Metabolic transformation of halogenated and other alkenes--a theoretical approach. Estimation of metabolic reactivities for in vivo conditions.

Olefinic hydrocarbons are metabolized in vivo by cytochrome P450-dependent monooxygenases to the corresponding epoxides. The maximum in vivo metabolic rate, which is an important toxicokinetic parameter, has been used to define the apparent rate constant (kapp) describing in vivo metabolic reactivity of alkenes. To derive kapp, the metabolic rate normalized per body weight was divided by the corresponding average alkene concentration in the body at saturation conditions of 90%. Toxicokinetic data obtained in rats for 13 compounds (ethene, 1-fluoroethene, 1,1-difluoroethene, 1-chloroethene, 1,1-dichloroethene, cis-1,2-dichloroethene, trans-1,2-dichloroethene, 1,1,2-trichloroethene, perchloroethene, propene, isoprene, 1,3-butadiene and styrene) have been used to calculate kapp values. A theoretical model, based on the assumption that in vivo epoxidation can be described as a cytochrome P450-mediated electrophilic reaction, has been developed. Using the olefinic hydrocarbons as an example it has been shown that kapp can be explained solely by the following molecular parameters: ionization potential, dipole moment and pi-electron density. These molecular parameters were calculated by a quantum chemical method or were taken from the literature. Furthermore, the model was tested also by predicting kapp for isobutene, an alkene which was not used for the model development. The predicted value of kapp agrees with the one derived experimentally, demonstrating that molecular parameters of halogenated and other alkenes can be used to predict in vivo metabolic reactivity. The model presented here is a first contribution to the ultimate goal to predict toxicokinetic parameters for in vivo conditions based on physicochemical parameters of enzymes and compounds exclusively.

Comparative mutagenicity of structurally related aliphatic epoxides in a modified Salmonella/microsome assay.

Four structurally related aliphatic epoxides (1,2-epoxypropane, 1,2-epoxyisobutane, cis- and trans-2,3-epoxybutane) have been tested in the Salmonella/microsome assay, modified for volatile substances, using the strains TA1535 and TA100. The aim of the study was to evaluate the effect of methylation on the mutagenicity of 1,2-epoxypropane in this vaporization assay, with and without exogenous metabolization. All substances induced a significant increase of revertants in the strains TA1535 and TA100. In terms of mutagenic potency, the following hierarchy was observed in the standard tester strain TA1535 and in the absence of rat S9: 1,2-epoxy-propane >> cis-2,3-epoxybutane > 1,2-epoxyisobutane > trans-2,3- epoxybutane. After exogenous metabolization, the mutagenic response of 1,2-epoxyisobutane was substantially reduced, while a moderate decrease of cis-2,3-epoxybutane was observed in the presence of S9, as compared with the response without S9. No influence of the S9 on the mutagenic response of trans-2,3-epoxybutane was noticed in both strains TA1535 and TA100, while an increased response with 1,2-epoxypropane was observed in TA100 but not in TA1535. The results suggest that the vaporization assay may provide more relevant information concerning mutagenic potencies of gaseous or volatile compounds than the common treat-and-plate or preincubation assays. Moreover, it appears that mutagenicity theories, based only upon inductive effects of side groups, may not suffice to explain differences in mutagenicity. Sterical factors or differential interactions with metabolizing enzymes could also be important in the evaluation of mutagenic effects.

Evaluation of DNA damage by alkaline elution technique after inhalation exposure of rats and mice to 1,3-butadiene.

The alkaline filter elution technique was used to evaluate single strand breaks (SSB), DNA-DNA (DDCL) and DNA-protein cross-links (DPCL) in liver and lung of male rats (Sprague-Dawley) and male mice (B6C3F1) after exposure to 2000 ppm 1,3-butadiene (BD) for 7 days (7 h/day and/or to 100, 250, 500, 1000) 2000 ppm BD for 7 h. SSB were detected in liver DNA of both species at 2000 ppm. Cross-links are more pronounced in mouse lung than in mouse liver. Elution rates of lung DNA from mice exposed for 7 h to different concentrations of BD revealed an increase in cross-links between 250 and 500 ppm, and a further increase in cross-links up to 2000 ppm. No such signs of genotoxicity could be observed for the lung of rats. Our data support the involvement of reactive metabolites (epoxybutene and especially diepoxybutane) in butadiene-induced carcinogenesis in the mouse but not to that extent in the rat.

Mutagenicity of 2-methylpropene (isobutene) and its epoxide in a modified Salmonella assay for volatile compounds.

The mutagenic properties of 2-methylpropene (MP) and 2-methyl-1,2- epoxypropane (MEP) were investigated in the Salmonella assay. A simple exposure system, consisting of gastight tissue culture flasks, was used. This method has the advantage that the volatile test chemical is present during the entire incubation period and that several concentrations of the investigated compound can be tested on a single day. MP is not mutagenic in strains TA100, TA102 and TA1535, and in the latter strain not even in the presence of metabolizing S9 mix. MEP is mutagenic in all the strains tested, as demonstrated by a clear dose-response relationship. Strain TA1535 seems to be most sensitive to MEP compared with the other bacterial strains studied. For this strain, the mutagenic activity of MEP decreased significantly in the presence of S9 mix, compatible with the epoxide being inactivated by epoxide hydrolase and by glutathione S-transferase, as reported previously. From the present study it can be concluded that the parent compound MP is not mutagenic, but that its primary metabolite MEP is a mutagenic substance. However, very high concentrations are necessary to induce a mutagenic effect and the epoxide is efficiently detoxified by different liver enzymes.

Pharmacokinetic interaction between 1,3-butadiene and styrene in Sprague-Dawley rats.

Gas uptake studies were carried out to evaluate kinetic interactions between 1,3-butadiene and styrene in Sprague-Dawley rats. The animals were co-exposed by inhalation to a mixture of 1,3-butadiene between 20 and 6000 ppm (v/v) and styrene between 0 and 500 ppm. The data demonstrate that metabolism of 1,3-butadiene was partially inhibited by styrene. The inhibition was competitive at atmospheric concentrations of styrene up to 90 ppm. Higher concentrations of styrene resulted in a small additional inhibition only. The apparent Michaelis-Menten constant for 1,3-butadiene, related to the average concentration in the organism of the animals, was Kmapp = 1.17 +/- 0.37 (mumol/l of tissue) and the corresponding atmospheric concentration at steady state was 560 ppm. The inhibition constant of styrene was found to be Ki = 0.23 +/- 0.30 (mumol/l of tissue). The maximal metabolic rate for 1,3-butadiene was 230 +/- 10 (mu/kg/h).

In vitro biotransformation of 2-methylpropene (isobutene): epoxide formation in mice liver.

Until now, no data are available concerning the biotransformation and toxicity of 2-methylpropene (or isobutene), a gaseous alkene widely used in industry (rubber, fuel additives, plastic polymers, adhesives, antioxidants). In this work, the biotransformation of 2-methylpropene (MP) has been studied, using total liver homogenates of mice, supplemented with a NADPH-generating system. In analogy to other olefins, 2-methylpropene is metabolized to its epoxide 2-methyl-1,2-epoxypropane (MEP), as proved by the identification by gas chromatography coupled with mass spectrometry. The epoxidation is cytochrome P-450 dependent, as shown by experiments in the absence of the NADPH-generating system and in the presence of various concentrations of metyrapone and SKF 525-A, two known inhibitors of the mono-oxygenases. A simple gas chromatographic headspace method has been developed for the quantitative determination of the epoxide formed. The formation of MEP is never linear in function of time and it reaches a maximum after 20 min. Thereafter is decreases continuously to undetectable levels. This observation can be explained by the immediate action of epoxide hydrolase and glutathione S-transferase, converting the epoxide to 2-methyl-1,2-propanediol and to the glutathione conjugate respectively. The involvement of both enzymes has been demonstrated by the addition of 3,3,3-trichloropropene oxide and indomethacin. These inhibitors of, respectively, epoxide hydrolase and glutathione S-transferase increase the epoxide formation in a significant way. The actual concentration of MEP is therefore not only dependent on its formation by cytochrome P-450 dependent mono-oxygenases, but also on its conversion by epoxide hydrolase and glutathione S-transferase, both very active in liver tissue.

Investigation of species differences in isobutene (2-methylpropene) metabolism between mice and rats.

Metabolism of isobutene (2-methylpropene) in rats (Sprague Dawley) and mice (B6C3F1) follows kinetics according to Michaelis-Menten. The maximal metabolic elimination rates are 340 mumol/kg/h for rats and 560 mumol/kg/h for mice. The atmospheric concentration at which Vmax/2 is reached is 1200 ppm for rats and 1800 ppm for mice. At steady state, below atmospheric concentrations of about 500 ppm the rate of metabolism of isobutene is direct proportional to its concentration. 1,1-Dimethyloxirane is formed as a primary reactive intermediate during metabolism of isobutene in rats and can be detected in the exhaled air of the animals. Under conditions of saturation of isobutene metabolism the concentration of 1,1-dimethyloxirane in the atmosphere of a closed exposure system is only about 1/15 of that observed for ethene oxide and about 1/100 of that observed for 1,2-epoxy-3-butene as intermediates in the metabolism of ethene or 1,3-butadiene.

Inhalation pharmacokinetics of 1,3-butadiene and 1,2-epoxybutene-3 in rats and mice.

Studies were conducted on inhalation pharmacokinetics of 1,3-butadiene and of its primary reactive metabolic intermediate 1,2-epoxybutene-3 in rats (Sprague-Dawley) and mice (B6C3F1). Investigations of inhalation pharmacokinetics of 1,3-butadiene revealed saturation kinetics of 1,3-butadiene metabolism in both species. For rats and mice linear pharmacokinetics apply at exposure concentrations below 1000 ppm 1,3-butadiene; saturation of 1,3-butadiene metabolism is observed at atmospheric concentrations of about 2000 ppm. The estimated maximal metabolic elimination rates were 400 mumole/hr/kg for mice and 200 mumole/hr/kg for rats. This shows that 1,3-butadiene is metabolized by mice at about twice the rate of rats. Investigations of inhalation pharmacokinetics of 1,2-epoxybutene-3 revealed major differences in metabolism of this compound between both species. No indication of saturation kinetics of 1,2-epoxybutene-3 metabolism could be observed in rats up to exposure concentrations of 5000 ppm, whereas in mice the saturation of epoxybutene metabolism became apparent at atmospheric concentrations of about 500 ppm. The estimated maximal metabolic rate for 1,2-epoxybutene-3 was 350 mumole/hr/kg in mice and greater than 2600 mumole/hr/kg in rats. When the animals are exposed to high concentrations of 1,3-butadiene, 1,2-epoxybutene-3 is exhaled by rats and mice. For rats 1,2-epoxybutene-3 concentration in the gas phase of the system reaches a plateau at about 4 ppm. For mice, 1,2-epoxybutene-3 concentration increases with exposure time until, at about 10 ppm, signs of acute toxicity are observed. Under these conditions hepatic nonprotein sulfhydryl compounds are virtually depleted in mice but not in rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhalation pharmacokinetics of isoprene in rats and mice.

Studies on inhalation pharmacokinetics of isoprene were conducted in rats (Wistar) and mice (B6C3F1) to investigate possible species differences in metabolism of this compound. Pharmacokinetic analysis of isoprene inhaled by rats and mice revealed saturation kinetics of isoprene metabolism in both species. For rats and mice, linear pharmacokinetics apply at exposure concentrations below 300 ppm isoprene. Saturation of isoprene metabolism is practically complete at atmospheric concentrations of about 1000 ppm in rats and about 2000 ppm in mice. In the lower concentration range where first-order metabolism applies, metabolic clearance (related to the concentration in the atmosphere) of inhaled isoprene per kilogram body weight was 6200 mL/hr for rats and 12,000 mL/hr for mice. The estimated maximal metabolic elimination rates were 130 mumole/hr/kg for rats and 400 mumole/hr/kg for mice. This shows that the rate of isoprene metabolism in mice is about two or three times that in rats. When the untreated animals are kept in a closed all-glass exposure system, the exhalation of isoprene into the system can be measured. This shows that the isoprene endogenously produced by the animals is systemically available within the animal organism. From such experiments the endogenous production rate of isoprene was calculated to be 1.9 mumole/hr/kg for rats and 0.4 mumole/hr/kg for mice. Our data indicate that the endogenous production of isoprene should be accounted for when discussing a possible carcinogenic or mutagenic risk of this compound.

Use of linear free energy relationships in toxicology: prediction of partition coefficients of volatile lipophilic compounds.

A relationship between the partition coefficient (P) and the boiling point (TBp, expressed in degrees Kelvin) was derived as TBp = alpha *ln(P) + beta. The relationship is based on the Clausius-Clapeyron equation and is valid for volatile lipophilic compounds, when one of the phases is air. Specific tissue/air and whole animal/air partition coefficients, as published in the literature, were used to validate the equation.

Concentration-dependent depletion of non-protein sulfhydryl (NPSH) content in lung, heart and liver tissue of rats and mice after acute inhalation exposure to butadiene.

The effects of different exposure concentrations of butadiene on the cellular non-protein sulfhydryl (NPSH) content of liver, lung and heart tissue were investigated in B6C3F1 mice and Sprague-Dawley rats. Groups of male animals of both species were exposed for 7 h to 10, 50, 100, 250, 500, 1000 and 2000 ppm butadiene. Immediately after exposure, NPSH content of liver, lung and heart tissue was determined according to a modified Ellman procedure. A comparison of both species shows that a dose-dependent NPSH depletion can be observed in mice for all tissues examined. In rats, liver NPSH content shows a major reduction at high exposure concentrations only. In mice, depletion of NPSH content of liver, lung and heart tissue starts at exposure concentrations of about 250 ppm butadiene. A reduction in NPSH content of about 80% is observed for lung tissue at 1000 ppm and for liver and heart tissue at exposure concentrations of 2000 ppm butadiene. The data on tissue concentrations of NPSH obtained after exposure of rats and mice to butadiene reflect the quantitative differences in butadiene metabolism and in biological effectivity of reactive butadiene intermediates between both species.

Increased alkylation of liver DNA and cell turnover in young versus old rats exposed to vinyl chloride correlates with cancer susceptibility.

To investigate the factors responsible for the high sensitivity of the livers of young rats to the carcinogenic stimulus of vinyl chloride (VC) adult and 11-day-old Wistar rats were exposed to [1,2-14C] VC. Adult rats received either a single 6-h exposure, or 2 single 6-h exposures separated by a treatment-free time interval of 15 h. Eleven-day-old rats received 2 single 6-h exposures, according to the same treatment schedule. The animals were sacrificed 1 h after the end of the corresponding exposure period; liver DNA was isolated, enzymatically hydrolyzed and analyzed by column chromatography. Incorporation of [14C]VC-derived radioactivity into the physiological deoxyribonucleosides (presumably reflecting the activity of DNA replication) was observed for all three sets of experiments. Virtually no difference in 14C-incorporation was observed between adult rats sacrificed immediately after one single 6-h exposure to [14C]VC and those which received a second exposure on the following day. In contrast, an about 8-fold increase in 14C-incorporation into the physiological purines of DNA of young versus adult rats was detected. This difference is indicative of a significantly elevated DNA synthesis/cell replication in the liver of young (11-d) rats. Radioactivity associated with 7-(2-oxoethyl)guanine was taken as an indicator of DNA alkylation by [14C]VC. Analysis of 7-(2-oxoethyl)guanine revealed that in adult animals the amount of this alkylation product formed is increased by a second exposure to VC. About 5-fold of the amount of 7-(2-oxoethyl)guanine present in adults could be determined in liver DNA of young (11-d) animals exposed under the same exposure conditions. Our results suggest that the high sensitivity of young rats to VC-induced hepatocarcinogenesis can reasonably be explained by enhanced DNA-alkylation and by increased cellular proliferation at an early age.

1,N6-etheno-2'-deoxyadenosine and 3,N4-etheno-2'-deoxycytidine detected by monoclonal antibodies in lung and liver DNA of rats exposed to vinyl chloride.

1,N6-Etheno-2'-deoxyadenosine (epsilon dAdo) and 3,N4-etheno-2'-deoxycytidine (epsilon dCyd) are formed in vitro by reaction of DNA with the electrophilic metabolites of vinyl chloride (VC), chloroethylene oxide and chloroacetaldehyde. To detect and quantitate these DNA adducts in vivo, we have raised a series of specific monoclonal antibodies (Mab). Among those, Mab EM-A-1 and Mab EM-C-1, respectively, were used for detection of epsilon dAdo and epsilon dCyd by competitive radioimmunoassay (RIA), following pre-separation of the etheno adducts from DNA hydrolysates by high performance liquid chromatography. At 50% inhibition of tracer-antibody binding, both Mab had a detection limit of 187 fmol and antibody affinity constants (K) of 2 x 10(9) l/mol. The levels of epsilon dAdo and epsilon dCyd were quantitated in the DNA of lung and liver tissue of young Sprague-Dawley rats exposed to 2000 p.p.m. of VC for 10 days. The epsilon dAdo/2'-deoxyadenosine and epsilon dCyd/2'-deoxycytidine molar ratios were 1.3 x 10(-7) and 3.3 x 10(-7), respectively, in lung DNA, and 5.0 x 10(-8) and 1.6 x 10(-7) in liver DNA. When hydrolysates of 3 mg of DNA were analyzed by RIA at 25% inhibition of tracer-antibody binding, epsilon dAdo and epsilon dCyd were not detected in liver DNA from untreated rats above the limiting epsilon dAdo/2'-deoxyadenosine and epsilon dCyd/2'-deoxycytidine molar ratios of 2.2 x 10(-8) and 3.1 x 10(-8), respectively.

Depletion of hepatic non-protein sulfhydryl content during exposure of rats and mice to butadiene.

B6C3F1 mice, Sprague-Dawley and Wistar rats were exposed to 1,3-butadiene in a closed exposure system. Exposure concentrations were kept above 2000 ppm to ensure saturation of butadiene metabolism in both species (Vmax conditions). Hepatic non-protein sulfhydryl (NPSH) content was determined in butadiene-exposed animals (and air-exposed controls) after exposures for 0, 7 and 15 h. Depletion of hepatic NPSH content was different for the species and strains investigated. In mice, hepatic NPSH content declined to about 20% after 7 h and was further depleted to about 4% at 15 h when signs of acute toxicity were observed. After a 7 h exposure of rats to butadiene, hepatic NPSH content was depleted to about 65% (Wistar) or 80% (Sprague-Dawley) of the corresponding controls but remained practically stable after a 15 h exposure to butadiene. The time-courses of depletion by butadiene of hepatic NPSH support previous findings on differences in butadiene metabolism between rats and mice and offer an additional explanation for the considerable species differences observed in the toxicity and carcinogenicity of this compound.

Species differences in butadiene metabolism between mouse and rat.

Studies on inhalation pharmacokinetics of 1,3-butadiene were conducted in mice (B6C3F1) and rats (Sprague-Dawley) to investigate the considerable differences in the susceptibility of both species to butadiene-induced carcinogenesis. In rats and mice metabolism of 1,3-butadiene to 1,2-epoxybutene-3 follows saturation kinetics. "Linear" (first-order) pharmacokinetics apply at exposure concentrations below 1000 ppm 1,3-butadiene. Saturation of butadiene metabolism is observed at atmospheric concentrations of about 2000 ppm butadiene. In the lower concentration range where first-order metabolism applies, metabolic clearance of inhaled 1,3-butadiene per kg body weight was 7300 ml (gas volume) x hr-1 for mice and 4500 ml x hr-1 for rats. The calculated maximal metabolic elimination rates (Vmax - conditions) were 400 mumol x hr-1 x kg-1 for mice and 220 mumol x hr-1 x kg-1 for rats. This shows that 1,3-butadiene is metabolized by mice at about twice the rate of rats, under conditions of both low and high exposure concentrations.

Inhalation pharmacokinetics of 1,2-epoxybutene-3 reveal species differences between rats and mice sensitive to butadiene-induced carcinogenesis.

Comparative investigations of inhalation pharmacokinetics of 1,2-epoxybutene-3 (vinyl oxirane, the primary reactive intermediate of butadiene) revealed major differences in metabolism of this compound between rats and mice. Whereas in rats no indication of saturation kinetics of epoxybutene metabolism could be observed up to exposure concentrations of 5000 ppm, in mice saturation of epoxybutene metabolism becomes apparent at atmospheric concentrations of about 500 ppm. The estimated maximal metabolic rate (Vmax) in mice for epoxybutene was only 350 mumol X h-1 X kg-1 (rats: greater than 2600 mumol X h-1 X kg-1). In the lower concentration range where first order metabolism applies (up to about 500 ppm) epoxybutene is metabolized by mice at higher rates compared to rats (metabolic clearance per kg body weight, mice: 24,900 ml X h-1, rats: 13,400 ml X h-1). Under these conditions the steady state concentration of epoxybutene in the mouse is about 10 times that in the rat. When mice are exposed to high concentrations of butadiene (greater than 2000 ppm; conditions of saturation of butadiene metabolism; closed exposure system) epoxybutene is exhaled by the animals, and its concentration in the gas phase increases with exposure time. At about 10 ppm epoxybutene signs of acute toxicity are observed. When rats are exposed to butadiene under similar conditions, the epoxybutene concentration reaches a plateau at about 4 ppm. Under these conditions hepatic non-protein sulfhydryl compounds are virtually depleted in mice but not in rats.(ABSTRACT TRUNCATED AT 250 WORDS)