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Biomedical subjects

J L Stevens

Publications and source records attributed to J L Stevens.

At least 91 records · Page 5Linked to original sources
PubMed1989

N-acetyl S-(1,2-dichlorovinyl)-L-cysteine produces a similar toxicity to S-(1,2-dichlorovinyl)-L-cysteine in rabbit renal slices: differential transport and metabolism.

Renal cortical slices were used to determine the toxicity of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (N-acetyl-DCVC) as well as to investigate the transport and metabolism of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and the N-acetyl derivative. N-Acetyl-DCVC produced dose- and time-dependent decreases in intracellular K+ content and lactate dehydrogenase activity. Histopathology demonstrated an initial S3 lesion followed by a lesion inclusive of all proximal tubules. N-Acetyl-DCVC was shown to be transported via the organic anion system by its ability to inhibit PAH transport by the cells and the ability of probenecid to decrease uptake (80%) and toxicity of N-acetyl-DCVC. DCVC, in contrast, was not transported by the organic anion system, but may be transported by one or more amino acid systems. N-Acetyl-DCVC must be deacetylated before undergoing metabolism by beta-lyase. This process must occur since covalent binding of a 35S-labeled reactive product from N-acetyl [35S]DCVC is observed within 1 hr. Both the uptake inhibitor, probenecid, and aminooxyacetic acid (AOAA), a beta-lyase inhibitor, decreased the covalent binding from N-acetyl [35S]DCVC (80 and 50%, respectively), but only AOAA inhibited the covalent binding of DCVC. AOAA also partially inhibited the toxicity of DCVC and N-acetyl-DCVC as determined by intracellular K+ content, lactate dehydrogenase activity, and histopathology. Despite the fact that a separate transport system and an additional enzymatic step (deacetylation) are required, N-acetyl-DCVC produces a lesion with similar intratubular specificity to that seen with DCVC. Therefore, the S3 specificity seen in vivo could be produced by either compound.

Acetylation↗
PubMed1989

In vitro and in vivo nephrotoxicity of the L and D isomers of S-(1,2-dichlorovinyl)-cysteine.

The toxicity of the optical isomers S-(1,2-dichlorovinyl)-L-cysteine (L-DCVC) and S-(1,2-dichlorovinyl)-D-cysteine (D-DCVC) was investigated in vivo and in vitro. In vitro studies, utilizing a rabbit renal cortical slice system, demonstrated toxicity due to both forms with the L-form being more toxic. Dose- and time-dependent decreases in intracellular K+ and LDH were observed. Both compounds produced an initial S3 lesion, L-DCVC at 10(-5) M (12 h), D-DCVC at 10(-4) M (8 h), followed by a lesion encompassing all proximal tubules. In vivo studies demonstrated elevated blood urea nitrogen values at 24 and 48 h with 25 mg/kg of either isomer. Histopathology indicated both D and L-DCVC produced a straight proximal tubular lesion by 48 h, the lesion produced by L-DCVC being more severe. The D and L isomers of DCVC were both shown to be toxic, the toxicity assessed in vitro corresponded well with the toxicity in vivo.

Animals↗
PubMed1988

Alterations in intermediate filament proteins in rat kidney proximal tubule epithelial cells.

Changes in the intermediate filament composition of rat kidney proximal tubule cells in culture have been investigated. The data suggest that differentiated tubular epithelial cells do not express vimentin, but vimentin expression is induced when the cells begin to proliferate in culture. The cultured cells are positive for both cytokeratins and vimentin by immunofluorescence microscopy. The data support the concept that the intermediate filament composition of proximal tubule epithelial cells can be altered during proliferation induced by nephrotoxic chemicals or by neoplastic transformation.

Animals↗
PubMed1988

The role of mitochondrial matrix enzymes in the metabolism and toxicity of cysteine conjugates.

The submitochondrial localization and identity of enzymes which metabolize cysteine conjugates were investigated. Glutamine transaminase K was purified from rat kidney mitochondrial soluble fraction and was shown to be a cysteine conjugate beta-lyase. The purified mitochondrial enzyme is similar to the cytosolic glutamine transaminase K whose beta-lyase activity with S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is regulated by concurrent transamination (Stevens, J. L., Robbins, J. D., and Byrd, R. A. (1986) J. Biol. Chem. 261, 15529-15537). However, beta-lyase activity in whole mitochondria is largely independent of regulation by cosubstrates for transamination suggesting that factors present in mitochondria are able to support the beta-lyase activity in the absence of added alpha-keto acid. Fractionation of mitochondria results in a loss of the independent beta-lyase activity. However, the majority of the beta-lyase activity can be recovered in the matrix if it is stimulated by the addition of alpha-keto-gamma-methiolbutyrate. The data suggest that the regulation of beta-elimination by the competing transamination pathway is different for each substrate and that multiple beta-lyases may exist in rat kidney. S-(2-Benzothiazolyl)-L-cysteine (BTC) is a poor substrate for purified glutamine transaminase K from mitochondria and cytosol, but BTC is as active as DCVC in crude mitochondrial matrix suggesting that other enzymes may be present. In contrast to DCVC, with BTC as substrate, the beta-lyase activity of the purified enzyme and enzyme(s) in the mitochondrial matrix is largely alpha-keto acid-independent. The existence of multiple enzymes is also supported by the observation that alpha-keto acids which are not substrates for purified glutamine transaminase K from mitochondria and cytosol do stimulate beta-lyase activity in the mitochondrial matrix fraction. Mitoplasts were found to be sensitive to DCVC toxicity consistent with the matrix localization of beta-lyase activity. The possible role in cysteine conjugate toxicity of matrix enzyme regulation by alpha-keto acids is discussed.

Aminooxyacetic Acid↗
PubMed1988

Immunohistochemical localization of glutamine transaminase K, a rat kidney cysteine conjugate beta-lyase, and the relationship to the segment specificity of cysteine conjugate nephrotoxicity.

Rat kidney glutamine transaminase K is a major rat kidney cysteine conjugate beta-lyase and is a key enzyme in the nephrotoxicity of some cysteine conjugates. However, it has not been demonstrated that the beta-lyase is present in the target cells. Furthermore, although all segments of the proximal tubule are affected by high doses of nephrotoxic cysteine conjugates, the S3 segment is the most sensitive. Because heterogeneous distribution of the beta-lyase could account for the enhanced sensitivity, antibody raised against rat kidney cysteine conjugate beta-lyase has been prepared and used to investigate the distribution of the enzyme in kidney and other tissues. The data show that the enzyme is highest in rat kidney, consistent with enzyme activity data. By immunohistochemical staining, no enzyme is present in the glomeruli or distal tubular elements of the kidney. The enzyme is present only in the target cells, the renal proximal tubular epithelium. However, the distribution of the beta-lyase within the proximal tubule is not consistent with the hypothesis that a higher concentration of the enzyme in the S3 segment accounts for the greater sensitivity of S3 to nephrotoxic cysteine conjugates compared to S1 and S2. Several alternative hypotheses are discussed.

Acetylcysteine↗
PubMed1987

The transport of S-cysteine conjugates in LLC-PK1 cells and its role in toxicity.

The transport of S-cysteine conjugates was studied in the kidney cell line, LLC-PK1, using the nephrotoxin, S-(1,2-dichlorovinyl)-L-cysteine (L-DCVC), as the model compound. The saturable uptake of this conjugate did not require sodium and was selectively inhibited by the amino acid transport system L-specific substrate, 2-amino-2-norbornane carboxylic acid, as well as a variety of other S-cysteine conjugates and neutral amino acids with large, nonpolar side chains. Kinetic studies suggested the existence of both low and high affinity transport systems with Km values that differed by 25-fold. Although these uptake systems showed no discernible differences in substrate specificity, the low affinity transport was more sensitive to trans-stimulation. L-DCVC uptake in subconfluent cultures was about 3-fold that of confluent cells, suggesting either adaptive regulation to cell growth or polarization of transport to the basolateral membrane. L-DCVC toxicity in LLC-PK1 cells was inhibited in the presence of nontoxic transport substrates but was potentiated when cells were preloaded with many of the same compounds, indicating that transport may be a rate-limiting factor in L-DCVC-induced toxicity under certain circumstances. The possible role of this system L-like uptake in the transport of S-cysteine conjugates in vivo is discussed.

Animals↗
PubMed1987

Mechanism of transport for toxic cysteine conjugates in rat kidney cortex membrane vesicles.

Cysteine conjugate transport plays a key role in the interorgan transport of xenobitoic metabolites which are formed via the mercapturic acid pathway. In the rat, transport of cysteine conjugates could be an important factor in the selective nephrotoxicity of some toxic cysteine conjugates. However, little information is available on the molecular mechanism(s) of cysteine conjugate transport in the rat kidney. Therefore, we have investigated the polarity and the molecular driving forces for the transport of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in isolated membrane vesicles from rat kidney cortex. Our data suggest that Na+-dependent transport on the lumenal side is responsible for the uptake of cysteine conjugates across the apical membrane. No Na+-stimulated transport was found on the basolateral side and uptake of DCVC in basolateral membrane vesicles was not saturable. Na+-dependent transport in brush border membrane vesicles was inhibited by a variety of neutral amino acids and cysteine conjugates, but not by polar amino acids. Therefore, the transporter is similar to the Na+-dependent neutral amino acid transporter of rat kidney brush border membranes. The system L-specific substrate, 2-amino-2-norbornane carboxylic acid, was not inhibitory. The Km for the Na+-stimulated transport system in brush border membrane vesicles was 225 microM and the Vmax was 782 pmol/15 sec/mg of protein. We propose that the driving force for the apical transport of cysteine conjugates may be the coupling of the lumenal transport to the Na+-gradient. The data are discussed with regard to a transepithelial transport model for cysteine conjugates and the role transport plays in the molecular mechanism of cysteine conjugate toxicity.

Animals↗
PubMed1986

A purified cysteine conjugate beta-lyase from rat kidney cytosol. Requirement for an alpha-keto acid or an amino acid oxidase for activity and identity with soluble glutamine transaminase K.

Cysteine conjugate beta-lyase has been purified from rat kidney cytosol. The enzyme is a 100,000-dalton dimer of two 55,000-dalton subunits and has an absorption maximum at 432 nm. The enzyme has phenylalanine alpha-keto-gamma-methiolbutyrate transaminase activity and appears to be identical to rat kidney cytosolic glutamine transaminase K. Metabolism of S-1,2-dichlorovinyl-L-cysteine (DCVC) by the purified enzyme was dependent on the presence of either alpha-keto-gamma-methiolbutyrate or a protein factor which is present in the cytosolic fraction of rat kidney cortex. The protein factor was identified as a flavin containing L-amino acid oxidase which oxidized DCVC to S-(1,2-dichlorovinyl)-3-mercapto-2-oxopropionic acid. S-(1,2-Dichlorovinyl)-3-mercapto-2-oxopropionic acid has not been previously reported as a metabolite of DCVC. The data also show that rat kidney cytosolic glutamine transaminase K catalyzes both a beta-elimination and a transamination reaction with DCVC when alpha-keto-gamma-methiolbutyrate is present and that amino acid oxidase and alpha-keto-gamma-methiolbutyrate stimulate the enzyme activity by providing amino acceptors. When incubations were done with DCVC as substrate in the presence of excess alpha-keto-gamma-methiolbutyrate, the beta-lyase catalyzed beta-elimination and transamination in a ratio of 1:1.3, respectively. Under conditions where most of the alpha-keto-gamma-methiolbutyrate was consumed, the beta-elimination predominated indicating that the S-1,2-dichlorovinyl-3-mercapto-2-oxopropionic acid pool was consumed by transamination after the alpha-keto-gamma-methiolbutyrate had been depleted. The data are discussed with regard to the importance of these pathways as regulators or participants in the toxicity of S-cysteine conjugates.

Amino Acid Oxidoreductases↗
PubMed1986

Cysteine conjugate beta-lyase in the gastrointestinal bacterium Eubacterium limosum.

A cysteine conjugate beta-lyase (beta-lyase) from the gastrointestinal bacterium Eubacterium limosum has been isolated and characterized. This organism has the highest specific activity for cysteine conjugate beta-lyase of the gastrointestinal bacteria studied. The beta-lyase was found to cleave the thioether linkage of S-alkyl- and S-aryl-L-cysteine conjugates. Stoichiometric amounts of 2-mercaptobenzothiazole, pyruvic acid, and ammonia were produced from the beta-lyase cleavage of S-(2-benzothiazolyl)-L-cysteine. The enzyme activity was inhibited by hydroxylamine, iodoacetic acid, or KCN. The enzyme appears to be a 75,000-Da dimer of two 38,000-Da subunits. A natural substrate, cystathionine, was cleaved by this enzyme, indicating that this beta-lyase has beta-cystathionase activity. These data suggest that a beta-cystathionase from E. limosum may be an important enzyme in the metabolism of a wide range of cysteine conjugates of xenobiotics to thiol-containing products.

Animals↗
PubMed1985

Isolation and characterization of a rat liver enzyme with both cysteine conjugate beta-lyase and kynureninase activity.

Cysteine conjugate beta-lyase is a name applied to enzymes which cleave the S-cysteine conjugates of some xenobiotics to pyruvate, ammonia, and a thiol. Recently, several laboratories have characterized these enzymes from kidney, liver, and bacterial sources in an effort to understand their role in the genesis of novel sulfur-containing metabolites of xenobiotics and in the toxicity of some S-cysteine conjugates. Kynureninase is an enzyme which plays a key role in the biosynthesis of nicotinamide ribonucleotides. This investigation demonstrates that rat hepatic cysteine conjugate beta-lyase is the same enzyme as kynureninase. Both activities copurify on ion exchange, hydroxylapatite, and molecular exclusion chromatography. The subunit composition of enzyme prepared by two different methods is identical, Mr = 55,000. The Km values for 3-OH-kynurenine and kynurenine are 13 and 400 microM, respectively. Kynurenine and 3-hydroxykynurenine inhibit cysteine conjugate beta-lyase activity. Inactivation of the enzyme by substrates which undergo beta-elimination results in loss of kynureninase activity, but kynurenine does not inactivate the enzyme. Both enzyme activities react with anti-cysteine conjugate beta-lyase antibody. Product inhibitors of kynureninase, anthranilate, and 3-hydroxyanthranilate are also inhibitors of cysteine conjugate beta-lyase. Heat inactivation at 70 degrees C produced coincident loss of both activities. The enzyme has an absorption maximum at 432 nm suggesting a bound pyridoxal phosphate. These data show that at least one cysteine conjugate beta-lyase is a pyridoxal phosphate enzyme with a biological function other than xenobiotic metabolism. The enzyme can catalyze two distinct types of reactions, i.e. beta-elimination and the kynureninase reaction.

Animals↗
PubMed1985

Cysteine conjugate beta-lyase activities in rat kidney cortex: subcellular localization and relationship to the hepatic enzyme.

Kidney cortex cysteine conjugate beta-lyase enzymes were characterized using S-(2-benzothiazolyl)-L-cysteine and S-(1,2-dichlorovinyl)-L-cysteine as substrates. The contribution of the hepatic form of cysteine conjugate beta-lyase to renal metabolism of these S-cysteine conjugates is not substantial. No cysteine conjugate beta-lyase activity was found in kidney cortex brush border membrane vesicles. Two cysteine conjugate beta-lyase activities with densities corresponding to the mitochondrial and soluble fractions were separated on Percoll gradients.

Animals↗
PubMed1984

Plasma nitroglycerin concentrations and hemodynamic effects of sublingual, ointment, and controlled-release forms of nitroglycerin.

After a sublingual test dose, 12 healthy men aged 21 to 29 yr were treated with controlled-release transdermal nitroglycerin skin patches designed to deliver 10 mg/day nitroglycerin and with nitroglycerin ointment (2%) (1-in amount from the tube spread over 50 cm2) for 24 hr in a double-blind crossover study. Assessment was by measurement of nitroglycerin in plasma, blood pressure, and pulse rate. The mean plasma concentration of nitroglycerin during ointment dosing was approximately 200% to 400% that during skin patch dosing. Levels during ointment dosing were closer to those from sublingual dosing than were those during skin patch dosing. Blood pressure and pulse rate changes were much the same during both transdermal treatments. Calculations showed that delivery of nitroglycerin from the skin patches would have to be over 40 to 80 cm2 of the skin to achieve nitroglycerin exposure of the order of that induced by 1 in of ointment spread over 50 cm2 or from sublingual dosing.

Administration, Oral↗
PubMed1981

Metabolism of haloforms to carbon monoxide. IV. studies on the reaction mechanism in vivo.

In vivo studies have been carried out in order to understand more fully the mechanism of haloform oxidation to carbon monoxide. A deuterium isotope effect on carbon monoxide production from chloroform was observed in both control and phenobarbital-treated rats. Diethyl maleate treatment decreased blood carbon monoxide concentrations produced from bromoform and chloroform and attenuated the effect of deuterium substitution on the metabolism of both compounds to carbon monoxide. Cysteine also decreased blood carbon monoxide concentrations seen after giving chloroform. A reaction mechanism similar to that proposed on the basis of in vitro data, which included a central role for dihalocarbonyl compounds in the formation of 2-oxothiazolidine-4-carboxylic acid, carbon monoxide, and carbon dioxide, is suggested for the in vivo metabolism of haloforms to carbon monoxide. These data indicate that carbon monoxide production may be a detoxification pathway for haloforms and that both the inhibition of carbon monoxide production from haloforms and the potentiation of haloform-hepatotoxicity by diethyl maleate are due to the depletion of glutathione.

Animals↗