Formation of S-[2-(N7-guanyl)ethyl] adducts by the postulated S-(2-chloroethyl)cysteinyl and S-(2-chloroethyl)glutathionyl conjugates of 1,2-dichloroethane

The formation of S-[2-(N7-guanyl)ethyl]glutathione (GEG) from dihaloethanes is postulated to occur through two intermediates: the S-(2-haloethyl)glutathione conjugate and the corresponding episulfonium ion. We report the formation of GEG when deoxyguanosine (dG) was incubated with chemically synthesized S-(2-chloroethyl)glutathione (CEG). The depurination of GEG was shown to be first order with a half-life of 7.4 +/- 0.4 h at 27 degrees C. Evidence is also presented for the formation of S-[2-(N7-guanyl)ethyl]-L-cysteine (GEC) in incubation mixtures containing dG and S-(2-chloroethyl)-L-cysteine (CEC), the corresponding cysteine conjugate of CEG. This finding demonstrates that this (haloethyl)cysteine conjugate does not require activation by enzymatic action of cysteine conjugate beta-lyase but, instead, can directly alkylate DNA. The half-life of the depurination of GEC was 6.5 +/- 0.9 h, which is no different from that of GEG. Of the two conjugates, CEC is a somewhat more active alkylating agent toward dG than CEG as N7-guanylic adduct was detected in reaction mixtures with lower concentrations of CEC than with CEG.     

Synthesis of an S-(2-Aminoethyl)-L-Cysteine Conjugate in S-(2-Aminoethyl)-L-Cysteine- Resistant Adenine-Auxotrophic Cells of Datura innoxia Mill

A line of S-(2-aminoethyl)-L-cysteine-resistant adenine-auxotrophiccells (Ad^–AEC^r strain) was isolated from adenine-auxotrophiccells (Ad^– strain) of Datura innoxia Mill by a stepwiseselection method. Ad^–AEC^r and Bl cells, which were clonedfrom the original Ad^–AEC^r cells, were able to grow activelyon medium that contained 10 mM S-(2-aminoethyl)-L-cysteine (AEC),whereas the growth of Ad^– cells ceased completely in thepresence of 0.5 mM AEC. The resistant phenotype has been maintainedfor at least 10 months in culture on medium without AEC. Levels of free lysine in Ad^–AEC^r and Bl cells were similarto that in Ad^– cells. By contrast, the level of free AECin Ad^–AEC cells was 10-fold lower than in Ad^– cellsand no free AEC was detectable in Bl cells. However, acid hydrolysisof extracts from Ad^–AEC^r and Bl cells resulted in a remarkableincrease in levels of detectable AEC. This result indicatesthat conjugated AEC is synthesized and accumulated in the AEC-resistantcells. The level of the AEC conjugate in Bl cells increasedwith increases in the concentration of AEC in the culture medium,while intracellular levels of AEC were so low as not to be detectablein the case of cells grown on medium supplemented with AEC atless than 1 mM. The AEC conjugate was also detected in Ad^–cells, but at lower levels than in the AEC-resistant cells.In addition, AEC was found to be incorporated into soluble proteinsin Ad^– cells. These results suggest that the resistance of AEC-resistant cellsof Datura innoxia is accomplished via acceleration of the synthesisof the AEC conjugate which prevents any increase in intracellularlevels of free AEC. ¹Present address: Institute for Biology and Chemistry, TsumuraCo.Ltd., Inashiki, Ibaraki, 300-03 Japan. ²Present address: North Kanto Shop, Sakata Seed Co. Ltd.,Saitama,347 Japan.     

Biosynthesis of S-(2-hydroxy-2-carboxyethylthio)-L-cysteine (3-mercaptolactate-cysteine disulfide) by the rat heart

Incubation of 3-mercaptopyruvate with rat heart homogenate resulted in the formation of S-(2-hydroxy-2-carboxy-ethylthio)-L-cysteine (HCETC, 3-mercaptolactate-cysteine disulfide), L-cysteine and 3-mercaptolactate with the concomitant decrease in glutamate and aspartate. These results indicate that a part of 3-mercaptopyruvate was converted to L-cysteine by transamination, a part was reduced to 3-mercaptolactate, and HCETC was formed from these two products. Another peak which corresponds to L-cysteine-glutathione disulfide on amino acid analysis was also produced during the incubation.     

S-(2-(acylamino)phenyl) 2,2-dimethylpropanethioates as CETP inhibitors

Studies on the relationship between the structure of the benzene moiety of S-(2-(acylamino)phenyl) 2,2-dimethylpropanethioates and CETP inhibitory activity were performed. Substituents on the benzene moiety influenced CETP inhibitory activity in a type and position dependent manner, and electron-withdrawing groups at the 4- or 5-position increased the activity. The most potent compound showed 50% inhibition of CETP activity in human plasma at a concentration of 2 microM.     

Metabolism of S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine to hydrogen sulfide and the role of hydrogen sulfide in S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine-induced mitochondrial toxicity

The nephrotoxic cysteine S-conjugate S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC) is metabolized by kidney homogenates and subcellular fractions to pyruvate and a reactive thiol, which is cytotoxic and partially decomposes to yield hydrogen sulfide and thiosulfate. Although hydrogen sulfide is a potent mitochondrial poison, the mitochondrial toxicity of CTFC is not attributable to hydrogen sulfide formation, as shown by different sites of inhibition of mitochondrial respiration by CTFC and hydrogen sulfide. The efficient mitochondrial oxidation of hydrogen sulfide apparently serves to protect mitochondria against the toxic effects of hydrogen sulfide generated from CTFC.     

芦笋抗S—（2—氨乙基）—L—半胱氨酸（AEC）变异体的筛选

S-(2-氨乙基)-L-半胱氨酸(AEC)可抑制芦笋愈伤组织的生长,此抑制作用可被赖氨酸或甲硫氨酸部分解除。用0.5mmol/L的AEC进行筛选,得到抗性愈伤组织AR10并再生植株。AR10愈伤组织经一年多的继代培养,在离开选择剂组培继代两代后仍保持对AEC的抗性。抗性系愈伤组织还表现出对2mmol/L的半胱氨酸具交叉抗性,对1mmol/L的赖氨酸加苏氨酸表现部分交叉抗性。AR10再生植株一部分保持对AEC的抗性,而一部分则无抗性。对抗性愈伤组织及其再生植株的氨基酸分析表明,愈伤组织内游离赖氨酸、苏氨酸、甲硫氨酸都有增加,而在再生植株内却发现半胱氨酸和赖氨酸的特异性增加,分别是对照植株的5.4和4.6倍。     

Thiiranium ion intermediates in the formation and reactions of S-(2-haloethyl)-l-cysteines

Thiiranium ions formed from the cysteine or glutathione conjugates of 1,2-dihaloethanes are believed to be responsible for the genotoxicity of the parent alkyl halides. The conversions of specifically deuterated β-hydroxyethyl sulfides to the corresponding β-haloethyl sulfides are studied to provide direct evidence for the involvement of thiiranium ions in the reactions of the cysteine conjugates of 1,2-dihaloethanes. S-(2-Hydroxyethyl-1, 1-d₂)-l-cysteine is converted to an equal mixture of the 1,1-d₂ and 2,2-d₂ isomers of the corresponding S-(2-haloethyl)-l-cysteines in concentrated hydrochloric, hydrobromic, or hydroiodic acids without detectable formation of the 2,2-d₂ isomer of the parent hydroxyethyl derivative. Dissolution of S-(2-hydroxyethyl)-l-cysteine in trifluoromethanesulfonic acid yields a compound with the NMR spectral properties of S-(l-cysteinyl)ethyl thiiranium trifluoromethanesulfonate. The organosoluble S-(2-hydroxyethyl-1,1-d₂) benzyl sulfide is converted to an equal mixture of the 1,1-d₂ and 2,2-d₂ isomers of S-(2-chloroethyl) benzyl sulfide by thionyl chloride or triphenylphosphine: carbon tetrachloride. These results demonstrate the involvement of thiiranium ion intermediates in the conversion of 2-hydroxyethyl sulfides to 2-haloethyl sulfides in halogen acids and a similar symmetrical intermediate in the chlorination reactions effected by thionyl chloride or triphenylphosphine: carbon tetrachloride.     

Synthesis and chromatographic properties of S-(1-carboxyethyl)-L-cysteine and S-(1-carboxypropyl)-L-cysteine

Details are reported for the synthesis of S-(1-carboxyethyl)-L-cysteine (1-CEC) and S-(1-carboxypropyl)-L-cysteine (1-CPC) from cysteine and 2-bromopropionic acid or 2-bromobutyric acid, respectively. Some analytical data and the behaviour of these two compounds on paper and ion-exchange chromatography are also reported, which allow their identification.     

Purification of S-2-hydroxyacylglutathione hydrolase (Glyoxalase II) from calf brain

S-2-Hydroxyacylglutathione hydrolase (Glyoxalase II) from calf brain has been purified 8333-times compared to 65,000 g supernatant of brain homogenate. The purification procedure employs Affi-Gel blue and preparative isoelectric focussing and offers a suitable method for the preparation of highly purified enzyme. Calf brain Glyoxalase II is a basic protein with a pl of 7.63 determined by isoelectric focusing. An evaluation of the relative molecular mass by gel filtration gave a value of about 23,000. During the purification procedure a constant Km value of about 0.325 mM was observed. A turnover number of 16,100 min-1 was calculated for the purified enzyme.     

Oxidative deamination of S-(1-carboxyethyl)-L-cysteine and S-(1-carboxypropyl)-L-cysteine by L-aminoacid oxidase

S-(1-carboxyethyl)-L-cysteine (1-CEC) and S-(1-carboxypropyl)-L-cysteine (1-CPC) are oxidatively deaminated by L-aminoacid oxidase with consumption of half a mole of oxygen per mole of substrate in the presence of catalase. This reaction gives rise to the corresponding alpha-ketoacids, identified by some chemical and chromatographic tests and by comparison with synthetic compounds. It has been possible, therefore, to demonstrate that S-(1-carboxyethyl)-thiopvruvic acid (1-CETP) and S-(1-carboxypropyl)-thiopvruvic acid (1-CPTP) are the main products of oxidative deamination of 1-CEC and 1-CPC.     

Comparative analysis of the behavior of gellan gum (S-60) and welan gum (S-130) in dilute aqueous solution

Optical rotation, circular dichroism, and microcalorimetric data clearly and consistently show that gellan gum, S-60 (Me₄N⁺ form), undergoes in water at 25° a rather sharp conformational transition upon increasing the concentration of added Me₄NCl. Similar data show that S-60 behaves anomalously upon addition of Ca²⁺ ions with, eventually, formation of aggregates and/or gels. The Me₄NCl-induced conformational change of S-60 is thermally reversible with no hysteresis. In contrast, with welan gum, S-130 (Me₄N⁺ form), no evidence could be found for a dependence of chain conformation of the main external variables considered. Comparison of the circular-dichroism spectra of the two polysaccharides suggests that S-130 in water might be present in a stiff conformation similar to that assumed by S-60 in aqueous Me₄NCl.     

Lysine excretion by S-(2-aminoethyl)L-cysteine resistant mutants of Bacillus subtilis

S-(2-Aminoethyl)L-cysteine (AEC) at 2 X 10(-1) mM concentration completely inhibited the growth of Bacillus subtilis. This inhibitory effect was readily reversed by 2 X 10(-2) mM L-lysine. Besides L-lysine, L-aspartic acid was only effective of all the natural amino acids tested in reversing the AEC-mediated growth inhibition. AEC resistant mutants of B. subtilis were isolated and found to excrete L-lysine in high yields.     

Purification of S-2-hydroxyacylglutathione hydrolase (glyoxalase II) from rat erythrocytes.

Glyoxalase II, a specific glutathione thiolesterase, has been purified 9100-fold from rat erythrocytes using a purification scheme which employs Affi-Gel blue as a hydrophobic affinity column and also employs a glutathione-affinity column prepared by coupling S-(p-chlorophenacyl)glutathione to Affi-Gel 202. This procedure offers a convenient method for the preparation of highly purified glyoxalase II. Also described is a convenient method for the preparation of S-lactoyl-glutathione, a substrate for glyoxalase II.     

Cytotoxicity of S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine in isolated rat kidney cells

S-(1,2-Dichlorovinyl)glutathione (DCVG) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) produced time- and concentration-dependent cell death in isolated rat kidney proximal tubular cells. AT-125 blocked and glycylglycine potentiated DCVG toxicity, indicating that metabolism by gamma-glutamyltransferase is required. S-(1,2-Dichlorovinyl)-L-cysteinylglycine, a putative metabolite of DCVG, also produced cell death, which was prevented by 1,10-phenanthroline, phenylalanylglycine, and aminooxyacetic acid, inhibitors of aminopeptidase M, cysteinylglycine dipeptidase, and cysteine conjugate beta-lyase, respectively. Aminooxyacetic acid and probenecid protected against DCVC toxicity, indicating a role for metabolism by cysteine conjugate beta-lyase and organic anion transport, respectively. DCVC produced a small decrease in cellular glutathione concentrations and did not change cellular glutathione disulfide concentrations or initiate lipid peroxidation. DCVC caused a large decrease in cellular glutamate and ATP concentrations with a parallel decrease in the total adenine nucleotide pool; these changes were partially prevented by aminooxyacetic acid. Both DCVG and DCVC inhibited succinate-dependent oxygen consumption, but DCVC had no effect when glutamate + malate or ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine were the electron donors. DCVC inhibited mitochondrial, but not microsomal, Ca2+ sequestration. These alterations in mitochondrial function were partially prevented by inhibition of DCVG and DCVC metabolism and were strongly correlated with decreases in cell viability, indicating that mitochondria may be the primary targets of nephrotoxic cysteine S-conjugates.