medium, was observed. The adduct from the reaction of tetrachloro-p-benzoquinone with ethanol was isolated and identified as trichloroethoxyquinone. Thus, the present paper describes a system in which the formation of tetrahalo-p-benzoquinone-type products by an oxidative heme-based catalyst could be unequivocally demonstrated.

Increased bioactivation of dihaloalkanes in rat liver due to induction of class theta glutathione S-transferase T1-1.

A characteristic feature of the class Theta glutathione S-transferase (GST) T1-1 is its ability to activate dichloromethane and dibromoethane by catalysing the formation of mutagenic conjugates. The level of the GSTT1 subunit within tissues is an important determinant of susceptibility to the carcinogenic effects of these dihaloalkanes. In the present study it is demonstrated that hepatic GST activity towards these compounds can be elevated significantly in female and male Fischer-344 rats by feeding these animals on diets supplemented with cancer chemopreventive agents. Immunoblotting experiments showed that increased activity towards the dihaloalkanes is associated with elevated levels of the GSTT1 subunit in rat liver. Sex-specific effects were observed in the induction of GSTT1 protein. Amongst the chemopreventive agents tested, indole-3-carbinol proved to be the most potent inducer of hepatic GSTT1 in male rats (6.2-fold), whereas coumarin was the most potent inducer of this subunit in the livers of female rats (3. 5-fold). Phenobarbital showed significant induction of GSTT1 only in male rat liver and had little effect in female rat liver. Western blotting showed that class Alpha, Mu and Pi GST subunits are not co-ordinately induced with GSTT1, indicating that the expression of GSTT1 is determined, at least in part, by mechanisms distinct from those that regulate levels of other transferases. The increase in amount of hepatic GSTT1 protein was also reflected by an increase in the steady-state level of mRNA in response to treatment with chemopreventive agents and model inducers. Immunohistochemical detection of GSTT1 in rat liver supported the Western blotting data, but showed, in addition to cytoplasmic staining, significant nuclear localization of the enzyme in hepatocytes from some treated animals, including those fed on an oltipraz-containing diet. Significantly, the hepatic level of cytochrome P-450 2E1, an enzyme which offers a detoxification pathway for dihaloalkanes, was unchanged by the various inducing agents studied. It is concluded that the induction of GSTT1 by dietary components and its localization within cells are important factors that should be considered when assessing the risk dihaloalkanes pose to human health.

Glutathione-dependent biotransformation of the alkylating drug thiotepa and transport of its metabolite monoglutathionylthiotepa in human MCF-7 breast cancer cells.

In this study, the role of glutathione S-transferase (GST) P1-1, the cellular reduced glutathione (GSH) status, and ATP-dependent efflux pumps in the cellular glutathione-dependent biotransformation of thiotepa and transport of the main metabolite monoglutathionylthiotepa in relation to cytotoxicity was studied in control and GST-P1-1-transfected MCF-7 cell lines. It was demonstrated that an enhanced cellular level of GST-P1-1 leads to an enhanced formation of monoglutathionylthiotepa, which is transported out of the cell into the medium. Monoglutathionylthiotepa was able to reversibly inhibit the activity of purified GST-P1-1, but only at nonphysiological concentrations, indicating that feedback inhibition of GST by its metabolites is not a relevant process in vivo. The GST activity, cellular GSH level, and/or ATP-dependent efflux of monoglutathionylthiotepa were modulated using ethacrynic acid, D,L-buthionine-S,R-sulfoximine, probenecid, and verapamil to understand the interplay between GSTs, glutathione conjugation, and efflux of glutathione conjugates in more detail. Inhibition of the GSH biosynthesis by D,L-buthionine-R,S-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase, significantly reduced the glutathione conjugation of thiotepa and potentiated the cytotoxicity of thiotepa. Pretreatment of cells with ethacrynic acid resulted in decreased formation of monoglutathionylthiotepa as a result of inhibition of GST in the GST-P1-1 transfectant. In addition, the intracellular amount of monoglutathionylthiotepa increased in both of the cell lines on exposure to ethacrynic acid, indicating that transport of the glutathione conjugate was partially inhibited by the glutathione conjugate of ethacrynic acid. Transport activity of monoglutathionylthiotepa could also be inhibited by probenecid and verapamil, inhibitors of organic anion transport, without influencing the biotransformation capacity of the cells. It was demonstrated that inhibition of glutathione conjugate efflux by probenecid and verapamil leads to enhanced cytotoxicity, which indicates that besides thiotepa, monoglutathionylthiotepa is also cytotoxic for the cells. Only enhanced biotransformation and subsequent transport of the glutathione conjugate into the medium (which occurs with the GST-P1-1 transfectant) results in enhanced viability. Therefore, it was concluded that only enhanced biotransformation of thiotepa represents a real detoxification pathway when the resulting conjugate is transported out of the cells. Altogether, the results indicate that it is not the overexpression of GST per se but the interplay between GSH/GST and glutathione conjugate efflux pumps that results in increased resistance to alkylating anticancer drugs such as thiotepa.

Disposition of 1,2-[14C]Dibromoethane in male Wistar rats.

In this study the disposition of 1,2-[14C]dibromoethane (1, 2-[14C]DBE) was investigated in male Wistar rats. 1,2-DBE is a cytotoxic and carcinogenic compound that has been used as an additive in leaded gasoline and as a fumigant. 1,2-[14C]DBE was administered orally or iv. Radioactivity was recovered (mostly within 48 hr after administration) in urine (75-82% of the dose), feces (3.2-4% of the dose), and expired air (0.53-7.2% of the dose). One hundred-sixty-eight hours after administration of 1,2-[14C]DBE, most of the radioactivity in tissues was found in the liver, lungs, and kidneys (<1% of the dose) and the red blood cells (0.3% of the dose). Identified urinary metabolites were S-(2-hydroxyethyl)mercapturic acid, thiodiacetic acid, and thiodiacetic acid sulfoxide, together accounting for, on average, 78% of the total amount of radioactivity in urine. In addition to S-(2-hydroxyethyl)mercapturic acid, thiodiacetic acid, and thiodiacetic acid sulfoxide, several compounds were anticipated as potential urinary metabolites of 1,2-DBE, i.e. S-(carboxymethyl)mercapturic acid, S-(2-hydroxyethyl)thioacetic acid, S-(2-hydroxyethyl)thiopyruvic acid, S-(carboxymethyl)thiopyruvic acid, S-(2-hydroxyethyl)thiolactic acid, and S-(carboxymethyl)thiolactic acid. All of the postulated urinary metabolites were synthesized and searched for in urine samples. None of these metabolites could be detected in urine, however. The data obtained in the present study might be useful for risk assessment and biomonitoring studies of 1,2-DBE and will also be used to further validate a physiologically based pharmacokinetic model for 1, 2-DBE in rats and humans that was recently developed.

Toxicological evaluation and risk assessment of chemical mixtures.

A major objective of combination toxicology is to establish whether a mixture of chemicals will result in an effect similar to that expected on the basis of additivity. This requires understanding of the basic concepts of the combined toxicological action of the compounds of the mixture: simple similar action (dose addition), simple dissimilar action (effect or response addition), and interaction (synergism, potentiation, antagonism). The number of possible combinations of chemicals is innumerable, and in vivo testing of these mixtures is unattainable from an ethical, economical, or pragmatic perspective. Prediction of the effect of a mixture based on the knowledge of each of the constituents requires detailed information on the composition of the mixture, exposure level, mechanism of action, and receptor of the individual compounds. Often, such information is not or is only partially available and additional studies are needed. Research strategies and methods to assess joint action or interaction of chemicals in mixtures such as whole mixture testing, physiologically based toxicokinetic modeling and isobologram and dose response surface analyses are discussed. Guidance is given for risk assessment of both simple and complex mixtures. We hypothesize that, as a rule, exposure to mixtures of chemicals at (low) non-toxic doses of the individual constituents is of no health concern. To verify the hypothesis is a challenge; to timely detect exceptions to the rule is the real challenge of major practical importance.