Styrene oxide in blood, hemoglobin adducts, and urinary metabolites in human volunteers exposed to (13)C(8)-styrene vapors

Styrene is used in the manufacture of plastics and polymers and in the boat-building industry. The major metabolic route for styrene in rats, mice, and humans involves conversion to styrene-7,8-oxide (SO). The purpose of this study was to evaluate blood SO, SO-hemoglobin (SO-Hb) adducts, and urinary metabolites in styrene-exposed human volunteers and to compare these results with data previously obtained for rodents. Four healthy male volunteers were exposed for 2 h during light physical exercise to 50 ppm (13)C(8)-styrene vapor via a face mask. Levels and time profiles of styrene in exhaled air, blood, and urine (analyzed by GC) and urinary excretion patterns of mandelic acid and phenylglyoxylic acid in urine (analyzed by HPLC) were comparable to previously published volunteer studies. Maximum levels of SO in blood (measured by GC-MS) of 2.5-12.2 (average 6.7) nM were seen after 2 h, i.e., in the first sample collected after exposure had ended. The styrene blood level in humans was about 1.5 to 2 times higher than in rats and 4 times higher than in mice for equivalent styrene exposures. In contrast the SO levels in human blood was approximately fourfold lower than in mice. The level of hydroxyphenethylvaline (determined by GC-MS-MS) in pooled blood collected after exposure was estimated as 0.3 pmol/g globin corresponding to a SO-Hb adduct increment of about 0.003 pmol/g and ppmh. NMR analyses of urine showed that a major portion (> 95%) of the excreted (13)C-derived metabolites was derived from hydrolysis of SO, while only a small percentage of the excreted metabolites (< 5%) was derived from metabolism via phenylacetaldehyde. Signals consistent with metabolites derived from other pathways of styrene metabolism in rodents (such as glutathione conjugation with SO or ring epoxidation) were not detected.     

Metabolism and hepatorenal toxicity due to repeated exposure to styrene in spontaneously hypertensive rats (SHR)

Groups of adult male rats (5 rats per group), either normotensive (WKY) or spontaneously hypertensive (SHR), were exposed by inhalation to 0, 821, and 3018 ppm styrene, 5 h per day for 3 consecutive days. After the exposure, the urines were collected for 24 h and the animals were then sacrificed. The various biochemical parameters of hepatorenal toxicity due to styrene as well as its urinary metabolites were measured. Hepatotoxicity due to styrene was not further increased at any exposure level due to hypertension. However, repeated exposure of SHR rats to 3018 ppm styrene showed significant increases in the urinary excretion of gamma-glutamyl transpeptidase, proteins, and volume of urine, compared to WKY treated rats, whereas no such changes were observed due to repeated exposure to 821 ppm styrene. Studies of in vivo metabolism of styrene at higher exposure level showed significant decrease in the urinary excretion of mandelic, phenylglyoxylic, and hippuric acids in SHR rats compared to WKY-treated rats, suggesting an inhibition of deactivation of styrene reactive intermediate involving the epoxide hydrase pathway due to hypertension. At the same time, a significant increase in the urinary excretion of a potential nephrotoxic metabolite of styrene (e.g., mercapturates or thioethers) was observed in SHR-treated rats when compared to WKY-treated rats. These results demonstrate that spontaneous hypertension has the potential to further increase the nephrotoxicity due to repeated exposure to styrene, and the metabolism of styrene plays an important role in modifying such toxicity in the hypertensive state.     

Styrene-oxide N-terminal valine haemoglobin adducts in reinforced plastic workers: possible influence of genetic polymorphism of drug-metabolising enzymes

Styrene is one of the most important organic chemicals used worldwide. In humans, styrene metabolism involves oxidation by cytochrome P450 monooxygenases (CYPs) to styrene-7,8-oxide, an epoxide thought to be responsible for the genotoxic effects of styrene exposure, and detoxification by means of epoxide hydrolase (mEH) and glutathione S-transferases (GSTs). The objective of this study was to investigate if genetic polymorphisms of metabolic enzymes modulate the level of urinary styrene metabolites and styrene oxide adducts with N-terminal valine of human globin (SO-Hb) in 75 workers occupationally exposed to styrene and 77 unexposed controls. The mean air concentration of styrene in the breathing zone of workers (30.4ppm) was higher than the threshold limit value of 20ppm recommended by the American Conference of Governmental Industrial Hygienists (ACGIH), and the biological exposure index adopted by the ACGIH for exposure to styrene prior to the next shift (MA+PGA=400mg/g creatinine) was exceeded, indicating that styrene exposure for this group of workers was higher than recommended. A highly significant correlation was observed between styrene concentration in the breathing zone and the MA+PGA in urine of workers (r=0.85, P<0.001). The levels of SO-Hb adducts in exposed workers were significantly increased as compared with controls, although no difference was observed between subjects stratified as high and medium exposure categories based on MA+PGA excretion. Regarding the effect of the genetic polymorphisms we found that the level of SO-Hb adducts might be modulated by the predicted mEH enzymatic activity in the exposed workers. From our data we conclude that SO-Hb adduct measurement is a complementary method to MA+PG measurement for assessing exposure to styrene at occupational and environmental levels, which reflects a more extensive exposure period.     

Analysis of styrene and its metabolites in blood and urine of workers exposed to both styrene and acetone

A purge-and-trap gas chromatographic (PT-GC) method for determining styrene concentrations in urine and blood samples has been used in the biological monitoring of workers exposed to styrene and acetone. Blood and urine samples were collected from 34 individuals exposed to both solvents at the end of a 4-h shift and measured for styrene in urine (Su), blood (Sb), and the two major urinary metabolites, mandelic acid (MA) and phenylglyoxylic acid (PGA). A second urine sample was taken at the beginning of the next shift. Environmental exposure was measured using passive personal monitoring and GC. Urinary excretion of MA and PGA was measured by high-performance liquid chromatography. The average exposures to styrene and acetone were 70.5 mg/m3 and 370.5 mg/m3, respectively. In end-of-shift samples there was a significant correlation between concentrations of Su and Sb and the metabolites PGA, MA (r = 0.714 and 0.788, p < 0.001 for Su and r = 0.644 and 0.566, p < 0.005 for Sb). A high correlation between Sb and Su (r = 0.732, p < 0.001) also existed. Poor correlations were found between Su and metabolites in samples collected at the beginning of the next shift (r = 0.491 and 0.474 for PGA and MA, respectively, p < 0.05). There was a better correlation between the biological parameters at the end of the shift and the environmental styrene (r = 0.841 for PGA, r = 0.834 for MA, r = 0.788 for Su, and r = 0.698 for Sb; p < 0.001) compared with those at the start of the shift (r = 0.81 for PGA, 0.675 for MA, and 0.650 for Su; p < 0.001). We found that the concentration of excreted metabolites decreased significantly when environmental concentrations of acetone increased (p < 0.05), particularly at the end of the shift. Although the best correlation with environmental styrene was obtained with the sum of PGA and MA at the end of the shift (r = 0.862, p < 0.001), urine and blood styrene were shown to be more useful biological monitoring indicators because their concentrations were not affected by acetone co-exposure.     

Metabolism of [14C]1,2,7,8-tetrachlorodibenzo-p-dioxin in a ruminating Holstein calf.

1. [UL-7,8-ring 14C]-1,2,7,8-tetrachlorodibenzo-p-dioxin (1278-TCDD) was administered orally to a ruminating Holstein bull calf (43.6 kg; 1.2 mg kg(-1) body weight). Urine and faeces were collected daily for 96 h, while blood was sampled at multiple time points. Tissues were removed for combustion analysis. 2. Each tissue contained < 0.65 of the dose at 96h. Tissues with highest levels of 1278-TCDD, as a percentage of administered dose, were the large and small intestine, rumen, liver and carcass. 3. Urinary excretion accounted for 10.6% of the dose, and faecal excretion accounted for 81.6% of the administered dose. The major urinary and faecal metabolites were isolated and characterized by mass spectrometry and 1H-NMR. 4. Plasma levels of 14C peaked at 24h, and decreased to near background at 96 h. Detectable plasmal levels of 1278-TCDD were observed by 2 h. 5. A hydroxylated metabolite of 1278-TCDD was detected in calf plasma, which has the potential to interfere with thyroid hormone homeostasis.     

Biotransformation of medetomidine in the rat

1. The metabolites of a novel alpha 2-adrenoceptor agonist, medetomidine, in rat urine after subcutaneous administration at two dose levels (80 micrograms/kg or 5 mg/kg), and after incubation with rat liver fractions, were characterized by h.p.l.c., 1H-n.m.r and mass spectrometry. 2. Hydroxylation of a methyl substituent was the main biotransformation in vitro. Hydroxylation occurred at a rate sufficient for high metabolic clearance. 3. The major urinary metabolites were the glucuronide of hydroxymedetomidine (about 35% of urinary metabolites) and medetomidine carboxylic acid (about 40%). 4. Medetomidine unchanged represented about 1% or 10% of the urinary excretion products, dependent on dose. 5. A metabolic pathway consisting of hydroxylation with subsequent glucuronidation, or further oxidation to carboxylic acid, is suggested.     

4-Vinylphenol excretion suggestive of arene oxide formation in workers occupationally exposed to styrene

The toxicity of styrene has often been attributed to the formation of reactive epoxide intermediate, styrene-7,8-oxide. It has been suggested that in addition, an arene oxide, styrene-3,4-oxide, is a metabolite of styrene. Styrene-3,4-oxide is easily converted to corresponding phenols. In this study the presence of 4-vinylphenol in the urine is verified by gas chromatography/mass spectrometry and its quantity compared to mandelic acid excretion. Both 4-vinylphenol and mandelic acid were detected in the urine samples of workers occupationally exposed to styrene. No 4-vinylphenol was found in urine samples of unexposed individuals. The correlation between mandelic acid and 4-vinylphenol was fairly good (r = 0.93); increasing excretion of mandelic acid was also accompanied by increasing amounts of 4-vinylphenol in the urine. The interindividual variation of the 4-vinylphenol/mandelic acid excretion ratio was small, the mean ratio being about 0.3%. The presence of 4-vinylphenol in the urine of workers exposed to styrene suggests that, in man, styrene is also metabolized via arene oxidation. However, when the arene oxidation of styrene is compared to vinyl group oxidation the latter appears to be at least quantitatively by far the more important metabolic pathway.     

Influence of genetic polymorphisms of styrene-metabolizing enzymes and smoking habits on levels of urinary metabolites after occupational exposure to styrene

Here we evaluate the influence of individual genetic polymorphisms of drug-metabolizing enzymes as well as body mass index (BMI) and lifestyle (smoking, alcohol consumption) on urinary metabolites after occupational exposure to styrene. Seventy-three workers exposed to styrene in a reinforced-plastics workplace were studied. The personal styrene exposure in the air and the urinary styrene metabolites mandelic acid and phenylglyoxylic acid were measured. The subjects' genetic polymorphisms in the genes that encode the styrene-metabolizing enzymes CYP2E1, CYP2B6, EPHX1, GSTM1, GSTT1 and GSTP1 were determined. Neither genotype nor lifestyle significantly affected urinary metabolites. There was, however, an interaction between the CYP2E1 genotype and smoking. Among non-smokers, urinary styrene metabolites were significantly decreased in subjects with c1/c1 alleles of CYP2E1 as compared with those with the c1/c2 genotype. There was no significant difference in urinary metabolites among smokers. When the combined influence of the CYP2B6 genotype and the predicted activity of EPHX1 were examined, urinary metabolites in subjects with low enzyme activity were lower than in those with medium or high activity after high styrene exposure (>or=50 ppm). The results suggest that genetic susceptibility and lifestyle should be considered in biological monitoring of exposure to styrene.     

Genotoxicity of styrene-7,8-oxide and styrene in Fisher 344 rats: a 4-week inhalation study

The cytogenetic alterations in leukocytes and the increased risk for leukemia, lymphoma, or all lymphohematopoietic cancer observed in workers occupationally exposed to styrene have been associated with its hepatic metabolisation into styrene-7,8-oxide, an epoxide which can induce DNA damages. However, it has been observed that styrene-7,8-oxide was also found in the atmosphere of reinforced plastic industries where large amounts of styrene are used. Since the main route of exposure to these compounds is inhalation, in order to gain new insights regarding their systemic genotoxicity, Fisher 344 male rats were exposed in full-body inhalation chambers, 6 h/day, 5 days/week for 4 weeks to styrene-7,8-oxide (25, 50, and 75 ppm) or styrene (75, 300, and 1000 ppm). Then, the induction of micronuclei in circulating reticulocytes and DNA strand breaks in leukocytes using the comet assay was studied at the end of the 3rd and 20th days of exposure. Our results showed that neither styrene nor styrene-7,8-oxide induced a significant increase of the micronucleus frequency in reticulocytes or DNA strand breaks in white blood cells. However, in the presence of the formamidopyridine DNA glycosylase, an enzyme able to recognize and excise DNA at the level of some oxidized DNA bases, a significant increase of DNA damages was observed at the end of the 3rd day of treatment in leukocytes from rats exposed to styrene but not to styrene-7,8-oxide. This experimental design helped to gather new information regarding the systemic genotoxicity of these two chemicals and may be valuable for the risk assessment associated with an occupational exposure to these molecules.     

Identification and determination of urinary metabolites of 2-isopropylnaphthalene in rabbits

Four unconjugated metabolites, which were produced through the oxidation of the isopropyl chain of 2-isopropylnaphthalene (2-IPN), were isolated from the urine of rabbits receiving 2-IPN orally and identified: 2-(2-naphthyl)propionic acid, 2-(2-naphthyl)-2-propanol, 2-(2-naphthyl)-1,2-propanediol, and 2-(2-naphthyl)-2-hydroxypropionic acid, together with a small amount of the unchanged compound. Further, the unconjugated metabolites, which were produced through the oxidation of the naphthalene ring, were isolated and identified: 2-isopropylnaphthols and 2-isopropyl-5,6 (or 7,8)-dihydronaphthalene-5,6 (or 7,8)-diol. The identification of these metabolites was made by means of TLC, GLC, MS, IR, GC/MS, and FT-NMR. The presence of glucuronides of metabolites B, C, D, F, and H was also suggested by TLC and GLC of the extract obtained after hydrolysis by beta-glucuronidase. In addition, quantitative determination of the metabolites indicated that the total urinary excretion of the metabolites except 2-isopropylnaphthols in 24 hr after administration was about 29% of the dose.     

Lack of relationship between glibenclamide metabolism and debrisoquine or mephenytoin hydroxylation phenotypes.

The pharmacokinetics of a single oral dose of 1.75 mg glibenclamide were studied in 15 healthy Caucasians including five poor metabolisers of debrisoquine and five poor metabolisers of S-mephenytoin. Plasma glibenclamide concentrations and the urinary concentrations of trans-4- and cis-3-hydroxyglibenclamide were analyzed by h.p.l.c. Thirty-six +/- 6% (mean +/- s.d., n = 15) of the given dose of glibenclamide was excreted in 48 h urine as hydroxylated metabolites, 27 +/- 4% as trans-4-hydroxyglibenclamide and 8 +/- 2% as cis-3-hydroxyglibenclamide. There were no differences in the plasma pharmacokinetics of glibenclamide or in the urinary excretion of the metabolites between poor and extensive metabolisers of debrisoquine, neither between the two mephenytoin hydroxylator phenotypes. The study thus indicates that the disposition of glibenclamide is not influenced by these two independent polymorphisms of drug oxidation.     

Studies of nephrotoxicity due to mixed exposure to styrene and toluene

Adult male Sprague-Dawley rats were treated simultaneously with 4 mmol styrene per kg i.p., twice a day at an interval of 4 h, and 10 mmol toluene per kg once a day, 5 days a week for 4 consecutive weeks. After the last day of treatment, the rats were placed in metabolism cages for collection of urines for 24 h and then were sacrificed. Such mixed exposure produced significant increases in the urinary excretion of gamma-glutamyl transpeptidase, glucose and proteins as compared to those with either solvent alone. An increase, but not significant, in the urinary excretion of N-acetyl-beta-D-glucosaminidase was also noticed due to such exposure. Electron microscopic examination of renal cortex 24 h after the mixed exposure showed the appearance of many vacuoles of various sizes surrounded by a single membrane, which were not seen in rats treated with either styrene or toluene alone. Metabolism studies showed only a significant increase in the urinary excretion of hippuric acid due to mixed exposure. These data indicate that under certain conditions, mixed subchronic exposure to styrene and toluene may have the potential to increase further the nephrotoxic response as compared to that of either solvent alone.     

Interest of genotyping and phenotyping of drug-metabolizing enzymes for the interpretation of biological monitoring of exposure to styrene

In the field of occupational and/or environmental toxicology, the measurement of specific metabolites in urine may serve to assess exposure to the parent compounds (biological monitoring of exposure). Styrene is one of the chemicals for which biological monitoring programs have been validated and implemented in environmental and occupational medicine. However, inter-individual differences in the urinary excretion exist both for the main end-products (mandelic acid and phenylglyoxylic acid) and for its specific mercapturic acids (phenylhydroxyethylmercapturic acids, PHEMA). This limits to a certain extent the use of these metabolites for an accurate assessment of styrene exposure. In a group of 26 volunteers selected with relevant genotypes, and exposed to styrene vapours (50 mg/m3, 8 h) in an inhalation chamber, we evaluated whether genotyping or phenotyping relevant drug-metabolizing enzymes (CYP2E1, EPHX1, GSTM1, GSTT1 and GSTP1) may help to explain the observed inter-individual variability in the urinary metabolite excretion. Peripheral blood lymphocytes were used for genotyping and as reporter cells for the phenotyping of CYP2E1 and EPHX1. The GSTM1 genotype was clearly the most significant parameter explaining the variance in urinary PHEMA excretion (6-fold lower in GSTM1 null subjects; P < 0.0001) so that systematic GSTM1 genotyping should be recommended routinely for a correct interpretation of PHEMA urinary levels. Variant alleles CYP2E1*6 (7632T>A) and His113EPHX1 were associated with a significant reduction of, respectively, the expression (P = 0.047) and activity (P = 0.022) of the enzyme in peripheral blood lymphocytes. In combination with GSTM1 genotyping, the phenotyping approach also contributed to improve the interpretation of urinary results, as illustrated by the combined effect of CYP2E1 expression and GSTM1 allelic status that explained 77% of the variance in PHEMA excretion and allows the recommendation of mercapturates as specific and reliable biomarkers of exposure to styrene.     

The uptake,distribution, metabolism,and excretion of methyl tertiary-butyl ether inhaled alone and in combination with gasoline vapor

The purpose of these studies was to evaluate the tissue uptake, distribution, metabolism, and excretion of methyl tertiary-butyl ether (MTBE) in rats and to determine the effects of coinhalation of the volatile fraction of unleaded gasoline on these parameters. Male F344 rats were exposed nose-only once for 4 h to 4, 40, or 400 ppm 14C-MTBE and to 20 and 200 ppm of the light fraction of unleaded gasoline (LFG) containing 4 and 40 ppm 14C-MTBE, respectively. To evaluate the effects of repeated inhalation of LFG on the fate of inhaled MTBE, rats were exposed for 7 consecutive days to 20 and 200 ppm LFG followed on d 8 by exposure to LFG containing 14C-MTBE. Three subgroups of rats were included for evaluation of respiratory parameters, rates and routes of excretion, and tissue distribution and elimination. MTBE and its chief metabolite, tertiary-butyl alcohol, were quantitated in blood and kidney (immediately after exposure), and the major urinary metabolites, 2-hydroxyisobutyric acid and 2-methyl-1,2- propanediol, were identified and quantified in urine. Inhalation of MTBE alone or as a component of LFG had no concentration-dependent effect on respiratory minute volume. The initial body burdens (IBBs) of MTBE equivalents achieved after 4 h of exposure to MTBE did not increase linearly with exposure concentration. MTBE equivalents rapidly distributed to all tissues examined, with the largest percentages distributed to liver. Between 40 and 400 ppm, there was a significant reduction in percentage of the IBB present in the major organs examined, both immediately and 72 h after exposure. At 400 ppm, the elimination rates of MTBE equivalents from tissues changed significantly. Furthermore, at 400 ppm there was a significant decrease in the elimination half-time of volatile organic compounds (VOCs) in breath and a significant increase in the percentage of the IBB of MTBE equivalents eliminated as VOCs in breath. LFG coexposure significantly decreased the percentage of the MTBE equivalent IBBs in tissues and increased rates of elimination of MTBE equivalents. The study results indicate that the uptake and fate of inhaled MTBE are altered upon increasing exposure levels from 4 to 400 ppm, suggesting that toxic effects observed previously upon repeated inhalation of concentrations of 400 ppm or greater may not necessarily be linearly extrapolated to effects that might occur at lower concentrations. Furthermore, coexposure to LFG, whether acute or repeated, decreases tissue burdens of MTBE equivalents and enhances the elimination rate of MTBE and its metabolites, thereby potentially reducing the toxic effects of the MTBE compared to when it is inhaled alone.     

Further analysis of sparteine oxidation in a Japanese population and comparison with data observed in different ethnic populations

1. Data on the oxidation polymorphism of sparteine (SP) studied in 84 unrelated Japanese subjects of whom two (2.4%) were classified as poor metabolizers (PMs) were re-evaluated. The data were obtained from 6-hour urinary excretion ratios of SP to 2- and 5-dehydrosparteines (DHS), after an oral dose of 100 mg of SP sulphate. 2. Urinary excretion of both SP and DHS correlated with the SP/DHS ratio (rs = 0.862 and -0.756, respectively, P less than 0.001). In addition, urinary excretion of 2-DHS, 5-DHS or total DHS discriminated between PMs and extensive metabolizers (EMs). There was also a highly significant correlation (rs = 0.669, P less than 0.001) between the urinary excretion of 2- and 5-DHS. 3. These re-evaluated results on the oxidation polymorphism of SP indicate that 2- and 5-DHS formation from SP shares a common metabolic pathway (presumably via the same P-450 isozyme), and that the SP/DHS ratio, conventionally used as a discriminating index between PMs and EMs, quantitatively reflects the capacity of 2- and 5-DHS formation. 4. The benefit of using a shorter (6 h) collection period for assessing the individual oxidation phenotype of SP and inter-ethnic comparison of SP oxidation is also discussed.     

Effects of styrene and its metabolites on different lung compartments of the mouse--cell proliferation and histomorphology

Styrene is not carcinogenic in rats but has caused pneumotoxicity and increased lung tumors after inhalation in mice. This study investigated whether styrene-7,8-oxide, ring-oxidized, and side-chain hydroxylated styrene metabolites induce cell proliferation, apoptosis, pathological changes, and glutathione depletion in mice lungs. Intraperitoneal treatment with phenylacetaldehyde and phenylacetic acid (3 x 100 mg/kg b.w./day) increased the levels of apoptosis and cell proliferation in the alveoli without producing any effects in the terminal bronchioli, the target site of tumor formation in mice. Only styrene-oxide (SO) at 3 x 100 mg/kg b.w./day and 4-vinyl-phenol (4-VP) at 3 x 35 and 3 x 20 mg/kg b.w./day, respectively, caused up to 19-fold increases in cell proliferation in the large/medium bronchi and terminal bronchioles; marginal increases in alveolar cell proliferation were noted with SO (1.6-fold) but not with 4-VP. These compounds also caused glutathione depletion in the bronchiolar epithelium and histomorphological changes of the bronchiolar epithelium in large and medium bronchi and terminal bronchioles. Changes were characterized by flattened cells and a loss of the typical bulging of the "dome-shaped" Clara cells, suggesting that Clara cells were primary target cells. The specific reactions of mouse lung to SO and 4-VP could serve as a verifiable hypothesis for the different response of rats and mice with regard to tumor formation.     

The importance of measured end-points in demonstrating the occurrence of interactions: a case study with methylchloroform and m-xylene.

Mixed exposures may result in significant changes in one biomarker of exposure without altering another biomarker, and this may have unknown significance in terms of exposure assessment and overall toxicity of the mixture. Results from a previous investigation showed that human exposure to methylchloroform (MC, 400 ppm) and m-xylene (XYL, 200 ppm) during 4 h did not result in any significant effect on blood concentrations of these solvents, suggesting the absence of interaction between MC and XYL. Those results were adequately described by conducting a physiologically-based toxicokinetic (PBTK) modeling of the MC-XYL interaction in humans; however, the model suggested that urinary excretion of MC metabolites would be reduced as a result of combined exposure, whereas that of XYL metabolites would not be modified. An experimental verification of this model prediction was then undertaken with rats. In this study, Sprague-Dawley rats (n, 5) were exposed during 4 h to MC (400 ppm) or XYL (200 ppm), alone or as a mixture. Results showed that combined exposure did not affect the blood concentration of MC whereas that of XYL was increased throughout the 2-h blood collection period following exposure. The excretion of MC metabolites during a period of 48 h following the onset of exposure, i.e., trichloroethanol (TCE: -71%) and trichloroacetic acid (TCA: -73%), were significantly reduced. Methylhippuric acid (MHA) was not affected by co-exposure to MC as expected from the PBTK model forecasts. These results exemplify the use of a priori PBPK modeling for designing interaction studies and choosing appropriate/sensitive end-points for demonstrating the occurrence of potential interactions.     

Biotransformation of medetomidine in the rat

1. The metabolites of a novel α₂-adrenoceptor agonist, medetomidine, in rat urine after subcutaneous administration at two dose levels (80 μg/kg or 5 mg/kg), and after incubation with rat liver fractions, were characterized by h.p.l.c., ¹H-n.m.r and mass spectrometry.

2. Hydroxylation of a methyl substituent was the main biotransformation in vitro. Hydroxylation occurred at a rate sufficient for high metabolic clearance.

3. The major urinary metabolites were the glucuronide of hydroxymedetomidine (about 35% of urinary metabolites) and medetomidine carboxylic acid (about 40%).

4. Medetomidine unchanged represented about 1% or 10% of the urinary excretion products, dependent on dose.

5. A metabolic pathway consisting of hydroxylation with subsequent glucuronidation, or further oxidation to carboxylic acid, is suggested.     

Metabolism of [14C]1,2,7,8-tetrachlorodibenzo-p-dioxin in a ruminating Holstein calf

1. [UL-7,8-ring ¹⁴C]-1,2,7,8-tetrachlorodibenzo-p-dioxin (1278-TCDD) was administered orally to a ruminating Holstein bull calf (43.6 kg; 1.2?mg kg^-1 body weight). Urine and faeces were collected daily for 96?h, while blood was sampled at multiple time points. Tissues were removed for combustion analysis. 2. Each tissue contained &;lt; 0.6% of the dose at 96h. Tissues with highest levels of 1278-TCDD, as a percentage of administered dose, were the large and small intestine, rumen, liver and carcass. 3. Urinary excretion accounted for 10.6% of the dose, and faecal excretion accounted for 81.6% of the administered dose. The major urinary and faecal metabolites were isolated and characterized by mass spectrometry and ¹H-NMR. 4. Plasma levels of ¹⁴C peaked at 24?h, and decreased to near background at 96?h. Detectable plasma levels of 1278-TCDD were observed by 2?h. 5. A hydroxylated metabolite of 1278-TCDD was detected in calf plasma, which has the potential to interfere with thyroid hormone homeostasis.     

Linoleate-dependent co-oxygenation of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol by rat cytosolic lipoxygenase.

1. Co-oxygenation of 14C-labelled benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol was studied in rat lung cytosol, using linoleic acid as a co-substrate. Covalently bound and soluble metabolites were quantified by radiometry and h.p.l.c., respectively. 2. The co-oxygenation resulted in the production of reactive metabolites capable of protein binding as well as a series of soluble derivatives. 3. Co-oxygenation of benzo(a)pyrene yielded primarily a significant amount of benzo(a)pyrene-6,12-dione while benzo(a)pyrene-7,8-dihydrodiol led to a significant amount of benzo(a)pyrene-trans-anti-tetrol. 4. Their production was abolished by addition of 25 microM of the lipoxygenase inhibitor and antioxidant NDGA. 5. It is postulated that the linoleic acid peroxyl radicals, formed by rat lung lipoxygenase, initiate the one-electron oxidation of benzo(a)pyrene to its quinones, and epoxidation of benzo(a)pyrene-7,8-diol to the ultimate carcinogenic benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide.