The pharmacokinetics of [14C]vinylidene chloride in rats following inhalation exposure

Studies on the pharmacokinetics of [¹⁴C]vinylidene chloride (VDC)in rats were undertaken to characterize the disposition of the inhaled chemical and to aid in assessing the hazard associated with VDC exposure. Male rats normally fed or previously fasted for 18 hr were exposed to 10 or 200 ppm of [¹⁴C]VDC vapor for 6 hr, and the elimination of ¹⁴C activity was followed for 72 hr after exposure. Following the exposure to 10 ppm of [¹⁴C]VDC, approximately 98% of the acquired body burden of [¹⁴C]VDC was metabolized to nonvolatile metabolites of VDC. Fasting had no effect on the metabolism of [¹⁴C]VDC at this exposure concentration. However, after exposure to 200 ppm of VDC only 92 to 96% of the body burden was metabolized with fasted rats showing a reduced capacity to metabolize VDC at this exposure concentration. Fasted rats exposed to 200 ppm of [¹⁴C]VDC sustained liver and kidney damage, which was not observed in fed rats at this exposure level or in any rats at 10 ppm. Centrilobular hepatic necrosis in fasted rats exposed to 200 ppm of [¹⁴C]VDC was associated with an increase in covalently bound ¹⁴C activity in the liver over that of fed rats. Two major urinary metabolites of VDC were identified as N-acetyl-S-(2-hydroxyethyl)cysteine and thiodiglycolic acid, indicating that a major pathway for detoxification of VDC is via conjugation with liver glutathione (GSH).     

Toxicokinetics of acrylamide in rats and humans following single oral administration of low doses

The rodent carcinogen acrylamide (AA) is formed during preparation of starch-containing foods. AA is partly metabolized to the genotoxic epoxide glycidamide (GA). After metabolic processing, the mercapturic acids N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA), rac-N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA) and rac-N-acetyl-S-(1-carbamoyl-moyl-2-hydroxyethyl)-L-cysteine (iso-GAMA) are excreted with urine. In humans, AAMA can be sulfoxidized to AAMA-sulfoxide. The aim of this study was to assess potential species-differences in AA-toxicokinetics in rats and humans after single oral administration of doses similar to the daily human dietary exposure. Male Fischer 344 rats (n = 5/dose group) were administered 20 and 100 μg/kg b.w. ¹³C₃-AA in deionized water via oral gavage. Human subjects (n = 3/gender) were orally administered 0.5 and 20 μg/kg b.w. ¹³C₃-AA with drinking water. Urine samples were collected in intervals for 96 and 94 h, respectively. Urinary concentrations of ¹³C₃-AAMA, ¹³C₃-GAMA and ¹³C₃-AAMA-sulfoxide were monitored by liquid chromatography-tandem mass spectrometry. The recovered urinary metabolites accounted for 66.3% and 70.5% of the 20 and 100 μg/kg b.w. doses in rats and for 71.3% and 70.0% of the 0.5 and 20 μg/kg b.w. doses in humans. In rats, ¹³C₃-AAMA accounted for 33.6% and 38.8% of dose and 32.7% and 31.7% of dose was recovered as ¹³C₃-GAMA; ¹³C₃-AAMA-sulfoxide was not detected in rat urine. In humans, ¹³C₃-AAMA, ¹³C₃-GAMA and ¹³C₃-AAMA-sulfoxide accounted for 51.7% and 49.2%, 6.3% and 6.4% and 13.2% and 14.5% of the applied dose, respectively. The obtained results suggest that the extent of AA bioactivation to GA in humans is lower than in rodents.     

Excretion of 2,3-dihydroxy-propionamide (OH-PA), the hydrolysis product of glycidamide,in human urine after single oral dose of deuterium-labeled acrylamide

A dose of 0.99 mg d₃-acrylamide (d₃-AA) (13.2 μg/kg body weight) was ingested by a healthy male volunteer. Urine samples were collected over a period of 46 h after the intake and analyzed for the hydrolysis product of glycidamide (GA), 2,3-dihydroxy-propionamide (OH-PA), a metabolite of the toxicologically relevant oxidative AA metabolism pathway; 5.4% of the administered d₃-AA dose was eliminated as OH-PA within 46 h after ingestion. Therefore, OH-PA represents a major metabolite of the oxidative metabolism pathway. Elimination kinetics of OH-PA is similar to the oxidative metabolites N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-cysteine (GAMA) and N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-cysteine (iso-GAMA). The major excretion of d₃-OH-PA took place between 8 and 22 h with the highest urinary d₃-OH-PA concentration (c _max) of 69.3 μg/L urine, 18 h (t _max) postdose. OH-PA (5.4%), together with the other known urinary metabolites of the oxidative pathway GAMA (4.6%) and iso-GAMA (0.8%), represents 10.8% of the total AA dose. The share of the oxidative pathway metabolites is much smaller than the share of the reductive pathway metabolite N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA) that represents 51.7% of the ingested d₃-AA dose. However, this new quantitative human data on OH-PA together with the previous data on the other oxidative pathway metabolites are of special importance when evaluating the carcinogenic potential of AA and when comparing human data with data from animal studies.     

Species Differences in the Urinary Disposition of Some Metabolities of Ethylene Oxide

Some aspects of the qualitative and quantitative urinary dispositionof some metabolites of ethylene oxide in three rodent species,mouse, rat, and rabbit, were examined by determining urinaryN-acetyl-S-(2-hydroxyethyl)-L-cys"teine, S-(2-hydroxyethyl)-L-cysteine,S-carboxy methyl-L-cysteine, and ethylene glycol after ethyleneoxide exposure by iv and inhalation routes. The animals weregiven ethylene oxide at 20 and 60 mg/kg, and urine samples werecollected at 6 and 24 hr. Important differences were observedbetween the three species in the urinary meta bolic dispositionof ethylene oxide. Mice excreted significant quantities of N-acetyl-S-(2-hydro-xyethyl)-L-cysteine,S-(2-hydroxyethyl)-L-cysteine, S-carboxymethyl-L- and ethyleneglycol (8.3, 5.8, 1.9, and 3.3% of the lower dose, respectively,in 24 hr), whereas in rats only N acetyl-S-(2-hydroxyethyl)-L-cysteine(31%) and ethylene glycol (6%) were apparent. In contrast, therabbits were found to excrete only ethylene glycol (2%). Thisstudy further reveals species- related differences in the urinaryexcretion of N-acetyl-S-(2-hydroxyethyl)-L-cysteine and ethylene glycol during the two collection periods. The observed differencesbetween the three species in the metabolic disposition of ethyleneoxide were found to be qualitatively independent of the routeof exposure, i.e., inhalation at 200 ppm or iv. These resultssuggest that care should be exercised when using an animal speciesas a model for human disposition of ethylene oxide.     

Mechanism of formation of mercapturic acids from (1-bromoethyl)benzene and (2-bromoethyl)benzene in the rat

1. Three hypotheses have been proposed for the mechanism of metabolism of alkylhalides to hydroxy-alkylmercapturic acids, two of which involve the intermediate step of dehydrohalogenation and formation of an epoxide.

2. After injection of (1-bromoethyl)benzene in rat, the only mercapturic acid appearing in the urine was N-acetyl-S-1 -phenylethylcysteine. After injecting (2-bromoethyl)benzene in the rat only N-acetyl-S-2-phenylethylcysteine and N-acetyl-S-(2-phenyl-2-hydroxyethyl)cysteine were found in the urine.

3. Since the principal mercapturic acid formed from both styrene and styrene oxide could not be detected in the urine of rats receiving either 1- or 2-bromoethyl benzene, the intermediate formation of styrene or styrene oxide from the arylalkylhalides does not occur.     

Biotransformation of trichloroethene: dose-dependent excretion of 2,2,2-trichloro-metabolites and mercapturic acids in rats and humans after inhalation

 Chronic bioassays with trichloroethene (TRI) demonstrated carcinogenicity in mice (hepatocellular carcinomas) and rats (renal tubular cell adenomas and carcinomas). The chronic toxicity and carcinogenicity is due to bioactivation reactions. TRI is metabolized by cytochrome P450 and by conjugation with glutathione. Glutathione conjugation results in S-(dichlorovinyl) glutathione (DCVG) and is presumed to be the initial biotransformation step resulting in the formation of nephrotoxic metabolites. Enzymes of the mercapturic acid pathway cleave DCVG to the corresponding cysteine S-conjugate, which is, after translocation to the kidney, cleaved by renal cysteine S-conjugate β-lyase to the electrophile chlorothioketene. After N-acetylation, cysteine S-conjugates are also excreted as mercapturic acids in urine. The object of this study was the dose-dependent quantification of the two isomers of N-acetyl-S-(dichlorovinyl)-L-cysteine, trichloroethanol and trichloroacetic acid, as markers for the glutathione- and cytochrome P450-mediated metabolism, respectively, in the urine of humans and rats after exposure to TRI. Three male volunteers and four rats were exposed to 40, 80 and 160 ppm TRI for 6 h. A dose-dependent increase in the excretion of trichloroacetic acid, trichloroethanol and N-acetyl-S-(dichlorovinyl)-L-cysteine after exposure to TRI was found both in humans and rats. Amounts of 3100 μmol trichloroacetic acid+trichloroethanol and 0.45 μmol mercapturic acids were excreted in urine of humans over 48 h after exposure to 160 ppm TRI. The ratio of trichloroacetic acid+trichloroethanol/mercapturic acid excretion was comparable in rats and humans. A slow rate of elimination with urine of N-acetyl-S-(dichlorovinyl)-L-cysteine was observed both in humans and in rats. However, the ratio of the two isomers of N-acetyl-S-(dichlorovinyl)-L-cysteine was different in man and rat. The results confirm the finding of the urinary excretion of mercapturic acids in humans after TRI exposure and suggest the formation of reactive intermediates in the metabolism of TRI after bioactivation by glutathione also in humans. Received: 22 June 1995 / Accepted: 5 October 1995     

1,2-Dichloropropane: investigation of the mechanism of mercapturic acid formation in the rat

1. Three mercapturic acid metabolites were identified in the urine of male and female Fischer 344 rats given 1,2-dichloropropane (DCP) orally (100mg/kg) or by inhalation exposure (100 ppm, 6h).

2. These compounds (N-acetyl-S-(2-hydroxypropyl)-L-cysteine, N-acetyl-S-(2-oxopropyl)-L-cysteine and N-acetyl-S-(1-carboxyethyl)-L-cysteine) were isolated from the urine following acidification and extraction with ethyl acetate. The extracts were derivatized with diazomethane and N,O-bis(trimethylsilyl)trifluoroacetamide and analysed by chemical ionization g.l.c.-mass spectrometry.

3. Further mechanistic studies were carried out with the stable isotope-labelled analogue, D₆-DCP (105mg/kg, orally). Analysis of the resulting mass spectra indicated retention of primarily three deuterium atoms in the 2-hydroxypropyl-mercapturic acid formed from D₆-DCP. Similar isotope retention was observed for the 2-oxopropyl-mercapturic acid metabolite.

4. These results do not support a sulphonium ion intermediate in the formation of the 2-hydroxypropyl-mercapturic acid metabolite of DCP. Instead, this metabolite is thought to arise via direct oxidation of DCP, either prior to or following conjugation with glutathione.     

Metabolism and pharmacokinetic profile of vinylidene chloride in rats following oral administration

Male rats (normally fed or previously fasted for 18 hr) were given a single oral dose of 1 or 50 mg/kg of [¹⁴C]vinylidene chloride (VDC) in corn oil and the routes and rates of elimination of ¹⁴C activity were then followed for 72 hr. After a single oral dose of 1 mg/kg of [¹⁴C]VDC, 78% of the dose was metabolized and excreted in urine and feces as nonvolatile metabolites of VDC. The remainder was exhaled as ¹⁴CO₂ (21%) and unchanged [¹⁴C]VDC (1–3%). Fasting prior to [¹⁴C]VDC administration did not significantly affect the fate of the 1-mg/kg dose of [¹⁴C]VDC. Conversely, after a 50-mg/kg dose of [¹⁴C]VDC, excretion of the parent compound via the lungs accounted for 19 and 29% of the dose in fed and fasted rats, respectively. Elimination of nonvolatile metabolites of VDC was slightly greater in fed than in fasted rats indicating a reduced capacity for metabolism of VDC in fasted rats. Fasting also resulted in an increased concentration of covalently bound [¹⁴C]VDC metabolites in the livers of rats given 50 mg/kg of [¹⁴C]VDC. Urinary radioactivity was separated by high-pressure liquid chromatography into four major metabolites. Two of the four urinary metabolites were identified as S-(2-hydroxyethyl)-N-acetylcysteine and thiodiglycolic acid by gas chromatography-mass spectrometry, indicating that a major pathway for detoxification of VDC is via conjugation with glutathione (GSH). The fate of VDC following oral administration to rats is depedent upon both the dose administered and the nutritional status of the animal. The diminished ability of fasted animals to metabolize the high dose of VDC correlates well with the previously observed enhancement of VDC-induced hepatotoxicity in fasted animals. Both the hepatotoxic response to VDC and the extent of its detoxification appear to be dependent on the concentration of GSH in the liver. When hepatic GSH is depleted (i.e., in fasted animals or at higher doses of VDC) a toxic response to VDC is elicited.     

Urinary Mercapturic Acids and a Hemoglobin Adduct for the Dosimetry of Acrylamide Exposure in Smokers and Nonsmokers

Acrylamide, used in the manufacture of polyacrylamide and grouting agents, is also present in the diet and tobacco smoke. It is a neurotoxin and a probable human carcinogen. Analytical methods were established to determine the mercapturic acids of acrylamide (N-acetyl-S-(2-carbamoylethyl)-L-cysteine, AAMA) and its metabolite glycidamide (N-(R/S)-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine, GAMA) by high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS), as well as the N-terminal valine adduct of acrylamide (N-2-carbamoylethylvaline, AAVal) released by N-alkyl Edman degradation of hemoglobin by gas chromatography-mass spectrometry (GC-MS). Twenty-four-hour urine samples from 60 smokers and 60 nonsmokers were analyzed for AAMA and GAMA, and blood samples were analyzed for AAVal. Smokers excreted 2.5-fold higher amounts of AAMA and 1.7-fold higher amounts of GAMA in their urine and had 3-fold higher levels of AAVal in their blood. All three biomarkers of acrylamide exposure were strongly correlated with the smoking dose as determined by the daily cigarette consumption, nicotine equivalents (the molar sum of nicotine, cotinine, trans-3′-hydroxycotinine, and their respective glucuronides) in urine, salivary cotinine, and carbon monoxide in expired breath. In nonsmokers, a weak but significant correlation between AAMA and the estimated dietary intake of acrylamide was found. It is concluded that all three biomarkers of acrylamide are suitable for the determination of exposure in both smokers and nonsmokers.     

The fate of S-propylcysteine in the rat

1. The metabolism of S-propylcysteine in the rat has been re-investigated. The previously known major metabolite has been isolated and identified as the mercapturic acid, N-acetyl-S-propylcysteine.

2. Several further metabolites have been isolated from the urine of rats treated with S-propyl[³⁵S]cysteine. These have been identified as S-propylcysteine-S-oxide, N-acetyl-S-(2-hydroxypropyl)cysteine, S-(propylthio)lactate, S-(2,3-dihydroxypropyl)cysteine and N-acetyl-S-(2-carboxyethyl)cysteine.

3. The metabolism of S-(2-hydroxypropyl)-, S-(3-hydroxypropyl)- and S-(2,3-dihydroxypropyl)[³⁵S]cysteine have been investigated in the rat. The results, integrated with those from the metabolism of S-propyl[³⁵S]cysteine, have enabled the pathways of S-propylcysteine to be deduced.

4. The oxidative metabolism of a number of S-alkyl cysteines is discussed.     

Thioethers as urinary metabolites of thiophene and monobromothiophenes

1. Thiophene and its two monobromo derivatives were administered to rats and the amounts of thioether excreted in urine were measured by an assay based on Ellman's reagent. This assay, which involves extraction and hydrolysis, was validated by determining extraction and hydrolysis efficiencies for several authentic thioethers including N-acetyl-S-(2-thienyl)-L-cysteine, a previously reported metabolite of thiophene and 2-bromothiophene.

2. The thioethers present in urine of animals dosed with thiophenes have been examined chromatographically. Contrary to previous reports, the present work indicates that S-substituted, N-acetyl-L-cysteines (mercapturic acids) are not important thioether metabolites of thiophene in rats, and the small quantity of such compounds formed does not include either of the two simple S-thienyl derivatives.

3. The two monobromo thiophenes form higher proportions of thioethers than does thiophene, and one of these thioethers, arising from 3-bromothiophene, was identified, chromatographically, as N-acetyl-S-(3-thienyl)-L-cysteine.     

Identification of urinary mercapturic acids formed from acrylate,methacrylate and crotonate in the rat

1. After administration to rats of methyl acrylate (I), methyl methacrylate (II) and methyl crotonate (III), urinary mercapturic acids were isolated and identified as the dicarboxylic acids N-acetyl-S-(2-carboxyethyl)cysteine (IV, R = H), N-acetyl-S-(2-carboxypropyl)cysteine (V, R = H) and N-acetyl-S-(1-methyl-2-carboxyethyl)cysteine (VI, R = H) and for a minor part as their monomethyl esters IV (R = CH₃), V (R = CH₃) and VI (R = CH₃).

2. After a single dose of the acrylates (I), (II) and (III) (0·14mmol/kg), the excretion of the thioethers amounted to 6·6 ± 0·6, 0·0, and 2·0 ± 0·6% dose respectively.

3. After 18?h previous administration of the carboxylesterase inhibitor tri-o-tolyl phosphate (0·34mmol/kg) the excretion of the thioethers amounted to 40·6 ± 2·1, 11·0 ± 3·3, and 16·0 ± 2·0% dose.

4. For methyl acrylate (I) the ratio of the excreted dicarboxylic acid and monomethyl ester was 20:1. After previous administration of tri-o-tolyl phosphate this ratio was 1 : 2.     

Biotransformation of derivatives of the fungicides pentachloronitrobenzene and hexachlorobenzene in mammals

1. Biotransformation of N-acetyl-S-(2, 3, 5, 6-tetrachlorophenyl)cysteme and N-acetyl-S-(pentachlorophenyl)cysteine-S-oxide, of the metabolites of both PCNB and HCB, namely N-acetyl-S-(pentachlorophenyl)cysteine, pentachlorothiophenol, penta-chlorothioanisole, 4-methylthio-tetrachlorothiophenol and tetrachloro-1,4-bis(methyl-thio)benzene, and of the PCNB metabolites, S, S'-(tetrachloro-p-phenyleni')dicysteine, and the isomeric tetrachlorothiophenols, has been studied in rabbits, rats and mice, as well as in vitro.

2. Biotransformation products such as thiophenols, thioanisoles, chlorinated benzenes, phenols and anisoles have been identified by g.l.c.     

N-Acetyl-S-(1-carbamoyl-2-hydroxy-ethyl)-L-cysteine (iso-GAMA) a further product of human metabolism of acrylamide: comparison with the simultaneously excreted other mercaptuic acids

The N-acetyl-S-(1-carbamoyl-2-hydroxy-ethyl)-l-cysteine (iso-GAMA) could be identified as a further human metabolite of acrylamide. In this study, we report the excretion of d₃-iso-GAMA in human urine after single oral administration of deuterium labelled acrylamide (d₃-AA). One healthy male volunteer ingested a dose of about 1 mg d₃-AA which is equivalent to a dose of 13 μg/kg bodyweight. Over a period of 46 h the urine was collected and the d₃-iso-GAMA levels analysed by LC-ESI-MS/MS. The excretion of iso-GAMA begins five hours after application. It rises to a maximum concentration (c _max) of 43 μg/l which was quantified in the urine excreted after 22 h (t _max). The excretion pattern is parallel to that of the major oxidative metabolite N-acetyl-S-(2-carbamoyl-2-hydroxy-ethyl)-l-cysteine (GAMA). Total recovery of iso-GAMA was about 1% of the applied dose. Together with N-acetyl-S-(2-carbamoylethyl)-l-cysteine (AAMA) and GAMA, 57% of the applied dose is eliminated as mercapturic acids. The elimination kinetics of the three mercapturic acids of AA are compared. We show that dietary doses of acrylamide (AA) cause an overload of detoxification via AAMA and lead to the formation of carcinogenic glycidamide (GA) in the human body.     

Toxicokinetics of acrylamide in primary rat hepatocytes: coupling to glutathione is faster than conversion to glycidamide

Acrylamide (AA), classified as class 2A carcinogen (probably carcinogenic to humans) by the International Agency for Research on Cancer (IARC), is formed during heating of food from reducing carbohydrates and asparagine by Maillard reaction chemistry. After dietary uptake, AA is in part metabolically converted into the proximate genotoxic phase I metabolite glycidamide (GA). GA reacts with nucleophilic base positions in DNA, primarily forming N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) adducts. In a competing phase II biotransformation pathway AA, as well as its phase I metabolite GA, is coupled to glutathione (GSH). The GSH coupling products are further biotransformed and excreted via urine as mercapturic acids (MA), N-acetyl-S-(2-carbamoylethyl)cysteine (AAMA), and N-acetyl-S-(2-hydroxy-2-carbamoylethyl)cysteine (GAMA). In the present study, hepatic biotransformation pathways and DNA adduct formation were studied in primary rat hepatocytes, incubated with AA (0.2–2,000 μM) for up to 24 h. Contents of AA-GSH, GA, AAMA, and GAMA were measured in the cell culture medium after solid phase extraction (SPE). N7-GA-Gua adducts in DNA of hepatocytes were determined by HPLC–ESI–MS/MS after lysis of the cells and neutral thermal hydrolysis. Formation of AA-GSH was linear with AA concentration and incubation time and became detectable already at 0.2 μM (4 h). In contrast to AA, GA was not detected before 16 h incubation at 10-fold higher AA concentration (2 μM). In summary, the rate of AA-GSH formation was found to be about 1.5–3 times higher than that of GA formation. N7-GA-Gua adducts were found only at the highest AA concentration tested (2,000 μM).     

Metabolism of the Hydroxyethylrutosides III. The Fate of Orally Administered Hydroxyethylrutosides in Laboratory Animals; Metabolism by Rat Intestinal Microflora in vitro

1. The absorption, metabolism and excretion of hydroxyethylrutosides in rat and other mammals have been studied. Following oral administration to rats of 3′,4′,7-tri-O-(β-hydroxyethyl)rutoside, 4′,7-di-O-(β-hydroxyethyl)ruto-side and 7-O-(β-hydroxyethyl)rutoside, significant levels of the administered compounds and their conjugates in bile were observed, but 3′,4′,5,7-tetra-O-(β-hydroxyethyl)rutoside was poorly absorbed. The major portion of the dose of each rutoside was excreted as the aglycone in faeces. Urinary excretion of all rutosides was low.

2. Levels of excretion of ¹⁴C in the urine of rabbits and rhesus monkeys given 3′,4′,7-tri-O-(β-hydroxy[¹⁴C₂]ethyl)rutoside and 3′,4′,5,7-tetra-O-(β-hydroxy[¹⁴C₂]ethyl)rutoside were similar (<2.5% of dose) to those observed in the rat.

3. The hydroxyethylrutosides and rutin are degraded to their aglycones by the rat intestinal microflora both in vivo and in vitro. The B rings of rutin and 7-O-(β-hydroxyethyl)rutoside give rise to the same phenolic acid metabolites. Mono-O-(β-hydroxyethyl)phloroglucinol is formed from the A ring of 7-O-(β-hydroxyethyl)rutoside. Excretion of these metabolites in urine is suppressed by concurrent administration of neomycin.

4. 3′,4′,7-Tri-O-(β-hydrcxyethyl)rutoside did not undergo cleavage to metabolites other than the aglycone after continuous administration to rats for periods of up to 5 months.     

Evidence for the active renal secretion of s-pentachlorophenyl-N-acetyl-L-cysteine by female rats

Male and female rats received 50 μmoles of pentachloronitrobenzene/kg by oral intubation daily for seven days. The final excreta were hydrolysed and analysed by electron capture GLC for the presence of pentachlorobenzenethiol and tetrachloro-1,4-benzenedithiol (derived from the equivalent N-acetylcysteine conjugates). No differences were found between the sexes for faeces and bile but the urinary excretion of both thiols by females was more than 10-fold greater than males. A similar result for urine was obtained following i.p. administration of a single 20 μmoles/kg dose of S-pentachlorophenyl -N-acetyl-L-cysteine (pentachlorophenyl mercapturate); in addition co-treatment with probenecid did not greatly change excretion by the males but considerably reduced excretion by females. The sex difference in the urinary levels of pentachlorophenyl N-acetylcysteine after 40 and 100 μmoles/kg doses of pentachloronitrobenzene was confirmed by h.p.l.c. of the mercapturate and again probenecid inhibited the excretion. Analysis of urine by TLC following a dose of [¹⁴C]hexachlorobenzene (8 μCi/kg; 0.67 μmoles/kg) showed that more radioactivity was associated with the mercapturate from female rats than males. The results suggest that S-pentachlorophenyl-N-acetyl-L-cysteine, a metabolite of hexachlorobenzene and pentachloronitrobenzene, may be excreted by an active renal secretion which is particularly developed in female F344 rats.     

Biotransformation of diethenylbenzenes. III: Identification of metabolites of 1,3-diethenylbenzene in rat

1. Biotransformation of 1,3-diethenylbenzene (1) in rat gave four major metabolites, namely, 3-ethenylphenylglyoxylic acid (2), 3-ethenylmandelic acid (3), N-acetyl-S-[2-(3-ethenylphenyl)-2-hydroxyethyl]-L-cysteine (4) and N-acetyl-S-[1-(3-ethenylphenyl)-2-hydroxyethyl]-L-cysteine (5) were isolated from urine and identified by n.m.r. and mass spectrometry.

2. Four minor metabolites, 3-ethenylbenzoic acid (6), 3-ethenylphenylacetic acid (7), 3-ethenylbenzoylglycine (8) and 2-(3-ethenylphenyl)ethanol (9) were identified by g.l.c.-mass spectrometric analysis of urine extract derivatized in two different ways.

3. All identified metabolites are derived from 3-ethenylphenyloxirane (10), a reactive metabolic intermediate. No product of any metabolic transformation of second ethenyl group has been identified. However, several minor unidentified metabolites were detected by g.l.c.-mass spectrometry.

4. Total thioether excretion in 24h urine after a single i.p. dose of 1 amounted to 28·3±3·5 dose (mean±SD). No significant differences in the thioether fraction were observed in the dose range 100-300mg/kg.

5. Thioether metabolites consisted mainly of mercapturic acids 4 and 5. The ratio of metabolites 5 to 4 was 62:38. Each mercapturic acid consisted of two diastereomers. Their ratio, as determined by quantitative ¹³C-n.m.r. measurement was 95:5 and 79:21 for mercapturic acids 4 and 5, respectively.     

Metabolism of a New Radioprotector; S-Acetyl-N-Glycyl Cysteamine. I. Absorption,Distribution and Excretion Metabolites in Mice Bearing Emt6 Tumours

1.The disposition of S-acetyl-N-glycyl cysteamine (I) labelled with ¹⁴C on the cysteamine group (label 1), the glycyl group (label 2) and the acetyl group (label 3) has been studied in mice bearing EMT6 tumours.

2.Label 1 was mainly excreted in urine (63.1% dose in 24 h). Label 2 elimination was both in urine (36.0% dose in 24h) and in expired air as ¹⁴CO₂ (12.1% dose in 24 h). Label 3 was essentially eliminated in expired air as ¹⁴CO₂ (55.4% dose in 24 h).

3.Tissue distribution studies of label 1 and label 2 showed that concentrations in tissues were higher than blood concentration as early as 10min after administration. Whichever label was used, only little radio-activity was found in EMT6 tumour and brain.

4.Analysis of the urinary elimination products showed the presence of unchanged I and of cystamine, N-acetylcystamine, N-acetyl-S-methyl cysteamine sulphoxide and taurine. I is a prodrug of cysteamine which is released after deacetylation and hydrolysis of the amide bond. A metabolic pathway is proposed for this new radioprotective agent.     

Acetamide--a metabolite of metronidazole formed by the intestinal flora.

Metronidazole is metabolized to acetamide in yields of between 8 and 15 per cent by cultures of rat cecal contents or Clostridium perfringens. The yield of acetamide is 6- to 9-fold greater than that of N-(2-hydroxyethyl)-oxamic acid which is also derived from metronidazole. When [2-¹⁴C] metronidazole is administered by gavage to conventional rats, 1.3 to 1.8 per cent of a 200 mg/kg dose is recovered as acetamide in the urine. An additional 0.9 to 2.4 per cent is recovered as acetamide in the feces. Acetamide is not detected, however, in either urine or feces when metronidazole is administered to germfree rats. The appearance of acetamide derived from metronidazole in conventional rats appears to be mediated by the intestinal microflora. The cleavage of the imidazole ring of metronidazole to yield both acetamide and N-(2-hydroxyethyl)-oxamic acid is consistent with nucleophilic attack at carbons 2 and 4 of a partially reduced nitroimidazole ring, which is then cleaved between positions 1 and 2 and between positions 3 and 4. Since acetamide has been shown previously to be a liver carcinogen for rats, its presence in the urine and the feces should be considered, together with other indirect evidence, when determining the possible risk of cancer to patients given metronidazole.