Search PubMed⌕ Search

Biomedical subjects

S Vamvakas

Publications and source records attributed to S Vamvakas.

At least 55 records · Page 3Linked to original sources
PubMed1995

Insulin-like growth factor I induced reduction in cysteine proteinase activity in freshly isolated proximal tubule cells of the rat.

The potential effects of insulin-like growth factor I (IGF-I) on lysosomal cysteine proteinases (cathepsin B, H and L+B activities) were investigated in the freshly isolated proximal tubule cells of rats. IGF-I significantly inhibited these enzyme activities after an incubation time of 80 min. This effect was associated with a dose-dependent increase in cellular protein content. The study suggests that, besides the established enhanced protein synthesis, IGF-I-induced cellular hypertrophy is mediated by a suppression of the proteolytic enzyme activity in proximal tubular cells.

Animals↗
PubMed1995

Angiotensin-II-induced cell hypertrophy: potential role of impaired proteolytic activity in cultured LLC-PK1 cells.

BACKGROUND: Angiotensin II-induced hypertrophy of both mesangial and tubular cells has been shown to be caused by enhanced protein synthesis. There are no data about its role on protein breakdown. Therefore, protein turnover and proteolytic activities were investigated in LLC-PK1 cells. METHODS: Protein turnover was measured by determining the incorporation and release of [14C]phenylalanine; collagenolytic and gelatinolytic activities were assayed by using fluorogenic peptidyl substrates. RESULTS: Angiotensin II (10(-8)-10(-6) M) exerted a dose-dependent inhibition of collagenolytic and gelatinolytic activities, associated with reduction of protein degradation rate. In addition angiotensin II stimulated protein synthesis in the cells. These combined effects on protein turnover resulted in an increase in both cell size and cell protein content (31.7% after 48 h). However, the rise of cell protein content was only partly (48.0%) prevented by the protein synthesis inhibitor cycloheximide (10(-5)M), which supports the role of decreased protein degradation in the angiotensin-II-induced cell hypertrophy. The angiotensin-II-induced effects on proteolytic activities as well as on cell protein content could be abolished by coincubation with the angiotensin II type I-receptor antagonist DuP 753 (10(-6)M). The calcium-channel blocker verapamil (10(-6)M) ameliorated the impairment of collagenolytic activity. On the contrary the calcium ionophore A23187 (10(-6)M) mimicked the action of angiotensin II on this enzyme activity (control 34.5 +/- 1.9; angiotensin II 24.0 +/- 2.0; A23187 23.0 +/- 2.2 and angiotensin II+verapamil, 33.8 +/- 2.6 pmol/min/micrograms DNA). The role of cytosolic [Ca2+] in the actions of angiotensin II could be finally shown by a dose-dependent rise which was partly blunted by verapamil. CONCLUSION: The angiotensin-II-induced hypertrophy in LLC-PK1 cells is caused not only by enhanced protein synthesis but also by reduced protein degradation. The concomitant decline of collagenolytic and gelatinolytic activities may contribute to the accumulation of extracellular matrix, and presumably also to cell hypertrophy. These effects are obviously mediated via angiotensin II type I receptors and seem to be [Ca2+] dependent.

Analysis of Variance↗
PubMed1994

Induction of poly(ADP-ribosyl)ation in the kidney after in vivo application of renal carcinogens.

Dichlorovinylcysteine, the key metabolite thought to be responsible for the nephrocarcinogenicity of trichloroethene and dichloroacetylene, induces DNA double-strand breaks followed by increased poly(ADP-ribosyl)ation of nuclear proteins in cultured renal cells (Vamvakas et al., 1992, Biochem. Pharmacol. 44, 1131-1138). Poly(ADP-ribosyl)ation represents a post-translational modification of nuclear proteins involved in DNA repair, DNA replication, and modulation of gene expression. The present study investigates the induction of DNA double-strand breaks and poly(ADP-ribosyl)ation in the renal cortex after in vivo administration of several renal carcinogens to male Wistar rats, and the temporal relationship between these two processes. Dichlorovinylcysteine caused a time-dependent increase in the amount of poly(ADP-ribosyl)conjugates in the kidney cortex, which was preceded by increased formation of DNA double-strand breaks. Potassium bromate and ferric nitrilotriacetate, whose nephrocarcinogenicity is thought to result from increased formation of reactive oxygen species, both induced poly(ADP-ribosyl)ation with the concomitant formation of DNA double-strand breaks. Dimethylnitrosamine, an indirect acting methylating agent, and trimethylpentane, a non-genotoxic renal carcinogen, failed to induce poly(ADP-ribosyl)ation or a significant increase in DNA double-strand breaks in the renal cortex. The results indicate that nephrocarcinogens capable of inducing DNA fragmentation also induce post-translational modification of renal proteins via increased poly(ADP-ribosyl)ation.

Acetylene↗
PubMed1993

Enhanced expression of the protooncogenes c-myc and c-fos in normal and malignant renal growth.

The protooncogenes c-myc and c-fos play an important role in growth and differentiation of renal tissue. They are highly expressed during embryogenesis in the mitotically active tubule epithelium, while in terminally differentiated tubule cells of the kidney the expression is completely shut off. Furthermore, induction of cell proliferation in cultured renal cells by addition of growth factors is preceded by enhanced expression of c-myc and c-fos. Increased expression of these protooncogenes is also obtained by treatment of kidney cells in culture with the potent nephrocarcinogen N-dimethylnitrosamine and also with the nephrotoxin and possibly nephrocarcinogen S-(1,2-dichlorovinyl)-L-cysteine. Finally, the expression of c-myc and c-fos is induced after unilateral nephrectomy during compensatory renal growth in the remaining kidney and also during regenerative cell proliferation after in vivo application of the strong nephrotoxins folic acid and mercury chloride.

Animals↗
PubMed1993

DNA adduct formation in Salmonella typhimurium, cultured liver cells and in Fischer 344 rats treated with o-tolyl phosphates and their metabolites.

2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is an electrophilic and a neurotoxic metabolite of o-tolyl phosphates. In a previous paper we reported that 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is mutagenic in Salmonella typhimurium TA100 and forms DNA adducts in incubations with nucleotides, nucleosides and isolated DNA. In the present study we compare DNA adduct formation using 32P-post-labelling assays in 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide-treated bacteria (S.typhimurium TA100) and hepatoma cells with DNA adducts formed in liver, kidney, lung and heart of tri-o-tolyl phosphate-exposed Fischer 344 male rats. In both bacteria and hepatoma cells two DNA adducts could be detected after treatment with 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide. The minor adduct co-chromatographed with synthetic N3-(o-hydroxy-benzyl)deoxyuridine 3' monophosphate after postlabelling. The major DNA adduct was a cytidine adduct, most likely N3-(o-hydroxybenzyl)deoxycytidine 3' monophosphate. Male Fischer 344 rats were treated orally for 10 days with tri-o-tolyl phosphate (50 mg/kg/day) and DNA was isolated from liver, kidney, lung, heart, brain and testes 1, 4, 7 and 28 days after giving the last dose. Analysis by 32P-postlabelling revealed that two adducts were present in the DNA isolated from liver, kidney, lung and heart on the first day after giving the last dose; DNA adducts were not detected in the brain and testes. The adduct pattern after in vivo treatment with tri-o-tolyl phosphate was identical with that found in bacteria and hepatoma cells treated with 2-phenoxy-4H-1,3,2-benzo-dioxaphosphorin 2-oxide, the major adduct being N3-(o-hydroxybenzyl)deoxycytidine 3' monophosphate and the minor N3-(o-hydroxybenzyl)deoxyuridine 3' monophosphate. Both DNA adducts persisted in the lungs for the entire observation period, whereas in the kidney only the cytidine adduct could be detected 28 days after the last dose of tri-o-tolyl phosphate. In liver and heart the adducts were detectable only on the first day after completion of the treatment. The results indicate that in addition to the well established neurotoxicity, some o-tolyl phosphates may have a carcinogenic potential.

Animals↗
PubMed1993

Nephrotoxic and genotoxic N-acetyl-S-dichlorovinyl-L-cysteine is a urinary metabolite after occupational 1,1,2-trichloroethene exposure in humans: implications for the risk of trichloroethene exposure.

Excretion of mercapturic acids in the urine is indicative of the formation of electrophiles in the metabolism of xenobiotics. The determination of these mercapturic acids thus may be a useful method to estimate the exposure. We identified the nephrotoxic and mutagenic mercapturic acids N-acetyl-S-(1,2-dichlorovinyl)-L- cysteine and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine in the urine of workers exposed to 1,1,2-trichloroethene. A method to quantify these mercapturic acids by gas chromatography-mass spectrometry-selected ion monitoring was developed and appreciable amounts (2.8-3.8 mumole/L were found in human urine samples. Because deacetylation determines notably the amount of the excreted mercapturic acids, the formation of the resulting cysteine S-conjugates was comparably measured in subcellular fractions of rodent and human kidneys; significant species differences in acylase activity were found. The formation of mutagenic and nephrotoxic metabolites during 1,1,2-trichloroethene metabolism mandates a revision of the risk assessment of trichloroethene exposure.

Acetylcysteine↗
PubMed1993

Glutathione-dependent bioactivation of xenobiotics.

Glutathione conjugation has been identified as an important detoxication reaction. However, in recent years several glutathione-dependent bioactivation reactions have been identified. Current knowledge on the mechanisms and the possible biological importance of these reactions are discussed. 1. Dichloromethane is metabolized by glutathione conjugation to formaldehyde via S-(chloromethyl)glutathione. Both compounds are reactive intermediates and may be responsible for the dichloromethane-induced tumorigenesis in sensitive species. 2. Vicinal dihaloalkanes are transformed by glutathione S-transferase-catalyzed reactions to mutagenic and nephrotoxic S-(2-haloethyl)glutathione S-conjugates. Electrophilic episulphonium ions are the ultimate reactive intermediates formed. 3. Several polychlorinated alkenes are bioactivated in a complex, glutathione-dependent pathway. The first step is hepatic glutathione S-conjugate formation followed by cleavage to the corresponding cysteine S-conjugates, and, after translocation to the kidney, metabolism by renal cysteine conjugate beta-lyase. Beta-Lyase-dependent metabolism of halovinyl cysteine S-conjugates yields electrophilic thioketenes, whose covalent binding to cellular macromolecules is responsible for the observed toxicity of the parent compounds. 4. Finally, hepatic glutathione conjugate formation with hydroquinones and aminophenols yields conjugates that are directed to gamma-glutamyltransferase-rich tissues, such as the kidney, where they undergo alkylation or redox cycling reactions, or both, that cause organ-selective damage.

Amino Acid Sequence↗
PubMed1992

Events that precede and that follow S-(1,2-dichlorovinyl)-L-cysteine-induced release of mitochondrial Ca2+ and their association with cytotoxicity to renal cells.

Previous studies showed that S-(1,2-dichlorovinyl)-L-cysteine perturbs intracellular Ca2+ homeostasis [Vamvakas et al., Mol Pharmacol 38: 455-461, 1990]. The objective of the present study was to investigate the cellular events that precede and that follow S-(1,2-dichlorovinyl)-L-cysteine-induced mitochondrial Ca2+ release. In incubations with isolated kidney mitochondria, S-(1,2-dichlorovinyl)-L-cysteine-induced Ca2+ efflux is preceded by increased oxidation of mitochondrial pyridine nucleotides and is prevented by ATP, an inhibitor of the hydrolysis of pyridine nucleotides, and by meta-iodobenzylguanidine, an acceptor of ADP-ribose moieties. In LLC-PK1 cells, elevation in the cytosolic Ca2+ concentration is followed by a several-fold increase in DNA double-strand breaks which is attributed to the activation of Ca2+- and Mg(2+)-dependent endonucleases. The formation of DNA double-strand breaks is followed by increased poly(ADP-ribosylation) of nuclear proteins. S-(1,2-Dichlorovinyl)-L-cysteine-induced cytotoxicity in LLC-PK1 cells is blocked by chelation of cytosolic Ca2+ with Quin-2, by inhibition of DNA fragmentation with aurintricarboxylic acid and by inhibition of increased poly(ADP-ribosyl)transferase activity by 3-aminobenzamide. These findings indicate that S-(1,2-dichlorovinyl)-L-cysteine bioactivation in renal cells may initiate the following cascade of events: increased oxidation and hydrolysis of mitochondrial pyridine nucleotides resulting in the modification of mitochondrial membrane proteins by pyridine nucleotide-derived ADP-ribose moieties, followed by Ca2+ release. Elevated Ca2+ concentrations may activate Ca(2+)-dependent endonucleases, which leads to DNA fragmentation followed by increased poly(ADP-ribosylation) of nuclear proteins and, finally, cytotoxicity.

Adenine Nucleotides↗
PubMed1992

Glutathione depletion, lipid peroxidation, DNA double-strand breaks and the cytotoxicity of 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone in rat renal cortical cells.

The mechanisms involved in the cytotoxicity of 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone, a model compound for hydroquinone derived mercapturic acids, were investigated in rat renal proximal tubule cells. 2-Bromo-3-(N-acetylcystein-S-yl)hydroquinone induced a time- and concentration-dependent decrease in cell viability and in the levels of cellular glutathione. Antioxidants such as N,N'-diphenyl-p-phenylene diamine and ascorbic acid and the iron chelator desferrioxamine very efficiently protected the cells from 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone without influencing glutathione depletion. The acetoxymethyl ester of the Ca2+ chelator Quin-2, the inhibitor of the Ca(2+)- and Mg(2+)-dependent endonucleases, aurintricarboxylic acid and the poly(ADP-ribose)-polymerase inhibitor 3-aminobenzamide also ameliorated 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone cytotoxicity. Moreover, 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone depleted Ca2+ from isolated kidney mitochondria, increased the amount of malondialdehyde in rat kidney cells and induced DNA double-strand breaks in renal cells in culture. These results suggest that renal cells oxidize 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone to the corresponding quinone; this soft electrophile reacts rapidly with glutathione, thus depleting cellular glutathione concentrations as indicated by the tentative identification of a 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone thioether in the incubation medium of renal cells treated with the mercapturate. As a result of the massive glutathione depletion, peroxidative mechanisms then cause an elevation of the cytosolic concentrations of ionized calcium; impairment of the ability of the mitochondria to sequester Ca2+ plays an important role in the elevation of the Ca2+ concentration. Finally, activation of Ca(2+)- and Mg(2+)-dependent endonucleases results in DNA damage and cell death.

Acetylcysteine↗
PubMed1990

Bioactivation of hexachlorobutadiene by glutathione conjugation.

Glutathione (GSH) conjugation reactions in the metabolism of hexachlorobutadiene (HCBD), in rats and mice, initiate a series of metabolic events resulting in the formation of reactive intermediates in the proximal tubular cells of the kidney. The GSH S-conjugate 1-(glutathion-S-yl)-1,2,3,4,4-pentachlorobutadiene (GPCB), which is formed by conjugation of HCBD with GSH in the liver, is not reactive and is eliminated from the liver in the bile or plasma, or both. GPCB may be translocated intact to the kidney and processed there by gamma-glutamyl transpeptidase and dipeptidases to the corresponding cysteine S-conjugate. Alternatively, gamma-glutamyl transpeptidase and dipeptidases present in epithelial cells of the bile duct and small intestine may catalyse the conversion of GPCB to cysteine S-conjugates. The kidney concentrates both GSH and cysteine S-conjugates and processes GSH conjugates to cysteine S-conjugates. A substantial fraction of HCBD cysteine S-conjugate thus concentrated in the kidney is metabolized by renal cysteine conjugate beta-lyase to reactive intermediates. The selective formation of reactive intermediates in the kidney most likely accounts for the organ-specific effects of HCBD. Alternatively, cysteine S-conjugates may be acetylated to yield excretable mercapturic acids.

Animals↗
PubMed1990

A mechanism of haloalkene-induced renal carcinogenesis.

Several halogenated alkenes are nephrotoxic; some others induce renal tubular adenocarcinomas in rodents after lifelong administration. A bioactivation mechanism accounting for the organ-selective tumor induction has been elucidated: conjugation of the parent compounds with glutathione (GSH), catalyzed by hepatic GSH S-transferases, results in the formation of haloalkyl and halovinyl glutathione S-conjugates. Formation of S-conjugates (identified by NMR and mass spectrometry) could be demonstrated with trichloroethene, tetrachloroethene, hexachlorobutadiene, perfluoropropene, trichlorotrifluoropropene, and dichloroacetylene in incubations with rat liver microsomes and in the isolated perfused rat liver. The GSH conjugates formed are eliminated from the rat liver with the bile and may be translocated to the kidney, intact or after metabolism to the corresponding cysteine S-conjugates that are metabolized in the kidney by renal tubular cysteine conjugate beta-lyase (beta-lyase) to reactive intermediates, most likely thioacylchlorides and thioketenes. Interaction of these potent electrophiles with DNA [demonstrated for intermediates formed from S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine] causes mutagenicity in bacteria, genotoxicity in cultured renal cells, and cytotoxicity in kidney cells. As an alternative to beta-lyase-catalyzed cleavage, the cysteine S-conjugates may be acetylated to the corresponding mercapturic acids, which have been identified in urine. The ability of the kidney to concentrate GSH and cysteine S-conjugates and the intensive metabolism of GSH S-conjugates to cysteine S-conjugates in this organ are evidently responsible for the organotropic carcinogenicity.

Acetylcysteine↗
PubMed1990

Perturbations of intracellular calcium distribution in kidney cells by nephrotoxic haloalkenyl cysteine S-conjugates.

The Ca2(+)-sensitive dye fura-2 was used to investigate the disturbances in intracellular Ca2+ concentration [( Ca2+]i) and distribution induced by the nephrotoxic cysteine S-conjugate S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and its homocysteine analog S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC) in LLC-PK1 cells. After 24-hr treatment with DCVC, the average [Ca2+]i increased from 88 +/- 23 nM to 415 +/- 92 nM. Digital image analysis revealed that the mitochondrial region, which was stained with rhodamine-123, contained lower Ca2+ concentration ([Ca2+]) than other cell areas. This distribution was different from the higher [Ca2+] in the nuclear and mitochondrial regions observed in control cells. In DCVHC-treated cells, there was also an increase in [Ca2+]i to 355 +/- 85 nM, but the increase in [Ca2+] was greater in the mitochondrial region, compared with the rest of the cell. After 72-hr treatment with DCVC or DCVHC, the average [Ca2+]i was 410 +/- 85 nM and 340 +/- 90 nM, respectively, and blebs with markedly higher [Ca2+] (600-1000 nM) than the rest of the cell appeared in both DCVC- and DCVHC-treated cells. Moreover, in DCVC-treated cells the mitochondria could not be stained with rhodamine-123, indicating severe mitochondrial damage and loss of membrane potential. All changes described above took place in viable (propidium iodide-negative) cells. The experiments demonstrate that severe perturbations of intracellular Ca2+ distribution, particularly in the mitochondrial region, precede bleb formation and cell death in the course of development of toxicity by DCVC and DCVHC.

Animals↗