Metabolism of some methyl 2-cyano-3-phenyl-acrylates (methyl alpha-cyanocinnamates) in rats.

1. The metabolism of 2-cyano-3-phenylacrylic acid (CPA) and some methyl 2-cyano-3-phenylacrylates (methyl alpha-cyanocinnamates) after i.p. administration to rats, was investigated. 2. The conjugation of CPA and methyl alpha-cyanocinnamates with L-cysteine, N-acetylcysteine and glutathione (GSH) in vitro was studied and the rate of hydrolysis of the double bond of the methyl alpha-cyanocinnamates determined. 3. CPA and the methyl alpha-cyanocinnamates caused a significant increase in urinary (24 h) thioether excretion, with no increase in SCN- excretion. 4. No thioethers, such as possible addition products of N-acetylcysteine with CPA or methyl alpha-cyanocinnamates, were isolated; such thioether compounds appeared to be very unstable. 5. Administration of the carboxylesterase inhibitor, tri-ortho tolyl phosphate, to rats resulted in increased urinary excretion of SCN- but no significant excretion of thioethers, after a single i.p. injection of methyl alpha-cyanocinnamates. 6. Following incubation of methyl alpha-cyanocinnamates with GSH under non-enzymic and enzymic conditions, the GSH was 100% recovered. However, incubation of CPA depleted GSH and N-acetylcysteine (non-enzymic). 7. A metabolic pathway for the metabolism of methyl alpha-cyanocinnamates to thioether adducts is proposed, which proceeds via the corresponding CPA. Ester hydrolysis and the carbon-carbon double bond hydrolysis are probably in vivo reactions of some importance.

The absorption of a new sustained release furazolidone formulation from the digestive tract of piglets and calves.

Chyme concentrations and total recoveries of furazolidone (5 mg/kg body-weight) were determined by a HPLC-method, after oral administration of two different furazolidone formulations to piglets (n = 6) and pre-ruminant calves (n = 8), provided with an ileal re-entrant canula. Additional blood samples were taken from the calves to measure the time dependent plasma levels of furazolidone. In the case of the normal crystalline preparation, the results indicate an almost complete absorption of the drug from the upper parts of the digestive tract. In both species, 96-99% of the dose had been absorbed by the time it reached the end of the ileum. The mean ileal recovery of the newly developed furazolidone formulation in calves and piglets was 14% and 38%, respectively. In calves the observed maximum plasma concentrations of furazolidone after oral application of the sustained release formulation were 14 times lower than with the normal crystalline preparation.

Percutaneous absorption of 14C-labelled 2-chlorobenzaldehyde in rats. Metabolism and toxicokinetics.

2-Chlorobenzaldehyde might be produced when a moist skin is exposed to the riot control agent CS. CS-hydrolysis to 2-chlorobenzaldehyde and malononitrile occurs both in vitro and in vivo. No quantitative data have thus far been reported with respect to the percutaneous absorption and the cutaneous biotransformation of 2-chlorobenzaldehyde. Percutaneous absorption, biotransformation and elimination of 14-C-labelled 2-chlorobenzaldehyde was investigated in the rat. Following IV (25 microliters/kg) and IP (37.5 microliters/kg) 14C-2-chlorobenzaldehyde administration to rats, the plasma radioactivity declined rapidly over a 24 h period with similar plasma radioactivity-time profiles. Following cutaneous administration (75 microliters/kg) in a closed glass-cup on the skin a slow skin penetration occurred as indicated by plasma radioactivity levels. A slow increase in plasma radioactivity was followed by a slow decline of radioactivity in plasma over a 3-day period. Most of the radioactivity was found in the urine with low levels in faeces and exhaled air. The cutaneously administered radioactivity was also partly recovered from the glass-cup. For the qualitative and quantitative determination of metabolites in urine, a thin layer chromatography-radioautography method was used. The metabolic patterns of urinary excreted metabolites following cutaneous application and systemic administration of 14C-2-chlorobenzaldehyde to rats were very similar. No parent compound was recovered from the rat urine. 2-Chlorohippuric acid was the principal urinary metabolite. Quantitatively, the urinary excretion of 14C-2-chlorobenzyl alcohol following cutaneous application differed substantially from that after the systemic administration. There was no evidence of storage in the skin or skin toxicity of 2-chlorobenzaldehyde following cutaneous application.

Glutathione conjugation and bacterial mutagenicity of racemic and enantiomerically pure cis- and trans-methyl epoxycinnamates.

This paper describes the ability of racemic, and enantiomerically pure cis- and trans-methyl epoxycinnamates (methyl 3-phenyl-2,3-epoxy-propanoates) to undergo glutathione conjugation and subsequent excretion as mercapturic acid and on the mutagenicities of these epoxy esters in the Ames assay. In incubation mixtures containing rat liver cytosol (9,000 g), the decrease of glutathione due to the epoxy esters occurred enzymatically. The highest glutathione depletion was found for the cis-epoxy cinnamic esters. Adult male rats administered a single i.p. dose of racemic trans- and cis-epoxy cinnamates (0.7 mmol/kg, n = 4) excreted thioethers in urine. Higher urinary thioether excretion was found after the cis-epoxy ester dosing. The structures of the thioether metabolites isolated from the urinary extracts were identified by TLC and confirmed by synthesis and mass spectrometry (FAB+). The thioethers appeared to be hydroxy mercapturic acids. The N-alkylating potential of the racemic epoxy esters was determined using 4-(p-nitrobenzyl)pyridine (= NBP). The trans-epoxy ester appeared to react much better with NBP than the cis-compound. Mutagenic effects of racemic trans-epoxy cinnamate as well as the enantiomerically pure trans-epoxy cinnamates were observed in the Ames test with S. typhimurium strains TA1535, TA1537, TA1538 and TA100 without metabolic activation. No mutagenic responses were detected using any of the epoxy cinnamates with S9 activation. By comparing the mutagenicity and the enzymatically catalyzed glutathione conjugation it follows that the activity of the respective enantiomeric methyl cinnamates goes in the opposite order. Glutathione conjugation plays a protective role in the detoxication in living organism of the potentially toxic methyl epoxy cinnamates.

Substituent effects during the rat liver aldehyde dehydrogenase catalyzed oxidation of aromatic aldehydes.

The influence of the steric hindrance of halogen substituents was investigated in vitro by measuring the activity of yeast aldehyde dehydrogenase (aldehyde: NAD(P)+ oxidoreductase, EC 1.2.1.5) and of aldehyde dehydrogenases in subcellular rat liver fractions with a series of ortho- and para-halo-substituted benzaldehydes as substrates. Upon an increase in the size of the halogen substituent (F, Cl, Br), the reactivity of yeast aldehyde dehydrogenase to ortho-substituted benzaldehydes decreased drastically. The same phenomenon was observed with the unspecific aldehyde dehydrogenases in three rat liver fractions; cytoplasm, mitochondria and microsomes. The corresponding para-halobenzaldehydes (F, Cl, Br, I) did not reveal large differences in reactivity to the various rat liver aldehyde dehydrogenases. The aldehyde dehydrogenases in the rat liver microsomal fraction exhibited most clearly the regiospecificity. Enzymatic oxidation of 4-bromobenzaldehyde was more than 30-times faster then the ortho-isomer. The findings in this investigation confirm the suggestion that the steric hindrance of bulky ortho-substituents of benzaldehydes account for the slowing down of the aldehyde dehydrogenase-catalyzed oxidation of benzaldehydes to corresponding benzoic acids. The enzymatic oxidation of microsomal aldehyde dehydrogenase is strongly influenced by steric effects of benzaldehydes, bearing a halogen in ortho-position. We think that the microsomal aldehyde dehydrogenase might be the principal enzyme responsible for oxidation of halobenzaldehydes in rat liver.

Bacterial mutagenicity of some methyl 2-cyanoacrylates and methyl 2-cyano-3-phenylacrylates.

The mutagenic potential of three alkyl 2-cyanoacrylate adhesives, three commercial alkyl 2-cyanoacrylate adhesives and three methyl 2-cyano-3-phenylacrylates, was assessed using the Salmonella/microsome mutagenicity assay. Compounds were tested with and without Aroclor 1254-induced rat-liver homogenate (S9 mix). The methyl 2-cyanoacrylate adhesives were mutagenic in the standard plate test with S. typhimurium strain TA100 with and without S9 activation. Methyl 2-cyano-3-(2-bromophenyl)acrylate revealed a direct mutagenic action to S. typhimurium strain TA1535. The compounds most toxic towards the bacterium S. typhimurium, were the methyl 2-cyanoacrylate adhesives (greater than 500 micrograms/plate). All alkyl 2-cyanoacrylate adhesives were tested in a modified spot test for volatile compounds with tester strain TA100. Mutagenic and toxic effects were observed with the three methyl 2-cyanoacrylate adhesives. It can be concluded from the results that the bacterial toxicity and mutagenicity of methyl 2-cyanoacrylate adhesives may be due to the methyl 2-cyanoacrylate monomer.

Glutathione conjugation of chlorobenzylidene malononitriles in vitro and the biotransformation to mercapturic acids in rats.

The glutathione conjugation of 2-chloro-, 3-chloro-, 4-chloro- and 2,6-dichlorobenzylidene malononitrile (chloroBMNs) was investigated in vitro. In incubation mixtures containing rat liver cytosol (9000 g), the decrease in the initial amount of glutathione due to the various chloroBMNs ranged from 40 to 60% and occurred both enzymatically and spontaneously at physiological conditions (37 degrees C, pH 7.4). 2,6-DichloroBMN, however, depleted glutathione largely spontaneously (38 +/- 3%). The steric hindrance of the two chlorosubstituents probably plays an important role during the glutathione-S-transferase catalyzed reaction. The hydrolysis of the chloroBMNs to the corresponding chlorobenzaldehydes and malononitrile was studied in a mixture of buffer pH 7.4 and ethanol. The rate of hydrolysis of 2,6-dichloroBMN was slower than those of the related chloroBMNs. This means that 2,6-dichloroBMN will be the most stable compound in the presence of water. Only IP administration of 2-chloroBMN (CS) to adult male Wistar rats gave enhancement of urinary thioether excretion. A thioether could be isolated and was identified as the N-acetyl-S-[2-chlorobenzyl]-L-cysteine. The quantity of this benzylmercapturic acid in the urine of rats amounted to 4.4% dose (0.07 mmol/kg, n = 12). After IP administration of 2-chloro- and 3-chlorobenzaldehyde to rats benzylmercapturic acid excretion in the urine was found to be 7.6 and 1.1% of the dose, respectively. Administration of the related 4-chloro- and 2,6-dichlorobenzaldehyde, however, resulted in no urinary mercapturic acid excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid onset of an increase in caffeine residence time in young women due to oral contraceptive steroids.

Oral contraceptive steroids increased the residence time of caffeine in 9 young women by a factor of 2. The effect was already manifested during the first cycle 2 weeks after starting oral contraceptive steroids (OCS) and was slightly increased in the second cycle, after 6 weeks on OCS. Toxic effects attributed to oral contraceptive steroids may in part be indirect and due to prolonged retention of absorbed toxic agents to which women are exposed.

PIP: Oral contraceptives (OCs) increased the residence time of caffeine in 9 young women by a factor of 2. The effect was already manifested during the 1st cycle 2 weeks after beginning OCs and was slightly increased in the 2nd cycle, after 6 weeks on OCs. Toxic effects attributed to OCs may in part be indirect and due to prolonged retention of absorbed toxic agents to which women are exposed.

2-Chlorobenzylmercapturic acid, a metabolite of the riot control agent 2-chlorobenzylidene malononitrile (CS) in the rat.

Adult male Wistar rats administered i.p. with 2-chlorobenzylidene malononitrile (CS) excreted one mercapturic acid in urine. The amount of mercapturic acid determined gaschromatographically was about 4% of the dose (0.07 mmol/kg, n = 12). The structure of the mercapturic acid methylester was identified by t.l.c. and confirmed by synthesis and mass-spectrography. The acid appeared to be 2-chlorobenzylmercapturic acid [N-acetyl-S-(2-chlorobenzyl)-L-cysteine]. CS and some of its metabolites were also tested in the Ames Salmonella/microsome assay. Both mutagenic and toxic effects were measured with strain TA 100 as the indicator organism. No mutagenic effects were found with any of the tested substances. At dosages of CS, higher than 1,000 micrograms/plate a bacteriotoxicity was revealed.

Mechanism of formation of mercapturic acids from aromatic aldehydes in vivo.

Male adult Wistar rats dosed i.p. with o-substituted benzaldehydes (o-F, o-Cl, and o-Br = V, VI, and VII) excreted mercapturic acids in urine. These acids were identified as N-acetyl-S-(ortho-substituted benzyl)cysteines (I, II, III). The total mercapturic acid excretion as % dose (2.0 mmol/kg, n = 4) was 1.2 +/- 0.4, 6.8 +/- 0.9, and 10.4 +/- 2.0 for V, VI, and VII. p-Cl-benzaldehyde administered in the same dose showed a non-significant urinary thioether excretion. The aim of the investigation was to prove in vivo a postulated metabolic pathway of substituted benzaldehydes via sulphate esters to mercapturic acids. After a single administration of the sodium salts of o- and p-Cl-benzylsulfuric acid a significant increase in mercapturic acid excretion of 21.2 +/- 1.8% and 14.5 +/- 1.2% of dose (2.0 mmol/kg, n = 4) was found. By pretreatment with pyrazole the mercapturic acid excretion increased after administration of o-Cl-benzyl alcohol (IX) whereas a significant decrease was found after administration of o-Cl-benzaldehyde (VI). After simultaneous administration of ethanol with IX and VI an increase in mercapturic acid excretion was observed. After previous administration of pentachlorophenol a significant decrease in urinary mercapturic acid excretion for IX and VI was found. These findings are in accordance with a metabolic pathway of substituted benzaldehydes via benzyl alcohols, subsequently sulphate esters to the corresponding benzylmercapturic acids.

Mercapturic acids as metabolites of aromatic aldehydes and alcohols.

After administration of substituted (CH3, OH, OCH3, F, CL, Br, NO2) benzaldehyde or benzyl alcohols in the rat an enhanced urinary thioether excretion was found in some cases. With p-substituted benzaldehyde only occasionally a slight increase could be shown, but with o-substituted aldehydes and alcohols thioether excretions amounted up to 13% of the dose. Mercapturic acids were isolated and identified by synthesis, mass-, and n.m.r.-spectrometry as the arylmethyl thioethers of N-acetylcysteine. Steric hindrance by o-substituents must be the main cause of a relative decrease in oxidation to the carboxylic acid and an increase of the importance of both the reduction of the aldehydes and the reaction of the alcohols, presumably to sulphuric acid esters, as intermediates for the alkylation of glutathione. Consequently, previous administration of pyrazole, an inhibitor of alcohol dehydrogenase, caused an even larger thioether excretion after injection of o-chlorobenzyl alcohol.