Kynurenine aminotransferase activity in human liver: identity with human hepatic C-S lyase activity and a physiological role for this enzyme

The C-S lyase enzymes are responsible for the generation of mutagenic and cytotoxic metabolites via aberrant drug-metabolising pathways in mammalian tissues. We have examined human hepatic cytosolic, mitochondrial and microsomal fractions for evidence of C-S lyase activity. The cytosolic enzyme was purified using fast protein liquid chromatography over FFQ Sepharose, Mono P and Superose 12. An homogeneous protein (monitored by SDS-PAGE) was obtained following purification, and an 11-fold increase in C-S lyase specific activity was observed. The molecular weight of the enzyme was found to be 37 kDa in denaturing conditions, 82.3 kDa in non-denaturing conditions, and the C-S lyase activity was shown to co-purify with kynurenine aminotransferase activity when the transaminase activity of the enzyme was examined with kynurenine as the substrate.     

Cellular energetics and glutathione status in NRK-52E cells: toxicological implications

Cellular energetics and redox status were evaluated in NRK-52E cells, a stable cell line derived from rat proximal tubules. To assess toxicological implications of these properties, susceptibility to apoptosis induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a well-known mitochondrial and renal cytotoxicant, was studied. Cells exhibited high activities of several glutathione (GSH)-dependent enzymes, including gamma-glutamylcysteine synthetase, GSH peroxidase, glutathione disulfide reductase, and GSH S-transferase, but very low activities of gamma-glutamyltransferase and alkaline phosphatase, consistent with a low content of brush-border microvilli. Uptake and total cellular accumulation of [14C]alpha-methylglucose was significantly higher when cells were exposed at the basolateral as compared to the brush-border membrane. Similarly, uptake of GSH was nearly 2-fold higher across the basolateral than the brush-border membrane. High activities of (Na(+)+K(+))-ATPase and malic dehydrogenase, but low activities of other mitochondrial enzymes, respiration, and transport of GSH and dicarboxylates into mitochondria were observed. Examination of mitochondrial density by confocal microscopy, using a fluorescent marker (MitoTracker Orange), indicated that NRK-52E cells contain a much lower content of mitochondria than rat renal proximal tubules in vivo. Incubation of cells with DCVC caused time- and concentration-dependent ATP depletion that was largely dependent on transport and bioactivation, as observed in the rat, on induction of apoptosis, and on morphological damage. Comparison with primary cultures of rat and human proximal tubular cells suggests that the NRK-52E cells are modestly less sensitive to DCVC. In most respects, however, NRK-52E cells exhibited functions similar to those of the rat renal proximal tubule in vivo.     

Renal injury and repair following S-1, 2 dichlorovinyl-L-cysteine administration to mice

S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a metabolite of a common environmental contaminant, trichloroethylene, is a selective proximal tubular nephrotoxicant. The objective of our study was to examine the dose-response relationship of renal injury and repair following DCVC administration. Male Swiss-Webster mice were injected with DCVC [15, 30, or 75 mg/kg ip in distilled water (10 ml/kg)] and the extent of nephrotoxicity and tissue repair was assessed over a 14-day period. The renal injury due to the low and medium doses of DCVC peaked at 36 and 72 h after dosing, respectively, and then regressed over time due to a timely and adequate tissue repair response. At the highest dose tissue repair was inhibited, thereby causing progression of renal injury, which led to acute renal failure and death of the mice. The possibility that compromised tissue repair was a result of the extensive nephrotoxic injury attendant to the high dose of DCVC was investigated via an equinephrotoxicity study in which separate groups of mice received 40 (LD40) and 75 (LD90) mg DCVC/kg, respectively. Bioactivation-based renal proximal tubular injury measured in these two groups over a time course was identical but there was a marked difference in mortality due to an early and robust tissue repair in the first group relative to the second group. These results support the concept that quantitative evaluation of renal tissue repair in parallel with injury is useful in the assessment of the likely toxic outcome associated with exposure to nephrotoxic drugs and toxicants.     

Toxic,halogenated cysteine S-conjugates and targeting of mitochondrial enzymes of energy metabolism

Several haloalkenes are metabolized in part to nephrotoxic cysteine S-conjugates; for example, trichloroethylene and tetrafluoroethylene are converted to S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC), respectively. Although DCVC-induced toxicity has been investigated since the 1950s, the toxicity of TFEC and other haloalkene-derived cysteine S-conjugates has been studied more recently. Some segments of the US population are exposed to haloalkenes either through drinking water or in the workplace. Therefore, it is important to define the toxicological consequences of such exposures. Most halogenated cysteine S-conjugates are metabolized by cysteine S-conjugate beta-lyases to pyruvate, ammonia, and an alpha-chloroenethiolate (with DCVC) or an alpha-difluoroalkylthiolate (with TFEC) that may eliminate halide to give a thioacyl halide, which reacts with epsilon-amino groups of lysine residues in proteins. Nine mammalian pyridoxal 5'-phosphate (PLP)-containing enzymes catalyze cysteine S-conjugate beta-lyase reactions, including mitochondrial aspartate aminotransferase (mitAspAT), and mitochondrial branched-chain amino acid aminotransferase (BCAT(m)). Most of the cysteine S-conjugate beta-lyases are syncatalytically inactivated. TFEC-induced toxicity is associated with covalent modification of several mitochondrial enzymes of energy metabolism. Interestingly, the alpha-ketoglutarate- and branched-chain alpha-keto acid dehydrogenase complexes (KGDHC and BCDHC), but not the pyruvate dehydrogenase complex (PDHC), are susceptible to inactivation. mitAspAT and BCAT(m) may form metabolons with KGDHC and BCDHC, respectively, but no PLP enzyme is known to associate with PDHC. Consequently, we hypothesize that not only do these metabolons facilitate substrate channeling, but they also facilitate toxicant channeling, thereby promoting the inactivation of proximate mitochondrial enzymes and the induction of mitochondrial dysfunction.     

Aminotransferase, L-amino acid oxidase and beta-lyase reactions involving L-cysteine S-conjugates found in allium extracts. Relevance to biological activity?

Several cysteine S-conjugates that occur in extracts of garlic and other plants of the allium family possess anti-oxidant properties, and many, including S-allyl-L-cysteine (SAC) and S-allylmercapto-L-cysteine (SAMC), are promising anti-cancer agents. To understand possible biochemical mechanisms contributing to the protective effects, the ability of selected allium-derived L-cysteine S-conjugates to undergo various enzyme-catalyzed transformations was investigated. SAC, SAMC, S-propylmercapto-L-cysteine and S-penta-1,3-dienylmercapto-L-cysteine were shown to be substrates of: (a) highly purified rat kidney glutamine transaminase K (GTK); (b) purified snake venom L-amino acid oxidase; and (c) a cysteine S-conjugate beta-lyase present in rat liver cytosol. S-Methylmercapto-L-cysteine was shown to be a substrate of GTK and L-amino acid oxidase, but not of the cysteine S-conjugate beta-lyase. Evidence is presented that a major enzyme responsible for the cysteine S-conjugate beta-lyase reactions in the rat liver cytosol is gamma-cystathionase. The possible role of gamma-cystathionase in generating sulfane sulfur from the disulfide-containing cysteine S-conjugates present in allium extracts, and the possible role of this sulfane sulfur in enzyme regulation, targeting of cancer cells and detoxification reactions is discussed. An interesting side finding of the present work is that rat liver mitochondria are more active than rat liver cytosol in catalyzing a cysteine S-conjugate beta-lyase reaction with the mitochondrial protoxicant S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) at physiological pH and at low substrate concentration.     

Human mitochondrial and cytosolic branched-chain aminotransferases are cysteine S-conjugate beta-lyases,but turnover leads to inactivation

The mitochondrial and cytosolic branched-chain aminotransferases (BCAT(m) and BCAT(c)) are homodimers in the fold type IV class of pyridoxal 5'-phosphate-containing enzymes that also contains D-amino acid aminotransferase and 4-amino-4-deoxychorismate lyase (a beta-lyase). Recombinant human BCAT(m) and BCAT(c) were shown to have beta-lyase activity toward three toxic cysteine S-conjugates [S-(1,1,2,2-tetrafluoroethyl)-L-cysteine, S-(1,2-dichlorovinyl)-L-cysteine, and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine] and toward beta-chloro-L-alanine. Human BCAT(m) is a much more effective beta-chloro-L-alanine beta-lyase than two aminotransferases (cytosolic and mitochondrial isozymes of aspartate aminotransferase) previously shown to possess this activity. BCAT(m), but not BCAT(c), also exhibits measurable beta-lyase activity toward a relatively bulky cysteine S-conjugate [benzothiazolyl-L-cysteine]. Benzothiazolyl-L-cysteine, however, inhibits the L-leucine-alpha-ketoglutarate transamination reaction catalyzed by both enzymes. Inhibition was more pronounced with BCAT(m). In the presence of beta-lyase substrates and alpha-ketoisocaproate (the alpha-keto acid analogue of leucine), no transamination could be detected. Therefore, with an amino acid containing a good leaving group in the beta position, beta-elimination is greatly preferred over transamination. Both BCAT isozymes are rapidly inactivated by the beta-lyase substrates. The ratio of turnover to inactivation per monomer in the presence of toxic halogenated cysteine S-conjugates is approximately 170-280 for BCAT(m) and approximately 40-50 for BCAT(c). Mitochondrial enzymes of energy metabolism are especially vulnerable to thioacylation and inactivation by the reactive fragment released from toxic, halogenated cysteine S-conjugates such as S-(1,1,2,2-tetrafluoroethyl)-L-cysteine. The present results suggest that BCAT isozymes may contribute to the mitochondrial toxicity of these compounds by providing thioacylating fragments, but inactivation of the BCAT isozymes might also block essential metabolic pathways.     

Transport of N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2)

N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine (Ac-DCVC) and S-(1,2-dichlorovinyl)-l-cysteine (DCVC) are the glutathione conjugation pathway metabolites of a common industrial contaminant and potent nephrotoxicant trichloroethylene (TCE). Ac-DCVC and DCVC are accumulated in the renal proximal tubule where they may be secreted into the urine by an unknown apical transporter(s). In this study, we explored the hypothesis that the apical transport of Ac-DCVC and/or DCVC may be mediated by the multidrug resistance associated protein 2 (Mrp2, ABCC2), which is known to mediate proximal tubular apical ATP-dependent transport of glutathione and numerous xenobiotics and endogenous substances conjugated with glutathione. Transport experiments using membrane vesicles prepared from mouse proximal tubule derived cells expressing mouse Mrp2 utilizing ATPase assay and direct measurements of Ac-DCVC/DCVC using liquid chromatography/tandem mass-spectrometry (LC/MS/MS) demonstrated that mouse Mrp2 mediates ATP-dependent transport of Ac-DCVC. Expression of mouse Mrp2 antisense mRNA significantly inhibited the vectorial basolateral to apical transport of Ac-DCVC but not DCVC in mouse proximal tubule derived cells endogenously expressing mouse Mrp2. The results suggest that Mrp2 may be involved in the renal secretion of Ac-DCVC.     

Role of tissue repair in survival from s-(1,2-dichlorovinyl)-L-cysteine-induced acute renal tubular necrosis in the mouse.

S-(1,2-Dichlorovinyl)-L-cysteine (DCVC), a model nephrotoxicant in mice, causes acute tubular necrosis and death at high doses. Our earlier studies revealed that renal tissue repair was critical for survival in mice with DCVC nephrotoxicity. The objective of this study was to investigate if increasing renal tissue repair could protect mice from the lethal outcome of DCVC. Male Swiss Webster (SW) mice were administered a low dose of DCVC (15 mg/kg, ip) 72 h before injection of a normally lethal dose of DCVC (75 mg/kg, ip); this resulted in 100% protection against the lethal effect of DCVC. Because DCVC caused approximately two fold decrease in cytosolic and mitochondrial beta-lyase activity, the possibility that DCVC protection may be caused by decreased bioactivation was examined. Mercuric chloride (HgCl2, 6 mg/kg), a nephrotoxicant with no effect on beta-lyase activity, was administered 96 h before a lethal dose of DCVC. This also resulted in 100% protection from the lethal effect of DCVC. In both studies total glutathione was unchanged at any time after the lethal dose of DCVC was administered, obviating the role of glutathione in protection. In both cases the augmented and sustained tissue repair induced by priming dose and documented by 3H-thymidine pulse labeling and immunocytochemistry for proliferating cell nuclear antigen resulted in 100% survival in spite of the extensive renal injury. These findings suggest that stimulation of renal tubular repair by the priming dose, through augmented cell division, and the resistance of new cells to mechanisms of progression of injury, underlies auto- and heteroprotection against DCVC. The molecular mechanisms may have potential application in pharmacotherapeutic intervention for treatment of acute renal failure.     

Diabetic mice are protected from normally lethal nephrotoxicity of S-1,2-dichlorovinyl-L-cysteine (DCVC): role of nephrogenic tissue repair

Streptozotocin (STZ)-induced diabetic (DB) rats are protected from nephrotoxicity of gentamicin, cisplatin and mercuric chloride, although the mechanisms remain unclear. Ninety percent of DB mice receiving a LD90 dose (75 mg/kg, ip) of S-1,2-dichlorovinyl-l-cysteine (DCVC) survived in contrast to only 10% of the nondiabetic (NDB) mice surviving the same dose. We tested the hypothesis that the mechanism of protection is upregulated tissue repair. In the NDB mice, DCVC produced steep temporal increases in blood urea nitrogen (BUN) and plasma creatinine, which were associated with proximal tubular cell (PTC) necrosis, acute renal failure (ARF), and death within 48 h. In contrast, in the DB mice, BUN and creatinine increased less steeply, declining after 36 h to completely resolve by 96 h. HPLC analysis of plasma and urine revealed that DB did not alter the toxicokinetics of DCVC. Furthermore, activity of renal cysteine conjugate beta-lyase, the enzyme that bio-activates DCVC, was unaltered in DB mice, undermining the possibility of lower bioactivation of DCVC leading to lower injury. [3H]-thymidine pulse labeling and PCNA analysis indicated an early onset and sustained nephrogenic tissue repair in DCVC-treated DB mice. BRDU immunohistochemistry revealed a fourfold increase in the number of cells in S-phase in the DB kidneys even without exposure to DCVC. Blocking the entry of cells into S-phase by antimitotic intervention using colchicine abolished stimulated nephrogenic tissue repair and nephro-protection. These findings suggest that pre-placement of S-phase cells in the kidney due to diabetes is critical in mitigating the progression of DCVC-initiated renal injury by upregulation of tissue repair, leading to survival of the DB mice by avoiding acute renal failure.