The metabolism of arylazoisoxazolones by rats and dogs

1. When rats were given a single oral dose of the lipid-soluble fungicide 4-(2-chlorophenylhydrazono)-3-methyl[4-(14)C]isoxazol-5-one ([(14)C]drazoxolon), about 75% of the label was excreted in the urine and 13% in the faeces in 96hr. An additional 7% of the radioactivity was recovered as (14)CO(2) in 48hr. 2. About 8% of the label was excreted by rats in the bile in 0-24hr. and an additional 6% was excreted by the same route in 24-48hr. 3. When dogs were given a single oral dose of [(14)C]drazoxolon about 35% of the label was excreted in the urine and a similar amount was excreted in the faeces in 96hr. 4. The major metabolites in the urine of the rat and the dog were identified as 2-(2-chloro-4-hydroxyphenylhydrazono)acetoacetic acid (dog, 14%), the corresponding ether glucosiduronic acid (dog, 12%; rat, 13%) and ester sulphate (rat, 65%). 5. When rats were given a single oral dose of 3-methyl-4-([U-(14)C]phenylhydrazono)isoxazol-5-one about 75% of the label was excreted in the urine and 15% in the faeces in 96hr. The major metabolite in the urine was identified as the ester sulphate conjugate of 2-(4-hydroxyphenylhydrazono)-acetoacetic acid. 6. Reduction of the azo link was of minor quantitative significance. 7. These results are discussed in their relation to species differences in the toxicity of these compounds.     

The biochemistry of aromatic amines: The metabolism of 2-naphthylamine and 2-naphthylhydroxylamine derivatives

1. 2-Naphthylhydroxylamine and 2-nitrosonaphthalene were present in urine of dogs but not of guinea pigs, hamsters, rabbits or rats dosed with 2-naphthylamine. N-Acetyl-2-naphthylhydroxylamine and its O-sulphonic acid and O-glucosiduronic acid were not detected in the urine of any of these species. 2. Bile from rats dosed with 2-naphthylamine contained (2-naphthylamine N-glucosid)uronic acid and 6- and 5,6-substituted derivatives of 2-acetamidonaphthalene. 2-Amino-1-naphthyl and 2-acetamido-1-naphthyl derivatives, 2-naphthylhydroxylamine and its N-acetyl derivative or conjugates of these were not detected. Bile from a dog dosed with 2-naphthylamine contained no 2-amino-1-naphthyl derivatives. 3. 2-Naphthylhydroxylamine was metabolized by the dog, rat and guinea pig to the same products as those formed by these species from 2-naphthylamine. Rabbits formed mainly 2-amino-1-naphthyl derivatives; these are minor metabolites of 2-naphthylamine in this species. 4. (N-Acetyl-2-naphthylhydroxylamine O-glucosid)uronic acid was excreted in the urine and the bile of rats and in the urine of guinea pigs and rabbits dosed with N-acetyl-2-naphthylhydroxylamine. 5. After the administration of 2-acetamidonaphthalene, (N-acetyl-2-naphthylhydroxylamine O-glucosid)uronic acid was detected in the urine of dogs, but not in the urine of other species. The dog excreted an acid-labile cysteine derivative of 2-acetamidonaphthalene, but only traces of the corresponding mercapturic acid. 6. After dosing with N-acetyl-2-naphthylhydroxylamine-O-sulphonic acid, rats excreted derivatives of 2-amino-1-naphthol. 7. 2-Nitrosonaphthalene, N-acetyl-2-naphthylhydroxylamine, N-acetyl-2-naphthylhydroxylamine-O-sulphonic acid, 2-naphthylhydroxylamine-N-sulphonic acid, N-benzyloxycarbonyl-2-naphthylhydroxylamine and N-benzyloxycarbonyl-2-naphthylhydroxylamine-O-sulphonic acid were synthesized.     

Some metabolites of 1-bromobutane in the rabbit and the rat

1. Rabbits and rats dosed with 1-bromobutane excrete in urine, in addition to butylmercapturic acid, (2-hydroxybutyl)mercapturic acid, (3-hydroxybutyl)mercapturic acid and 3-(butylthio)lactic acid. 2. Although both species excrete both the hydroxybutylmercapturic acids, only traces of the 2-isomer are excreted by the rabbit. The 3-isomer has been isolated from rabbit urine as the dicyclohexylammonium salt. 3. 3-(Butylthio)lactic acid is formed more readily in the rabbit; only traces are excreted by the rat. 4. Traces of the sulphoxide of butylmercapturic acid have been found in rat urine but not in rabbit urine. 5. In the rabbit about 14% and in the rat about 22% of the dose of 1-bromobutane is excreted in the form of the hydroxymercapturic acids. 6. Slices of rat liver incubated with S-butylcysteine or butylmercapturic acid form both (2-hydroxybutyl)mercapturic acid and (3-hydroxybutyl)mercapturic acid, but only the 3-hydroxy acid is formed by slices of rabbit liver. 7. S-Butylglutathione, S-butylcysteinylglycine and S-butylcysteine are excreted in bile by rats dosed with 1-bromobutane. 8. Rabbits and rats dosed with 1,2-epoxybutane excrete (2-hydroxybutyl)mercapturic acid to the extent of about 4% and 11% of the dose respectively. 9. The following have been synthesized: N-acetyl-S-(2-hydroxybutyl)-l-cysteine [(2-hydroxybutyl)mercapturic acid] and N-acetyl-S-(3-hydroxybutyl)-l-cysteine [(3-hydroxybutyl)mercapturic acid] isolated as dicyclohexylammonium salts, N-toluene-p-sulphonyl-S-(2-hydroxybutyl)-l-cysteine, S-butylglutathione and N-acetyl-S-butylcysteinyl-glycine ethyl ester.     

Intrahepatic conversion of a glutathione conjugate to its mercapturic acid. Metabolism of 1-chloro-2,4-dinitrobenzene in isolated perfused rat and guinea pig livers

Because of the low hepatic activity of gamma-glutamyl-transferase in the rat, the liver is generally considered to play only a minor role in the degradation of glutathione conjugates, a limiting step in mercapturic acid formation. Recent findings indicate, however, that the liver has a prominent role in glutathione catabolism, particularly in species other than rat. To examine the contributions of liver to mercapturic acid biosynthesis, mercapturate formation was compared in isolated perfused livers from rats and guinea pigs dosed with either 0.3 or 3.0 mumol of 1-chloro-2,4-dinitrobenzene (CDNB). Chemically synthesized glutathione conjugate, mercapturic acid, and intermediary metabolites of CDNB were used as standards in the high performance liquid chromatography analysis of bile and perfusate samples. Biliary excretion accounted for almost all of the recovered metabolites. A marked species difference was observed in the pattern of CDNB metabolism. Rat livers dosed with 0.3 mumol of CDNB excreted 55% of total biliary metabolites as the glutathione conjugate and 8.2% as the mercapturic acid, whereas guinea pig livers excreted only 4.8% as the glutathione conjugate and 47% as the mercapturate. Mercapturic formation was also dose-dependent, with a larger fraction formed at the 0.3- versus the 3.0-mumol dose (8.2 versus 3.7% in the rat; 47 versus 19% in the guinea pig). Hepatic conversion of the glutathione conjugate to the mercapturic acid was markedly inhibited in both species after retrograde intrabiliary infusion of acivicin, an inhibitor of gamma-glutamyltransferase activity. These findings provide direct evidence for intrahepatic biosynthesis of mercapturic acids. Thus, glutathione conjugates synthesized within hepatocytes are secreted into bile and broken down to cysteine conjugates; the latter are then presumably reabsorbed by the liver, N-acetylated to form the mercapturic acid and re-excreted into bile.     

Pharmacokinetics,Tissue Distribution,and Anti-Lipogenic/Adipogenic Effects of Allyl-Isothiocyanate Metabolites

Allyl-isothiocyanate (AITC) is an organosulfur phytochemical found in abundance in common cruciferous vegetables such as mustard, wasabi, and cabbage. Although AITC is metabolized primarily through the mercapturic acid pathway, its exact pharmacokinetics remains undefined and the biological function of AITC metabolites is still largely unknown. In this study, we evaluated the inhibitory effects of AITC metabolites on lipid accumulation in vitro and elucidated the pharmacokinetics and tissue distribution of AITC metabolites in rats. We found that AITC metabolites generally conjugate with glutathione (GSH) or N-acetylcysteine (NAC) and are distributed in most organs and tissues. Pharmacokinetic analysis showed a rapid uptake and complete metabolism of AITC following oral administration to rats. Although AITC has been reported to exhibit anti-tumor activity in bladder cancer, the potential bioactivity of its metabolites has not been explored. We found that GSH-AITC and NAC-AITC effectively inhibit adipogenic differentiation of 3T3-L1 preadipocytes and suppress expression of PPAR-γ, C/EBPα, and FAS, which are up-regulated during adipogenesis. GSH-AITC and NAC-AITC also suppressed oleic acid-induced lipid accumulation and lipogenesis in hepatocytes. Our findings suggest that AITC is almost completely metabolized in the liver and rapidly excreted in urine through the mercapturic acid pathway following administration in rats. AITC metabolites may exert anti-obesity effects through suppression of adipogenesis or lipogenesis.     

The metabolism of 2,6-di-tert.-butyl-4-hydroxymethylphenol (Ionox 100) in the dog and rat

1. A single oral dose of [(14)C]Ionox 100 to rats is almost entirely eliminated in 11 days: 89.1-107.2% of the (14)C is excreted and 0.29+/-0.02% of the dose is present in the carcass plus viscera after removal of the gut. Rats exhibit an individual variation in the elimination pattern, 15.6-70.8% of (14)C being excreted in the urine and 75.2-27.0% in the faeces during 11 days. 2. After the oral administration of [(14)C]Ionox 100 to dogs, 87.1-90.3% of the (14)C is excreted in the faeces and urine during 4 days. 3. Dogs and rats do not show a species difference in this pattern of elimination. 4. The rate of elimination from dogs and rats given a single dose of Ionox 100 is not affected by the size of the dose and the presence of triglyceride fat in the diet. 5. Ionox 100 is completely metabolized in dogs and rats: unchanged Ionox 100 is absent from the urine and faeces, and from the carcass when elimination is complete. In rats, 3,5-di-tert.-butyl-4-hydroxybenzoic acid accounts for 50-85% of a dose of Ionox 100 and (3,5-di-tert.-butyl-4-hydroxybenzoyl beta-d-glucopyranosid)uronic acid for 47-10%; in dogs, the unconjugated acid accounts for 85% and the ester glucuronide for 10-12%. 3,5-Di-tert.-butyl-4-hydroxyhippuric acid is not formed. Other metabolites, which have been detected in small quantity in the faeces and urine of animals dosed with Ionox 100, have not been identified. 6. 3,5-Di-tert.-butyl-4-hydroxybenzoic acid and (3,5-di-tert.-butyl-4-hydroxybenzoyl beta-d-glucopyranosid)uronic acid are also the major metabolites of Ionol (2,6-di-tert.-butyl-p-cresol) in rats. 7. The elimination of Ionox 100 metabolites from rats is faster than that of Ionol and its metabolites. Unlike Ionol, unchanged Ionox 100 could not be detected in the bodies of these animals.     

The metabolism of 3,5-di-tert.-butyl-4-hydroxytoluene in the rat and in man

1. The major metabolites of 3,5-di-tert.-butyl-4-hydroxytoluene (BHT) in the rat are 3,5-di-tert.-butyl-4-hydroxybenzoic acid (BHT-acid), both free (9% of the dose) and as a glucuronide (15%), and S-(3,5-di-tert.-butyl-4-hydroxybenzyl)-N-acetylcysteine. 2. The mercapturic acid does not appear to derive from the usually accepted enzyme mechanism, and may involve a non-enzymic reaction between BHT free radical and cysteine. 3. The ester glucuronide and mercapturic acid found in rat urine are also the major metabolites in rat bile and must be responsible for the enterohepatic circulation. 4. Free BHT-acid is the main component in rat faeces. 5. In man, BHT-acid, free and conjugated, is a minor component in urine, and the mercapturic acid is virtually absent. The bulk of the radioactivity is excreted as the ether-insoluble glucuronide of a metabolite in which the ring methyl group and one tert.-butyl methyl group are oxidized to carboxyl groups, and a methyl group on the other tert.-butyl group is also oxidized, probably to an aldehyde group. 6. These differences in metabolism by the rat and by man are sufficient to account for the difference in excretion by the two species.     

Compensatory Expression of MRP3 in the Livers of MRP2-Deficient EHBRs Is Promoted by DHA Intake

We have hypothesized a suppressive mechanism against dietary docosahexaenoic acid (22:6n-3; DHA)-induced tissue lipid peroxidation, in which the degradation products, including their conjugates, are excreted into the urine by xenobiotic or organic anion transporters. In this study, we employed parent-strain Sprague-Dawley rats (SDRs), together with their mutant strain, Eisai hyperbilirubinuria rats (EHBRs). EHBRs are deficient in multidrug resistance-associated protein (MRP) 2, and show defective urinary excretion of numerous xenobiotics and organic anions. Both strains of rats were fed a diet containing DHA at 8.4% of total energy for 31 d. In the livers of the DHA-fed rats, the level of free malondialdehyde (MDA) + 4-hydroxy-2-alkenals (HAE) fell, and conversely glutathione S-transferase (GST) activity increased in MRP2-deficient EHBRs as compared to the SDRs, suggesting that the glutathione (GSH)-conjugation reaction for the aldehydes generated on DHA intake was accelerated in the MRP2-deficient EHBRs. Since the gene expression of liver MRP3 in the MRP2-deficient EHBRs was amplified to compensate for DHA intake, it is thought that the transport of MRP3 substrates into the bloodstream, rather than MRP2-mediated excretion of its substrates into the bile, was promoted. Indeed, excretion of mercapturic acid (acetylcysteine conjugates derived metabolically from the conjugate of each aldehyde with GSH) into the urine increased significantly in MRP2-deficient EHBRs fed DHA.     

The comparative metabolism of 2,6-dichlorothiobenzamide (prefix) and 2,6-dichlorobenzonitrile in the dog and rat

1. A single oral dose of either [(14)C]Prefix or 2,6-dichlorobenzo[(14)C]nitrile to rats is almost entirely eliminated in 4 days: 84.8-100.5% of (14)C from [(14)C]Prefix is excreted, 67.3-79.7% in the urine, and 85.8-97.2% of (14)C from 2,6-dichlorobenzo-[(14)C]nitrile is excreted, 72.3-80.7% in the urine. Only 0.37+/-0.03% of the dose of [(14)C]Prefix and 0.25+/-0.03% of the dose of 2,6-dichlorobenzo[(14)C]nitrile are present in the carcass plus viscera after removal of the gut. Rats do not show sex differences in the pattern of elimination of the respective metabolites of the two herbicides. The rates of elimination of (14)C from the two compounds in the 24hr. and 48hr. urines are not significantly different (P >0.05) from one another. 2. After oral administration to dogs, 85.9-106.1% of (14)C from [(14)C]Prefix is excreted, 66.6-80.9% in the urine, and 86.8-92.5% of (14)C from 2,6-dichlorobenzo[(14)C]nitrile is excreted, 60.0-70.1% in the urine. Dogs do not show sex differences in the pattern of eliminating the metabolites of either Prefix or 2,6-dichlorobenzonitrile. 3. Dogs and rats do not show species differences in the patterns of elimination of the two herbicides. 4. Prefix and 2,6-dichlorobenzonitrile are completely metabolized; unchanged Prefix and 2,6-dichlorobenzonitrile are absent from the urine and faeces, and from the carcasses when elimination is complete. In the hydrolysed urine of rats dosed with either [(14)C]Prefix or 2,6-dichlorobenzo[(14)C]nitrile, 2,6-dichloro-3-hydroxybenzonitrile accounts for approx. 42% of the (14)C, a further 10-11% is accounted for by 2,6-dichlorobenzamide, 2,6-dichlorobenzoic acid, 2,6-dichloro-3- and -4-hydroxybenzoic acid and 2,6-dichloro-4-hydroxybenzonitrile collectively, and 25-30% by six polar constituents, of which two are sulphur-containing amino acids. 5. In the unhydrolysed urines of rats dosed with either [(14)C]Prefix or 2,6-dichlorobenzo[(14)C]nitrile, there are present free 2,6-dichloro-3- and -4-hydroxybenzonitrile, their glucuronide conjugates, ester glucuronides of the principal aromatic acids that are present in the hydrolysed urines, and two sulphur-containing metabolites analogous to mercapturic acids or premercapturic acids. 6. Prefix is thus extensively transformed into 2,6-dichlorobenzonitrile: R.CS.NH(2)-->R.CN+H(2)S, where R=C(6)H(3)Cl(2). However, the competitive reaction: R.CS.NH(2)+H(2)O-->R.CO.NH(2)+H(2)S takes place to a very limited extent.     

The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium. II. Activation by the isolated perfused rat liver.

In this investigation Salmonella typhimurium strain TA 1530 and TA 1535 were combined with isolated perfused rat liver. Samples of perfusate and bile produced were tested for mutagenicity after treatment with 1,2-dichloroethane (DCE), 1,2-dibromoethane (DBE) or 2-chloroethanol. The results are in good agreement with our previous experiments which indicate that both DEC and DBE are activated through conjugation with glutathione (GSH). Most GSH conjugates are normally excreted in bile. Following liver perfusion the bile was highly mutagenic after DCE and DBE treatments, while 2-chloroethanol did not have this effect. The highest mutagenic effect was seen 15--30 min after the addition of DCE or DBE. The production of mutagenic bile also occurred in mice treated in vivo with DCE. One possible metabolic endproduct of a GSH conjugate is the corresponding mercapturic acid. Thus synthetic N-acetyl-S-(2-chloroethyl)-L-cysteine was tested on TA 1535 and found to be as mutagenic as S-(2-chloroethyl)-L-cysteine in the concentration range 0.2--0.6 mumol/plate. Differences and similarities in the metabolism of DCE and vinyl chloride are discussed on the basis of these results.     

Metabolism of [14C]methamphetamine in man, the guinea pig and the rat

1. The metabolites of (+/-)-2-methylamino-1-phenyl[1-(14)C]propane ([(14)C]methamphetamine) in urine were examined in man, rat and guinea pig. 2. In two male human subjects receiving the drug orally (20mg per person) about 90% of the (14)C was excreted in the urine in 4 days. The urine of the first day was examined for metabolites, and the main metabolites were the unchanged drug (22% of the dose) and 4-hydroxymethamphetamine (15%). Minor metabolites were hippuric acid, norephedrine, 4-hydroxyamphetamine, 4-hydroxynorephedrine and an acid-labile precursor of benzyl methyl ketone. 3. In the rat some 82% of the dose of (14)C (45mg/kg) was excreted in the urine and 2-3% in the faeces in 3-4 days. In 2 days the main metabolites in the urine were 4-hydroxymethamphetamine (31% of dose), 4-hydroxynorephedrine (16%) and unchanged drug (11%). Minor metabolites were amphetamine, 4-hydroxyamphetamine and benzoic acid. 4. The guinea pig was injected intraperitoneally with the drug at two doses, 10 and 45mg/kg. In both cases nearly 90% of the (14)C was excreted, mainly in the urine after the lower dose, but in the urine (69%) and faeces (18%) after the higher dose. The main metabolites in the guinea pig were benzoic acid and its conjugates. Minor metabolites were unchanged drug, amphetamine, norephedrine, an acid-labile precursor of benzyl methyl ketone and an unknown weakly acidic metabolite. The output of norephedrine was dose-dependent, being about 19% on the higher dose and about 1% on the lower dose. 5. Marked species differences in the metabolism of methamphetamine were observed. The main reaction in the rat was aromatic hydroxylation, in the guinea pig demethylation and deamination, whereas in man much of the drug, possibly one-half, was excreted unchanged.     

Metabolism of a glutathione conjugate of 2-hydroxyoestradiol by rat liver and kidney preparations in vitro

Adult male rat liver and kidney preparations were incubated with (2-hydroxyoestradiol-1-yl)[(35)S]glutathione. The glutamic acid and glycine residues were removed by enzymes present in the kidney microsomal fraction; the liver preparations had no effect. The resulting 2-hydroxyoestradiol-cysteine conjugate was acetylated at the alpha-amino group by both liver and kidney homogenates fortified with acetyl-coenzyme A, but not significantly in the absence of this coenzyme, or by liver or kidney slices. These results suggest that an oestrogen-glutathione conjugate, if formed in vivo, would be converted into the corresponding mercapturic acid before excretion.     

The metabolism of labelled hexadecyl sulphate salts in the rat, dog and human.

The metabolic fate of [1-14-C]hexadecylsulphate and hexadecyl[35-S]sulphate, administered intravenously as the sodium and trimethylammonium salt to dogs and orally as the erythromycin salt to dogs, rats and humans, was studied. Studies with rats indicated that the compounds were well absorbed and rapidly excreted in the urine. However, after oral administration of the 14-C-and 35-S-labelled hexadecyl sulphate erythromycin salt to dogs, considerable amounts of radioactivity were excreted in the faeces as unmetabolized hexadecyl sulphate. Studies with two humans showed that orally administered erythromycin salt of [1-14C]hexadecyl sulphate was well absorbed in one person but poorly absorbed in the other. Radioactive metabolites in urine were separated by t.l.c. in two solvent systems. The main metabolite of hexadecyl sulphate in the dog, rat and human was identified as the sulphate ester of 4-hydroxybutyric acid. In addition, psi-[14-C]butyrolactone as a minor metabolic product of [1-14-C]hexadecyl sulphate was also isolated from the urine of rat, dog and man. However, there was still another metabolite in dog urine, which comprised about 20% of the total urinary radioactivity and carried both 14-C and 35-S labels. This metabolite was absent from rat urine. The metabolite in dog urine was isolated and subsequently identified by t.l.c. and g.l.c. and by isotope-dilution experiments as the sulphate ester of glycollic acid. Small amounts (about 5% of the total recovered radioactivity in excreta) of labelled glycollic acid sulphate were also found in human urine after ingestion of erythromycin [1-14-C]hexadecyl sulphate.     

Mutagenicity of tetrachloroethene in the Ames test--metabolic activation by conjugation with glutathione

The mutagenicity of tetrachloroethene (tetra) and its S conjugate, S-(1,2,2-trichlorovinyl)glutathione (TCVG) was investigated using a modified Ames preincubation assay. TCVG was a potent mutagen in presence of rat kidney particulate fractions containing high concentrations of gamma-glutamyl transpeptidase (GGT) and dipeptidases. Purified tetra was not mutagenic without exogenous metabolic activation or under conditions favoring oxidative metabolism. Preincubation of tetra with purified rat liver glutathione (GSH) S-transferases in presence of GSH and rat kidney fractions resulted in a time-dependent formation of TCVG as determined by (HPLC) analysis and in an unequivocal mutagenic response in the Ames test. Experiments with tetra in the isolated perfused rat liver demonstrated TCVG formation and its excretion with the bile; bile collected after the addition of tetra to the isolated perfused liver was unequivocally mutagenic in bacteria in the presence of kidney particulate fractions. The mutagenicity was reduced in all cases by the GGT inhibitor serine borate or the beta-lyase inhibitor aminooxyacetic acid. These results support the suggestion that cleavage of the GSH S conjugate formed from tetra by the enzymes of the mercapturic acid pathway and by beta-lyase may be involved in the nephrocarcinogenic effects of this haloalkene in rats.     

Reversed-phase chromatography of urinary metabolites of paracetamol using ion suppression and ion pairing

High-performance liquid chromatography (HPLC) has proven particularly useful for the study of paracetamol metabolism. Two alternative methods were developed using reversed-phase C₁₈ columns. A rapid ion suppression technique was used for the analysis of free paracetamol, paracetamol mercapturic acid and cysteine conjugate in urine samples obtained from isolated perfused rat kidney preparations, which has conveniently demonstrated the oxidative metabolic capacity of the kidney towards paracetamol. A somewhat longer, but higher resolution, ion-pair HPLC procedure was developed for the analysis of paracetamol metabolites in urine samples from experimental animals. The ion-pairing solvent was composed of tetrabutylammonium hydroxide, Tris and EDTA buffered to pH 7.2 with phosphoric acid. Gradient programming was further used to enhance resolution. Using this system two new metabolites, the sulphate and glucuronide conjugates of 3-thiomethyl-paracetamol were detected and routinely determined along with other known paracetamol metabolites, viz. free paracetamol, paracetamol sulphate, glucuronide, mercapturic acid, and cysteine conjugates, 3-methoxyparacetamol glucuronide and sulphate, p-aminophenol and its O-glucuronide and O-sulphate conjugates. Phenolic O-substituted glucuronide and sulphate conjugates of N-hydroxyparacetamol were also separated.     

The metabolism of N-triphenylmethylmorpholine in the dog and rat

1. Rats were given N-triphenyl[(14)C]methylmorpholine, triphenyl[(14)C]carbinol, N-triphenylmethyl[G-(3)H]morpholine or [G-(3)H]morpholine as single oral doses; the routes of excretion were examined. 2. Dogs were given single oral doses of N-triphenyl[(14)C]methylmorpholine. 3. (14)C-labelled metabolites were excreted mainly in the faeces in both rats and dogs; no (14)CO(2) was expired and less than 3% remained in the carcass and skin after 96hr. 4. (3)H-labelled metabolites were excreted rapidly in urine; part of the label was found in the expired gases and over 10% remained in the carcass and skin after 96hr. 5. Differences in excretion pattern between the sexes were noticed in rats but not in dogs. 6. N-Triphenylmethylmorpholine was rapidly hydrolysed to form triphenylcarbinol and morpholine in the stomach; morpholine was absorbed rapidly and excreted largely unchanged, though some was degraded, since some of the (3)H was found in water. 7. Triphenylcarbinol was absorbed only slowly and was oxidized to p-hydroxyphenyldiphenylcarbinol. 8. Both triphenylcarbinol and its p-hydroxy derivative were found in urine, bile and faeces in the free form and conjugated with glucuronic acid. The proportion of conjugates was higher in rat bile than in faeces. 9. Traces of o-hydroxyphenyldiphenylcarbinol and m-hydroxyphenyldiphenylcarbinol were detected as metabolites both free and conjugated.     

Metabolism of straight saturated medium chain length (C9 to C12) dicarboxylic acids

A method utilizing thin-layer chromatography, high performance liquid chromatography, and mass spectrometry was developed for the quantification of C9, C10, C11, and C12 dicarboxylic acids in serum, urine, and feces of human volunteers and rats after oral administration of the acids. The method allowed good resolution and measurement of the dicarboxylic acids at nanogram levels. In humans, excretion was independent of the dosage; about 60% of C9, 17% of C10, 5% of C11, and 1% of C12 were excreted in the urine during the first 12 hours after administration. The concentration of the acids in serum peaked between 2 and 3 hours. Excretion was also independent of dosage in rats. About 2.5% of C, 2.1% of C10, 1.8% of C11, and 1.6% of C12 were excreted in the urine over a period of 5 days. The serum concentration and the urinary excretion of the diacids reached a maximum at the second day after the oral dose. In both humans and rats, the dicarboxylic acids found in serum and urine were 2, 4, or 6 carbon atoms shorter than the corresponding administered diacid. This indicates that there was beta-oxidation of the ingested diacids to some extent. The administration of [1,9-14C]azeliac acid and of [10,11-3H]dodecandioic acid confirmed the occurrence of beta-oxidation, and led to elucidation of the fate of the ingested diacids that were not excreted as such in the urine.     

The metabolism of 2-chloro-1-(2′,4′-dichlorophenyl)vinyl diethyl phosphate (Chlorfenvinphos) in the dog and rat

1. A single oral dose of [(14)C]Chlorfenvinphos to rats is quantitatively eliminated in 4 days. Rats do not show a sex difference in the elimination pattern and show only a small degree of biological variation in the total excretion data. Of the label 87.2% is excreted in the urine (67.5% in the first day after dosage), 11.2% in the faeces and 1.4% in the expired gases; less than 0.9% of (14)C is present in the gut and contents after 4 days. 2. After oral administration of [(14)C]Chlorfenvinphos to dogs, 94.0% (91.8-97.6%) of the (14)C is excreted in the urine and faeces during 4 days. Dogs do not show a sex difference in the pattern of elimination, and excretion of radioactivity in the urine is very rapid: 86.0% of (14)C during 0-24hr. 3. Chlorfenvinphos is completely metabolized in rats and dogs: unchanged Chlorfenvinphos is absent from the urine and from the carcass, when elimination is complete. In rats, 2-chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate accounts for 32.3% of a dose of Chlorfenvinphos, [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid]uronic acid for 41.0%, 2,4-dichloromandelic acid for 7.0%, 2,4-dichlorophenylethanediol glucuronide for 2.6% and 2,4-dichlorohippuric acid for 4.3%; in dogs, 2-chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate accounts for 69.6%, [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid] uronic acid for 3.6%, 2,4-dichloromandelic acid for 13.4% and 2,4-dichlorophenylethanediol glucuronide for 2.7%. 4. Dogs and rats show a species difference in the rate of excretion of (14)C in the urine, and in the proportions of the metabolites, with the exception of 2,4-dichlorophenylethanediol glucuronide, that are excreted in the urine. Alternative explanations for the latter species difference are suggested. 5. 2-Chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate and 2,4-dichlorophenacyl chloride probably lie on the main metabolic pathway of Chlorfenvinphos, since, in common with that insecticide, they give rise to [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid]uronic acid and 2,4-dichloromandelic acid as major metabolites in the urine. 6. The proposed scheme for the metabolism of Chlorfenvinphos represents a detoxication mechanism.     

Kinetics of 8-2 fluorotelomer alcohol and its metabolites, and liver glutathione status following daily oral dosing for 45 days in male and female rats

Fluorotelomer alcohols (FTOHs) are raw materials used in the manufacture of polymeric and surfactant products. Based on previous findings from single oral dosing in rats with radiolabeled 8-2 FTOH, glutathione (GSH) depletion and/or the presence of perfluorinated/polyfluorinated acids and aldehyde metabolites was hypothesized to account for the hepatocellular lesions observed in male rats from a 90-day subchronic oral dosing study. Further, the reported nephropathy in female rats from the subchronic experiment was hypothesized to have been initiated by a thiol metabolite produced by degradation of GSH conjugates. In the current investigation, the kinetics of 8-2 FTOH and its metabolites along with liver GSH status were evaluated in the rat following daily oral dosing with 8-2 FTOH for 45 days at 5 and 125 mg/kg/day. Liver GSH stores 1-2 h after dosing were unaffected, suggesting that GSH depletion is not likely a relevant mode of action in the liver. The tissue metabolite data indicate that the liver toxicity mode of action is likely associated with elevated levels of perfluoroalkyl acids found in males, since other polyfluorinated metabolites and 8-2 FTOH were present in livers from female rats at comparable or higher levels. Detection of the N-acetyl cysteine conjugate of the unsaturated parent telomer alcohol in urine from female rats and not male rats provides some evidence to support the mechanistic basis for the observed kidney effects. Further, the increasing levels of perfluorooctanoic acid (PFOA) in plasma from female rats over the 45-day dosing phase, while unexpected, may reflect an increased net absorption of 8-2 FTOH, slow elimination of intermediates in the metabolic pathway between 8-2 FTOH and PFOA, or altered kidney clearance. The results of this study have enhanced our understanding of 8-2 FTOH kinetics and metabolism and potential modes of action in the rat, which will guide the design of future studies for FTOHs and our need to define the mechanistic basis for the observed effects.