Johannes G. Filser

A physiologically based pharmacokinetic model for butadiene and its metabolite butadiene monoxide in rat and mouse and its significance for risk extrapolation

The gas 1,3-butadiene (BU) is an important industrial chemical and an environmental air pollutant. BU has been shown to be a weak carcinogen in the rat but a potent carcinogen in the B6C3F1 mouse. This species difference makes risk extrapolation to humans difficult and the underlying mechanism should be clarified before meaningful risk extrapolation to humans can be made. One possible explanation for the species differences in cancer response is that there are quantitative species differences in the formation of genotoxic epoxides. To investigate this possibility a physiologically based pharmacokinetic (pbpk) model for BU together with its first reactive metabolite l,2-epoxybutene-3 (butadiene monoxide, BMO) was developed. Previously reported values on hepatic glutathione (GSH) turnover, depletion of hepatic GSH in rodents exposed to BU, and in vitro metabolic data of BU and BMO were included in the model, which incorporates intrahepatic first-pass hydrolysis of BMO and the ordered sequential, ping-pong mechanism to describe the enzyme kinetics of BMO-GSH conjugation. In vitro studies were carried out to obtain tissue: air partition coefficients of BU and BMO in rat tissue homogenates. The simulated pharmacokinetics of BU, BMO, and GSH agreed with previously published experimental observations in rat and mouse obtained in closed and open chamber experiments. According to the model, the internal dose of BMO (expressed either as the concentration in mixed venous blood or as the area under the concentration-time curve) is approximately 1.6 times higher in the mouse than in the rat for exposure to BU below 1000 ppm. At higher exposure levels, GSH depletion occurs in the mouse, but not in the rat, after about 6–9 h. This GSH depletion results in up to 2–3 times higher internal doses in the mouse than in the rat. The clear but relatively small species difference in body burdens of BMO indicated from our model can only partly explain the marked species difference in cancer response between mice and rats exposed to BU.

Species differences in butadiene metabolism between mice and rats evaluated by inhalation pharmacokinetics

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 7300ml×h−1 (rat: 4500 ml×h−1). Maximal metabolic elimination rate (Vmax) was 400 μmol×h−1 ×kg−1 (rat: 220 μmol ×h−1×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.

Experimental data from closed chamber gas uptake studies in rodents suggest lower uptake rate of chemical than calculated from literature values on alveolar ventilation

Experimental data obtained in vivo with the closed-chamber gas uptake technique have been reported for a series of volatile chemicals. Pharmacokinetic analyses of these data have been performed either by using a two-compartment model or physiological models. In the former the transfer rate of chemical from ambient air to body is defined by the clearance of uptake. In the latter models the transfer rate depends on alveolar ventilation, cardiac output, and blood: air partition coefficient. In this communication we describe the quantitative relationship between clearance of uptake and alveolar ventilation, cardiac output, and blood: air partition coefficient. Theoretical values of clearance of uptake were calculated for a variety of volatile chemicals using literature data on alveolar ventilation, cardiac output, and blood: air partition coefficient. For most chemicals the experimentally determined values in rats and mice were about 60% of the theoretical values. This suggests that the inhalatory uptake rate of chemical may be overestimated if literature values of alveolar ventilation are used in physiological pharmacokinetic models for rodents.

Species-specific pharmacokinetics of styrene in rat and mouse

The pharmacokinetics of styrene were investigated in male Sprague-Dawley rats and male B6C3F1 mice using the closed chamber technique. Animals were exposed to styrene vapors of initial concentrations ranging from 550 to 5000 ppm, or received intraperitoneal (i.p.) doses of styrene from 20 to 340 mg/kg or oral (p.o.) doses of styrene in olive oil from 100 to 350 mg/kg. Concentration-time courses of styrene in the chamber atmosphere were monitored and analyzed by a pharmacokinetic two-compartment model. In both species, the rate of metabolism of inhaled styrene was concentration dependent. At steady state it increased linearly with exposure concentration up to about 300 ppm; more than 95% of inhaled styrene was metabolized and only small amounts were exhaled unchanged. At these low concentrations transport to the metabolizing enzymes and not their metabolic capacity was the rate limiting step for metabolism. Pharmacokinetic behaviour of styrene was strongly influenced by physiological parameters such as blood flow and especially the alveolar ventilation rate. At exposure concentrations of styrene above 300 ppm the rate of metabolism at steady state was progressively limited by biochemical parameters of the metabolizing enzymes. Saturation of metabolism (Vmax) was reached at atmospheric concentrations of about 700 ppm in rats and 800 ppm in mice, Vmax being 224 μmol/(h·kg) and 625 μmol/(h·kg), respectively. The atmospheric concentrations at Vmax/2 were 190 ppm in rats and 270 ppm in mice. Styrene accumulates preferentially in the fatty tissue as can be deduced from its partition coefficients in olive oil∶air and water∶air which have been determined in vitro at 37°C to be 5600 and 15. In rats and mice exposed to styrene vapors below 300 ppm, there was little accumulation since the uptake was rate limiting. The bioaccumulation factor body:air at steady state (K′st*) was rather low in comparison to the thermodynamic partition coefficient body:air (Keq) which was determined to be 420. K′st* increased from 2.7 at 10 ppm to 13 at 310 ppm in the rat and from 5.9 at 20 ppm to 13 at 310 ppm in the mouse. Above 300 ppm, K′st* increased considerably with increasing concentration since metabolism became saturated in both species. At levels above 2000 ppm K′st* reached its maximum of 420 being equivalent to Keq. Pretreatment with diethyldithiocarbamate, administered intraperitoneally (200 mg/kg in rats, 400 mg/kg in mice) 15 min prior to exposure of styrene vapours, resulted in effective inhibition of styrene metabolism, indicating that most of the styrene is metabolized by cytochrome P450-dependent monooxygenases. In order to simulate chronic exposure rats and mice were exposed to 150 and 500 ppm styrene on 5 consecutive days (6 h/day). On day 6, inhalation kinetics were studied. No change in the rate of styrene metabolism was detected compared to non-pretreated controls. Intraperitoneal administration of styrene to rats and mice led to concentration-time courses in the atmosphere of the closed chamber with agreed with those predicted by the applied pharmacokinetic model. After p.o. administration of styrene to rats and mice concentration time-courses showed considerable inter-animal variability. The pharmacokinetic model was extended by a first order absorption from the gastrointestinal tract with half-lives of 0.87 h (rat) and 0.41 h (mouse) to obtain reasonable fits through the measured data. The pharmacokinetic parameters of inhaled styrene were extrapolated allometrically from rat to mouse and from rat and mouse to man. A good agreement was obtained with experimentally determined values indicating similar pharmacokinetic behaviour of styrene in these species.

Tissue distribution of DNA adducts in male Fischer rats exposed to 500 ppm of propylene oxide: quantitative analysis of 7-(2-hydroxypropyl)guanine by 32P-postlabelling

7-(2-Hydroxypropyl)guanine (7-HPG) constitutes the major adduct from alkylation of DNA by the genotoxic carcinogen, propylene oxide. The levels of 7-HPG in DNA of various organs provides a relevant measure of tissue dose. 7-Alkylguanines can induce mutation through abasic sites formed from spontaneous depurination of the adduct. In the current study the formation of 7-HPG was investigated in male Fisher 344 rats exposed to 500 ppm of propylene oxide by inhalation for 6 h/day, 5 days/week, for up to 20 days. 7-HPG was analyzed using the 32P-postlabelling assay with anion-exchange cartridges for adduct enrichment. In animals sacrificed directly following 20 days of exposure, the adduct level was highest in the respiratory nasal epithelium (98.1 adducts per 10(6) nucleotides), followed by olfactory nasal epithelium (58.5), lung (16.3), lymphocytes (9.92), spleen (9.26), liver (4.64), and testis (2.95). The nasal cavity is the major target for tumor induction in the rat following inhalation. This finding is consistent with the major difference in adduct levels observed in nasal epithelium compared to other tissues. In rats sacrificed 3 days after cessation of exposure, the levels of 7-HPG in the aforementioned tissues had, on the average, decreased by about one-quarter of their initial concentrations. This degree of loss closely corresponds to the spontaneous rate of depurination for this adduct (t 1/2 = 120 h), and suggests a low efficiency of repair for 7-HPG in the rat. The postlabelling assay used had a detection limit of one to two adducts per 10(8) nucleotides, i.e. it is likely that this adduct could be analyzed in nasal tissues of rats exposed to less than 1 ppm of propylene oxide.

Pharmacokinetics of isoprene in mice and rats

Pharmacokinetic analysis of isoprene inhaled by male Wistar rats and male B6C3F1 mice showed saturation kinetics in both species. Below atmospheric concentrations of 300 ppm in rats and in mice the rate of metabolism is directly proportional to the concentration. The low accumulation of isoprene in the body at low atmospheric concentrations suggests transport limitation of the metabolism. Only small amounts of isoprene taken up are exhaled as unchanged substance (15% in rats and 25% in mice). Its half life in rats is 6.8 min and in mice 4.4 min. At concentrations above 300 ppm the rate of metabolism does not increase further in proportion to the atmospheric concentration. It finally approaches maximal values of 130 mumol/(h X kg) body weight at atmospheric concentrations above 1500 ppm in rats, and 400 mumol/(h X kg) body weight at concentrations above 2000 ppm in mice. This indicates limited production of the two possible mono-epoxides of isoprene at high concentrations. Isoprene is endogenously produced and is systemically available. Its production rate is 1.9 mumol/(h X kg) in rats, and 0.4 mumol/(h X kg) in mice, respectively. Part of the endogenous isoprene is exhaled by the animals but it is metabolized to a greater extent: the rate of metabolism of endogenously produced and systemically available isoprene is 1.6 mumol/(h X kg) (rats) and 0.3 mumol/(h X kg) (mice).

Metabolism and pharmacokinetics of vinyl acetate

The hydrolysis of vinyl acetate (formation of acetic acid) has been studied in vitro with rat liver and lung microsomes, rat and human plasma and purified esterases (such as acetylcholine esterase, butyrylcholine esterase, carboxyl esterase). Characterization of the kinetic parameters revealed that rat liver microsomes and purified carboxyl esterase (from porcine liver) displayed the highest activity.In order to establish the rate of metabolism of vinyl acetate in vivo, rats were exposed in closed desiccator jar chambers, and gas uptake kinetics were studied. The decay of vinyl acetate was dose-dependent, indicating possible saturation of metabolic pathway(s). The maximal clearance (at lower concentrations) of vinyl acetate from the system (30 000 ml/h per kg body weight) was similar to the maximal ventilation rate in this species. This indicated that under conditions when metabolic enzymes are not saturated the metabolic rate is mainly determined by pulmonary uptake.The exposure of rats to vinyl acetate resulted in a transient exhalation of significant amounts of acetaldehyde into the closed exposure system. This indicates the presence of this metabolic intermediate of vinyl acetate in the organism in vivo.

Propylene oxide: mutagenesis, carcinogenesis and molecular dose.

The results from mutagenic and carcinogenic studies of propylene oxide (PO) and the current efforts to develop molecular dosimetry methods for PO-DNA adducts are reviewed. PO has been shown to be active in several bacterial and mammalian mutagenicity tests and induces site of contact tumors in rodents after long-term administration. Quantitation of N7-(2-hydroxypropyl)guanine (7-HPG) in nasal and hepatic tissues of male F344 rats exposed to 500 ppm PO (6 h/day; 5 days/week for 4 weeks) by inhalation was performed to evaluate the potential of high concentrations of PO to produce adducts in the DNA of rodent tissues and to obtain information necessary for the design of molecular dosimetry studies. The persistence of 7-HPG in nasal and hepatic tissues was studied in rats killed three days after cessation of a 4-week exposure period. DNA samples from exposed and untreated animals were analyzed for 7-HPG by two different methods. The first method consisted of separation of the adduct from DNA by neutral thermal hydrolysis, followed by electrophoretic derivatization of the adduct and gas chromatography-high resolution mass spectrometry (GC-HRMS) analysis. The second method utilized 32P-postlabeling to quantitate the amount of this adduct in rat tissues. Adducts present in tissues from rats killed immediately after cessation of exposure were 835.4 +/- 80.1 (respiratory), 396.8 +/- 53.1 (olfactory) and 34.6 +/- 3.0 (liver) pmol adduct/mumol guanine using GC-HRMS. Lower values, 592.7 +/- 53.3, 296.5 +/- 32.6 and 23.2 +/- 0.6 pmol adduct/mumol guanine were found in respiratory, olfactory and hepatic tissues of rats killed after three days of recovery. Analysis of the tissues by 32P-postlabeling yielded the following values: 445.7 +/- 8.0 (respiratory), 301.6 +/- 49.2 (olfactory) and 20.6 +/- 1.8 (liver) pmol adduct/mumol guanine in DNA of rats killed immediately after exposure cessation and 327.1 +/- 21.7 (respiratory), 185.3 +/- 29.2 (olfactory) and 15.7 +/- 0.9 (liver) pmol adduct/mumol guanine after recovery. Current methods of quantitation did not provide evidence for the endogenous formation of this adduct in control animals. These studies demonstrated that the target tissue for carcinogenesis has much greater alkylation of DNA than liver, a tissue that did not exhibit a carcinogenic response.

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 μmol×h−1×kg−1 (rats: >2600 μmol× h−1×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: 24900 ml×h−1, rats: 13400 ml×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 (>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. We conclude that in addition to the higher rate of metabolism of butadiene in mice, limited detoxification and consequently accumulation of its primary reactive intermediate epoxybutene may be a major determinant for the higher susceptibility of mice to butadiene-induced carcinogenesis.

Changes in the classification of carcinogenic chemicals in the work area

Abstract Carcinogenic chemicals in the work area are currently classified into three categories in section III of the German List of MAK and BAT Values (list of values on maximum workplace concentrations and biological tolerance for occupational exposures). This classification is based on qualitative criteria and reflects essentially the weight of evidence available for judging the carcinogenic potential of the chemicals. It is proposed that these categories – IIIA1, IIIA2, IIIB – be retained as Categories 1, 2, and 3, to correspond with European Union regulations. On the basis of our advancing knowledge of reaction mechanisms and the potency of carcinogens, these three categories are supplemented with two additional categories. The essential feature of substances classified in the new categories is that exposure to these chemicals does not contribute significantly to risk of cancer to man, provided that an appropriate exposure limit (MAK value) is observed. Chemicals known to act typically by nongenotoxic mechanisms and for which information is available that allows evaluation of the effects of low-dose exposures, are classified in Category 4. Genotoxic chemicals for which low carcinogenic potency can be expected on the basis of dose-response relationships and toxicokinetics, and for which risk at low doses can be assessed are classified in Category 5.The basis for a better differentiation of carcinogens is discussed, the new categories are defined, and possible criteria for classification are described. Examples for Category 4 (1,4-dioxane) and Category 5 (styrene) are presented.