L-cysteine Conjugates Research Articles

Characterization of Catecholaldehyde Adducts with Carnosine and l-Cysteine Reveals Their Potential as Biomarkers of Catecholaminergic Stress.

Monoamine oxidase (MAO) catalyzes the oxidative deamination of dopamine and norepinephrine to produce 3,4-dihydroxyphenylacetaldehyde (DOPAL) and 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), respectively. Both of these aldehydes are potently cytotoxic and have been implicated in pathogenesis of neurodegenerative and cardiometabolic disorders. Previous work has demonstrated that both the catechol and aldehyde moieties of DOPAL are reactive and cytotoxic via their propensity to cause macromolecular cross-linking. With certain amines, DOPAL likely reacts via a Schiff base before oxidative activation of the catechol and rearrangement to a stable indole product. Our current work expands on this reactivity and includes the less-studied DOPEGAL. Although we confirmed that antioxidants mediated DOPAL’s reactivity with carnosine and N-acetyl-l-lysine, antioxidants had no effect on reactivity with l-cysteine. Therefore, we propose a non-oxidative mechanism where, following Schiff base formation, the thiol of l-cysteine reacts to form a thiazolidine. Similarly, we demonstrate that DOPEGAL forms a putative thiazolidine conjugate with l-cysteine. We identified and characterized both l-cysteine conjugates via HPLC-MS and additionally identified a DOPEGAL adduct with carnosine, which is likely an Amadori product. Furthermore, we were able to demonstrate that these conjugates are produced in biological systems via MAO after treatment of the cell lysate with norepinephrine or dopamine along with the corresponding nucleophiles (i.e., l-cysteine and carnosine). As it has been established that metabolic and oxidative stress leads to increased MAO activity and accumulation of DOPAL and DOPEGAL, it is conceivable that conjugation of these aldehydes to carnosine or l-cysteine is a newly identified detoxification pathway. Furthermore, the ability to characterize these adducts via analytical techniques reveals their potential for use as biomarkers of dopamine or norepinephrine metabolic disruption.

Radiodefluorination of 3-Fluoro-5-(2-(2-[18F](fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile ([18F]SP203), a Radioligand for Imaging Brain Metabotropic Glutamate Subtype-5 Receptors with Positron Emission Tomography, Occurs by Glutathionylation in Rat Brain

Metabotropic glutamate subtype-5 receptors (mGluR5) are implicated in several neuropsychiatric disorders. Positron emission tomography (PET) with a suitable radioligand may enable monitoring of regional brain mGluR5 density before and during treatments. We have developed a new radioligand, 3-fluoro-5-(2-(2-[(18)F](fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile ([(18)F]SP203), for imaging brain mGluR5 in monkey and human. In monkey, radioactivity was observed in bone, showing release of [(18)F]-fluoride ion from [(18)F]SP203. This defluorination was not inhibited by disulfiram, a potent inhibitor of CYP2E1. PET confirmed bone uptake of radioactivity and therefore defluorination of [(18)F]SP203 in rats. To understand the biochemical basis for defluorination, we administered [(18)F]SP203 plus SP203 in rats for ex vivo analysis of metabolites. Radio-high-performance liquid chromatography detected [(18)F]fluoride ion as a major radiometabolite in both brain extract and urine. Incubation of [(18)F]SP203 with brain homogenate also generated this radiometabolite, whereas no metabolism was detected in whole blood in vitro. Liquid chromatography-mass spectrometry analysis of the brain extract detected m/z 548 and 404 ions, assignable to the [M + H](+) of S-glutathione (SP203Glu) and N-acetyl-S-l-cysteine (SP203Nac) conjugates of SP203, respectively. In urine, only the [M + H](+) of SP203Nac was detected. Mass spectrometry/mass spectrometry and multi-stage mass spectrometry analyses of each metabolite yielded product ions consistent with its proposed structure, including the former fluoromethyl group as the site of conjugation. Metabolite structures were confirmed by similar analyses of SP203Glu and SP203Nac, prepared by glutathione S-transferase reaction and chemical synthesis, respectively. Thus, glutathionylation at the 2-fluoromethyl group is responsible for the radiodefluorination of [(18)F]SP203 in rat. This study provides the first demonstration of glutathione-promoted radiodefluorination of a PET radioligand.

Thiolated polymers: Evaluation of the influence of the amount of covalently attached l-cysteine to poly(acrylic acid)

It was the aim of this study to investigate the influence of the amount of thiol groups being covalently attached to poly(acrylic acid) 450 kDa on its properties. Five different PAA 450– l-cysteine conjugates (PAA 450–Cys) were synthesized bearing 53.0 (PAA I), 113.4 (PAA II), 288.8 (PAA III), 549.1 (PAA IV) and 767.0 (PAA V) μmol immobilized thiol groups per gram polymer. Mucoadhesion studies utilizing the rotating cylinder method, tensile studies and disintegration studies were performed. Self-crosslinking properties were measured by the increase in viscosity. Permeation studies were performed on rat small intestine and Caco-2 monolayers using sodium fluorescein as model drug. Following residence times on the rotating cylinder could be identified: PAA I 3.1; PAA II 5.2; PAA III 22.0; PAA IV 33.8; PAA V 53.7; control 1.3 [h]. The disintegration time of all PAA 450–Cys tablets was strongly dependent on the degree of thiolation of the polymer. Self-crosslinking studies showed that the different PAA 450–Cys conjugates (3% m/v) in phosphate buffer, pH 6.8, formed intramolecular disulfide bonds. In case of Caco-2 monolayer transport studies following P app-values could be identified: PAA I 9.8; PAA II 10.1; PAA III 11.1; PAA IV 8.9; PAA V 8.2; control 6.4 [ P app × 10 −6, cm s −1]. Mucoadhesive and self-crosslinking properties are strongly dependent on the degree of thiolation of the polymer and with respect to transport studies, an optimum amount of covalently attached l-cysteine could be identified.

Theoretical evaluation of two plausible routes for bioactivation of S-(1,1-difluoro-2,2-dihaloethyl)-L-cysteine conjugates: thiirane vs thionoacyl fluoride pathway.

The selective nephrotoxicity of halogenated alkenes has been attributed to a glutathione (GSH) S-conjugate pathway involving enzymatic hydrolysis to the cysteine S-conjugate and beta-lyase bioactivation to thiolates, which are presumed to give rise to the ultimate mutagenic or cytotoxic reactive species. Studies have shown that the brominated S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates are mutagenic in the Ames test, whereas the nonbrominated analogues are nonmutagenic. While careful experimentation has contributed much to current understanding, the ultimate reactive species responsible for the differing mutagenic effects remain unknown. Computational methods were applied to the investigation of two proposed metabolic pathways leading from the thiolate to either a thiirane or thionoacyl fluoride intermediate, both electrophilic species presumed capable of binding to proteins or DNA. Studied were six F-, Cl-, and Br-substituted 2,2-dihalo-1,1-difluoroethane-1-thiolates (2,2-dihalo-DFETs). Pathway preference was determined for each thiolate by comparison of reaction energy profiles and activation energies. At all but the lowest level of ab initio theory, a thionoacyl fluoride pathway was predicted for 2,2-difluoro-DFET, while a thiirane pathway was energetically preferred for the brominated 2,2-dihalo-DFETs. These results offer a clear mechanism-based rationale for distinguishing 2,2-difluoro-DFET from the brominated 2,2-dihalo-DFETs, while the results are less clear for the 2,2-dichloro and 2-chloro-2-fluoro-DFETs, which at the highest level of ab initio treatment had a relatively small energy preference (2.4 kcal/mol) for the thiirane pathway. The predicted clear preference for a thiirane pathway for the brominated 2,2-dihalo-DFETs is not consistent with a recently proposed pathway involving alpha-thiolactone formation through a thionoacyl fluoride intermediate [Finkelstein, M. B., et al. (1995) J. Am. Chem. Soc. 117, 9590-9591], but is supported by results of a recent study providing experimental evidence for thiirane formation from the brominated 2,2-dihalo-DFETs [Finkelstein, M. B., et al. (1996) Chem. Res. Toxicol. 9, 227-231].

Novel sources of mammalian C-S lyase activity.

The C-S lysis of L-cysteine conjugates is one biotransformation pathway which is responsible for the generation of mutagenic and cytotoxic metabolic species. Thirteen cysteine S-conjugates were synthesized in our laboratories and incubated with aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) enzymes from porcine heart tissue. The C-S lyase (CSL) activity for each enzyme-substrate combination was determined. ASAT and ALAT were shown to exhibit CSL activity and it was also demonstrated that this activity was inhibited in the presence of the pyridoxal phosphate-dependent enzyme inhibitor amino(oxyacetic acid) confirming the pyridoxal phosphate-dependent mechanism by which C-S lysis is known to take place. This finding has potentially important implications for the risk assessment of compounds which produce L-cysteine conjugates during their biotransformation.

Cysteine conjugate beta-lyase-catalyzed bioactivation of bromine-containing cysteine S-conjugates: stoichiometry and formation of 2,2-difluoro-3-halothiiranes.

1,1-Dichloroalkene-derived S-(1-chloroalkenyl)-L-cysteine conjugates, but not 1,1-difluoroalkene-derived S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates, are mutagenic in the Ames test. Recent studies have showed, however, that bromine-containing, 1,1-difluoroalkene-derived S-(2-bromo-2-halo-1,1-difluoroethyl)-L-cysteine conjugates are mutagenic [Finkelstein, M. B., et al. (1994) Chem. Res. Toxicol. 7, 157-163] and that alpha-thiolactones are formed as reactive intermediates and glyoxylate as a terminal product [Finkelstein, M. B., et al. (1995) J. Am. Chem. Soc. 117, 9590-9591]. The present studies were undertaken to examine the stoichiometry of cysteine conjugate beta-lyase-catalyzed product formation from a panel of bromine-containing and bromine-lacking cysteine S-conjugates and to search for additional metabolites. The cysteine S-conjugates were incubated with rat renal homogenates, and pyruvate:product (glyoxylate, bromide, fluoride, dihaloacetate, trihaloethene) ratios were measured. Pyruvate:glyoxylate ratios for S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine, S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine, and S-(2,2-dibromo-1,1-difluoroethyl)-L-cysteine ranged from 1:0.13 to 1:0.16. With S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine and S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine, pyruvate:bromide ratios were 1:1, but with the dibrominated conjugate S-(2,2-dibromo-1,1-difluoroethyl)-L-cysteine, the pyruvate:bromide ratio was 1:1.2. All bromine-containing cysteine S-conjugates gave less than complete conversion to fluoride. A search for additional metabolites led to the consideration of 2,2-difluoro-3-halothiiranes as putative intermediates. 2,2-Difluoro-3-halothiiranes may arise by internal displacement of bromide and cyclization of 2-bromo-2-halo-1,1-difluoroethanethiolates, which are beta-elimination products of cysteine S-conjugates. Such halogenated thiiranes may eliminate sulfur to give 1,1-difluoro-2-haloethenes. GC/MS analysis showed that trifluoroethene, 2-chloro-1,1-difluoroethene, and 2-bromo-1,1-difluoroethene were terminal products of S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine, S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine, and S-(2,2-dibromo-1,1-difluoroethyl)-L-cysteine, respectively. The bromine-lacking conjugate S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine did not yield glyoxylate or trifluoroethene as products, but the formation of chlorofluoroacetate was confirmed. The pyruvate:chlorofluoroacetate ratio was 1:0.38, indicating that other products are formed. This is the first report of the stoichiometry of the beta-lyase-catalyzed biotransformation of haloalkene-derived cysteine S-conjugates and of the formation of 2,2-difluoro-3-halothiiranes as reactive intermediates in the biotransformation of bromine-containing cysteine S-conjugates.

Structure-mutagenicity and structure-cytotoxicity studies on bromine-containing cysteine S-conjugates and related compounds.

Glutathione and cysteine S-conjugates of several haloalkenes are nephrotoxic and cytotoxic. Chloroalkene-derived S-(1-chloroalkenyl)-L-cysteine conjugates, but not fluoroalkene-derived S-(2,2-dihalo-1,1-difluorethyl)-L-cysteine conjugates, are mutagenic in the Ames test, although both types of S-conjugates are cytotoxic and nephrotoxic. Recent studies showed that bromine-containing S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates are mutagenic in the Ames test, thus challenging the generalization that S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates are not mutagenic. Hence a series of bromine-containing and bromine-lacking S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates was prepared, and their mutagenicity was assessed in the Ames test with Salmonella typhimurium TA2638 as the test strain. In addition, several indices of cytotoxicity, including cytotoxicity in LLC-PK1 cells, induction of Ca2+ release from pig kidney mitochondria, and DNA double-strand breaks in LLC-PK1 cells, were measured. The bromine-containing S-conjugates S-(2-bromo-2-chloro-1,1-difluoroethyl)-L- cysteine (BCD-FC), S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine (BTFC), and S-(2,2-dibromo-1,1-difluoroethyl)-L-cysteine (DBDFC) were mutagenic in the Ames test, whereas S-(2-chloro-1,1,2-trifluorethyl)-L-cysteine (CTFC), S-(2,2-dichloro-1,1-difluoroethyl)-L-cysteine (DCDFC), and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFC), which lack bromine, were not. BCDFC, BTFC, CTFC, DBDFC, and TFC were cytotoxic in LLC-PK1 cells, and their cytotoxicity was blocked by the cysteine conjugate beta-lyase inhibitor (aminooxy)acetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

In VitroMethods for the Assessment of L-Cysteine Conjugate Toxicity

L-Cysteine conjugates are normally metabolised via N-acetylation to produce a mercapturic acid. However, a recently identified metabolic route (C-S lysis) may lead to the generation of an unstable thiol which has been demonstrated to be responsible for toxicity in various mammalian species.Human Chang liver cells were challenged with a number of established L-cysteine conjugates. The cellular toxicity of these compounds was determined using a range of assay procedures, which provided differing information, depending on the assay method used. These observations were then investigated in order to establish which system would provide the most reliable indication of C-S lyase toxicity and whether any information on the mechanism of action could be obtained by these assay methods.

Examination of the structure-toxicity relationships of [formula omitted] of halogenated alkenes and their corresponding mercapturic acids in rat renal tissue slices

Rat kidney slices were produced using a modified version of a mechanical tissue slicer. The slices were incubated with various concentrations of l-cysteine conjugates and mercapturic acids of halogenated alkenes in a submersion incubation system. The slices showed a time- and concentration-dependent toxicity to the nephrotoxic conjugates. The five l-cysteine conjugates tested: S-(1,2- dichlorovinyl)- l-cysteine (1,2-DCVC), S-(1,1,2,2- tetrafluoroethyl)- l-cysteine (TFEC), S-(1- chloro-1,2,2-trifluoroethyl)- l- cysteine (CTFEC), S-(1,1- dichloro-2,2- difluoroethyl)- l-cysteine (DCDFEC) and S-(1,1, dibromo-2,2- difluoroethyl)- l-cysteine (DBDFEC) were more toxic compared to the corresponding mercapturic acids. Comparing the in vitro toxicity data with the in vivo data for the same compounds results in similar ranking for the relative nephrotoxicity of the conjugates.

High-performance liquid chromatography-fluorescence assay of pyruvic acid to determine cysteine conjugate β-lyase activity: Application to S-1,2-dichlorovinyl- l-cysteine and S-2-benzothiazolyl- l-cysteine

An HPLC—fluorescence assay has been developed for the determination of the activity of rat renal cytosolic cysteine conjugate β-lyase. The method is based on isocratic HPLC separation and fluorescence detection of pyruvic acid, derivatized with o-phenylenediamine (OPD), and is shown to be rapid, specific, and very sensitive. The assay has been evaluated with two model substrates for rat renal cytosolic β-lyase, notably S-1,2-dichorovinyl- l-cysteine (DCVC) and S-2-benzothiazolyl- l-cysteine (BTC). Equimolar formation of pyruvic acid and 2-mercaptobenzothiazole, a chromophoric thiol, indicated that pyruvic acid formation actually reflects the β-elimination activity of β-lyase during the β-elimination of BTC. From this it follows that the pyruvic acid assay can be applied to the measurement of the β-elimination activity of this enzyme, independent of the presence of chromophoric groups or radiolabels in substrates. Due to the large linear range and the very high sensitivity of the present HPLC-fluorescence assay (detection limit, 7.5 pmol of pyruvic acid), both good and poor substrates of β-lyase can be measured. Enzyme kinetic data are presented for the model substrates BTC and DCVC and for four structurally related S-2,2-difluoroethyl- l-cysteine conjugates.

Kynurenine aminotransferase/human hepatic C-S lyase: preliminary structure-activity relationship studies

Partially purified human hepatic cytosolic and mitochondrial fractions have been investigated for evidence of C-S lyase (CSL) activity. CSL activity has been characterized with synthetic aliphatic and aromatic L-cysteine conjugates. Preliminary structure-activity relationship studies have shown that aliphatic and aromatic L-cysteine conjugates are substrates. Partially purified human hepatic cytosolic and mitochondrial fractions have been investigated for evidence of C-S lyase (CSL) activity. CSL activity has been characterized with synthetic aliphatic and aromatic L-cysteine conjugates. Preliminary structure-activity relationship studies have shown that aliphatic and aromatic L-cysteine conjugates are substrates.

Glutathione conjugate mediated toxicities

Glutathione (γ-glutamyl- l-cysteinylglycine; GSH) is present in high concentrations in most living cells and participates in a variety of vital cellular reactions. In particular, GSH protects cells from potentially toxic electrophiles formed via the metabolism of xenobiotics, and such reactions have long been associated with the process of detoxication (Baumann and Preusse. 1879; Jaffe, 1879). Compounds that form GSH conjugates are processed by γ-glutamyl transpeptidase (γ-GT) and dipeptidases to cysteine S-conjugates, which are usually excreted in urine as their corresponding mercapturic acids ( S-substituted N-acetyl- l-cysteine conjugates). In addition. GSH peroxidase activity, whether catalyzed by the selenium-dependent GSH peroxidase or by the GSH S-transferases, serves to detoxify hydrogen peroxide and organic hydroperoxides. However, in recent years, evidence indicating that GSH conjugation plays an important role in the formation of toxic metabolites from a variety of chemicals has accumulated. Thus, several classes of compounds are converted, via conjugation with GSH, into either cytotoxic, genotoxic, or mutagenic metabolites. The purposes of the symposium on “Glutathione Conjugate Mediated Toxicities” presented at the 1990 Society of Toxicology Annual Meeting were to discuss recent findings in this rapidly moving field, to present ideas on the mechanisms and modulation of GSH conjugate-dependent toxicities, to present a consensus on the broader significance of this work, and to identify directions for future research. This paper summarizes these presentations. GSH conjugation reactions are involved in the bioactivation of several classes of xenobiotics, and four types of GSH-dependent bioactivation reactions can be identified: (1) directly toxic GSH conjugates may be formed from vicinal dihaloalkanes via formation of electrophilic sulfur mustards: (2) cysteine conjugate β-lyase-dependent bioactivation is involved in the selective nephrotoxicity of haloalkenes; (3) GSH conjugates of hydroquinones and isothiocyanates may serve as transport and targeting metabolites; and (4) GSH-dependent reactions may be involved in the release of toxic agents from precursor organic thiocyanates and nitrosoguanidines ( N-methyl- N′-nitro- N-nitrosguanidine).

Brain, kidney and liver 203Hg-methyl mercury uptake in the rat: relationship to the neutral amino acid carrier.

To investigate the effect of L-neutral amino acids on tissue levels of methyl mercury in the adult animal, rats were infused into the external jugular vein with solutions containing a) 0.05 mM 203Hg-MeHgCl and saline, b) 0.05 mM 203Hg-MgHgCl-0.1 mM L-cysteine, c) 0.05 mM 203Hg-MeHgCl-0.1 mM L-cysteine-0.1 mM L-cysteine-0.1 mM L-methionine, d) 0.05 mM 203Hg-MeHgCl-0.1 mM L-leucine, or e) 0.05 mM 203Hg-MeHgCl-0.1 mM L-cysteine-0.1 mM L-leucine, Groups of animals were sacrificed at 3 min. 7 hr, and 96 hr. Brain, kidney, and liver 203Hg radioactivity was measured by means of gamma-scintillation spectrometry. Brain 203Hg concentrations L-cysteine treated animals were significantly higher compared with saline treated animals (P less than 0.05) at 3 min., 7 hr and 96 hr. The coinjection or coinfusion of methyl mercury with L-cysteine and L-methionine abolished the L-cysteine-mediated brain 203Hg uptake (P less than 0.05), at each sacrifice time. Kidney and liver 203Hg concentrations were not significantly different in any of the treatment groups compared with controls, irrespective of the sacrifice time. Furthermore, the percentage of diffusible 203Hg (non-protein bound) at each sacrifice time was not statistically different irrespective of the treatment assigned. These results suggest that methyl mercury L-cysteine conjugates in the plasma may share a common transport step with the L-neutral amino acid carrier transport system and indicate the presence in brain capillaries of a transport system capable of selectively mediating methyl mercury uptake across the capillary endothelial cell membrane.

Glutathione- and cysteine-mediated cytotoxicity of allyl and benzyl isothiocyanate

Allyl isothiocyanate has been reported to be a bladder carcinogen in male rats. On the other hand, benzyl isothiocyanate is an anti carcinogen. These contradicting properties led us to investigate the cytotoxicity of these compounds in RL-4 rat hepatocytes. Since conjugation with glutathione plays an important role in the metabolism of these isothiocyanates, the glutathione and l-cysteine derivatives were also tested for cytotoxicity (electron microscopy, trypan blue exclusion, cell attachment, and inhibition of cell division). Both types of conjugates caused considerable toxicity: allyl isothiocyanate conjugates gave effects comparable to the parent compound, but benzyl isothiocyanate was more toxic than its conjugates. Addition of excess glutathione (>4m m) to the free isothiocyanates as well as their conjugates abolished cytotoxicity up to the highest concentration tested (250 μ m). Addition of excess l-cysteine (5 to 20 m m) lowered the effects but did not abolish them. The reaction of thiols with isothiocyanates was readily reversible: 15 min after dissolving the conjugates in buffer, pH 7.4, an equilibrium was established in which 9 to 15% of the conjugates was converted to free isothiocyanate. Two hours after addition of 1 m m of the l-cysteine conjugates to medium containing 5 m m glutathione, 80% of the total conjugates was present as the glutathione derivatives. The glutathione conjugates were similarly converted to l-cysteine conjugates. Glutathione conjugates are not able to enter the cell, thus their toxicity is presumably due to the release of free isothiocyanate, and in the presence of excess glutathione no toxocity was observed. l-cysteine derivatives are able to cross presence of excess glutathione no toxicity was observed. l-cysteine derivatives are able to cross the cell membrane, thus excess l-cysteine diminishes cytotoxicity, since less free isothiocyanate is present outside the cells, but does not completely protect the cells. Glutathione and cysteine can be regarded as transporting agents for the isothiocyanates through the body. Initial detoxification can be followed by release of the reactive compound at some other site.