Rat Kidney Cytosol Research Articles

Glutathione S-transferases: two-dimensional electrophoretic protein markers of lead exposure.

Glutathione S-transferases (GST) are a family of detoxification isoenzymes that catalyze the conjugation of xenobiotics and their metabolites with reduced glutathione. Lead exposure in rats is known to induce GST isoenzymes in the liver and kidney. These changes in expression have potential use as biomarkers of lead exposure. Because two-dimensional electrophoresis (2-DE) enables one to analyze both protein abundance changes and chemical changes in protein structure, 2-DE was used to determine the effect of in vivo lead exposure on GST isoform expression in rat kidney cytosols. Male Sprague-Dawley rats were exposed to inorganic lead, and proteins were separated by conventional ISO-DALT and NEPHGE-DALT techniques and blotted for immunological identification. Lead exposure caused detectable inductions in both GSTP1 and GSTM1 and quantifiable charge modification in GSTP1. These preliminary data confirm the utility of 2-D electrophoretic GST analysis as indicative of lead exposure and toxicity and support its use for further elaboration of lead's effects on renal protein expression.

Alteration of acetohexamide reductase activities in kidney microsomes and cytosol of cadmium-treated rats

We examined the alteration of acetohexamide reductase activities in kidney microsomes and cytosol of cadmium (Cd)-treated rats. Acetohexamide reductase activity in kidney microsomes of male rats was markedly decreased by treatment with Cd at a dose of 1.23 mg/kg body weight. However, the decreased enzyme activity was completely restored by repeated treatment with testosterone propionate. Therefore, it is reasonable to assume that the treatment with Cd indirectly affect the androgen-dependent acetohexamide reductase activity in kidney microsomes of male rats, possibly by depressing androgen production. In the case of female rats, unlike male rats, the microsomal enzyme activity was little detectable, and was unaffected by the treatment with Cd. Furthermore, Cd treatment had no significant effect on acetohexamide reductase activity in kidney cytosol of male or female rats.

Purification of a heat-stable activator protein for ADP-ribosylation factor-dependent phospholipase D

A heat-stable activator for ADP-ribosylation factor (ARF)-dependent phospholipase D (PLD) was purified to near homogeneity from rat kidney cytosol by a sequential column chromatography. The purified activator has a molecular mass of 23 kDa on SDS-PAGE. Using a partially purified ARF-dependent PLD from rat kidney, the activator synergistically stimulates PLD with ARF in time- and dose-dependent manner. In the absence of ARF, the activator has little or no effect. The purified activator also stimulates PLD under several conditions including permeabilized cell system, suggesting that the activator is a physiologically relevant regulator of PLD.

Cysteine conjugate beta-lyase-dependent biotransformation of the cysteine S-conjugates of the sevoflurane degradation product 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A).

2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (1, Compound A) is a fluoroalkene formed by the base-catalyzed degradation of sevoflurane that is nephrotoxic in rats. Fluoroalkene 1 is a structural analog of other nephrotoxic haloalkenes that undergo glutathione S-conjugate formation and cysteine S-conjugate beta-lyase-dependent bioactivation to reactive intermediates. The present experiments were designed to study the beta-lyase-dependent biotransformation of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine (4) and S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine (5) by 19F NMR and UV spectroscopy and GC/MS. Incubation of cysteine S-conjugate 4 with rat kidney cytosol or a pyridoxal model system showed the formation of inorganic fluoride, pyruvate, and 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid (9), the expected products of a beta-lyase-catalyzed reaction. The ratio of fluoride to pyruvate ranged from 2.3 to 2.5. The amount of acid 9 formed in the rat kidney cytosol and the pyridoxal model system was, however, less than 5% of the amount of pyruvate formed. Incubation of conjugate 4 with rat kidney cytosol and analysis by 19F NMR spectroscopy showed resonances that were assigned to 3,3,3-trifluorolactic acid (10); the formation of acid 10 was observed in the pyridoxal model only after prolonged incubation (> 18 h). Lactic acid 10 was identified as a degradation product of acid 9. Cysteine S-conjugate 5 was not stable in pH 7.4 buffer and underwent a rapid cyclisation reaction (t1/2 approximately 5 min) to form 2-[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3-thiazol e-4 -carboxylic acid (14). These data show that fluoroalkene 1-derived cysteine S-conjugates are substrates for renal beta-lyase and that acid 9 is formed as a terminal product. Acid 9 is, however, unstable and affords lactic acid 10 as a degradation product.

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity in rat kidney cytosol.

The effect of regucalcin, a novel Ca2+-binding protein, on Ca2+/calmodulin-dependent cyclic adenosine monophosphate (AMP) phosphodiesterase activity in the cytosol of rat renal cortex was investigated. Regucalcin with physiologic concentration (10[-7] M) in rat kidney had no effect on cyclic AMP phosphodiesterase activity in the absence of CaCl2 and calmodulin. However, the activatory effect of both CaCl2 (10 microM) and calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was markedly inhibited by the addition of regucalcin (10[-8] to 10[-6] M) in the enzyme reaction mixture. The inhibitory effect of regucalcin on the enzyme activity was also seen in the presence of CaCl2 (5-50 microM) or calmodulin (5-50 U/ml) with increasing concentrations. The presence of trifluoperazine (10 microM), an antagonist of calmodulin, caused a partial inhibition of Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity. This inhibition was further enhanced by the addition of regucalcin (10[-7] M). The inhibitory effect of regucalcin (10[-7] M) was not seen in the presence of 20 microM trifluoperazine. Moreover, the activatory effect of calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was not entirely seen, when calmodulin was added 10 min after incubation in the presence of CaCl2 (10 microM) and regucalcin (10[-7] M). The present results demonstrates that regucalcin has an inhibitory effect on Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activation in the cytosol of rat renal cortex.

High affinity thyroid hormone-binding protein in human kidney: kinetic characterization and identification by photoaffinity labeling.

The binding of thyroid hormones and its regulation of NADPH and NADP+ were studied in human kidney cytosol, and a 38-kDa polypeptide (p38) was identified by photoaffinity labeling of cytosol with underivatized [125I]T3, SDS-PAGE, and autoradiography. The cytosolic thyroid hormone binding and p38 photolabeling were strongly activated by NADPH (maximum at 10(-7) M), whereas other nucleotides were less effective or ineffective. NADP+ did not activate T3 binding and p38 photolabeling, provided it was protected from conversion to NADPH by the addition of an exogenous oxidizing enzymatic system (oxidized glutathione plus glutathione reductase). Furthermore, NADP+ inhibited NADPH activation (half-maximum inhibitory effect at approximately 2 x 10(-5)M), and oxidation of NADPH to NADP+ induced dissociation of bound T3. The equilibrium dissociation constant (Kd) of the NADPH-activated cytosolic T3-binding sites was 0.3 nM, similar to the Kd of the nuclear T3 receptors. The kidney contained 200 times more cytosolic NADPH-activated thyroid hormone-binding sites than nuclear T3 receptors. Nonradioactive iodothyronines competed with [125I]T3 for both NADPH-activated binding and p38 photolabeling, with the following order of decreasing affinity: D-isomer of T3 > T3 > T4 > triiodothyroacetic acid > 3'-isopropyl-3,5-diiodothyronine > rT3. NADPH-activated T3 binding and photolabeled p38 were also detected in human heart and liver cytosols, but not in pancreas, cultured fibroblast and erythrocyte cytosols, or plasma. Rat kidney cytosol contained a 35-kDa photolabeled polypeptide homolog to human p38. The native molecular mass of the human photolabeled protein was 50 kDa, whereas that of the rat protein was 60 kDa, as determined by nondenaturing polyacrylamide gel electrophoresis. Two-dimensional PAGE of photolabeled p38 indicated an isoelectric point of 5.3. These findings describe the molecular properties of a NADPH/NADP+-regulated thyroid hormone-binding protein not previously identified in human and rat kidney cytosol.

Cloning and Expression of a Rat Kidney Cytosolic Glutamine Transaminase K That Has Strong Sequence Homology to Kynurenine Pyruvate Aminotransferase

An α-keto acid-dependent cysteineS-conjugate β-lyase of rat kidney was identified as cytosolic glutamine transaminase K by Stevenset al.(J. Biol. Chem.261, 15529–15537, 1986). Subsequently, a rat kidney protein with both glutamine transaminase K- and cysteineS-conjugate β-lyase activities was cloned and sequenced by Perryet al.(Mol. Pharmacol.43, 660–665, 1993). This protein was later shown to be identical with kynurenine pyruvate aminotransferase (Moscaet al., FEBS Lett.353, 21–24, 1994). Thus, kynurenine pyruvate aminotransferase possesses both glutamine transaminase K- and cysteineS-conjugate β-lyase-type activities. We have also cloned a cytosolic glutamine transaminase K from a rat kidney cDNA library and expressed the full-length clone in COS1 cells. The transfected cells exhibit marked increases in activities of both glutamine transaminase K and cysteineS-conjugate β-lyase. The enzyme cloned in the present work is a homodimer. Each subunit has a molecular mass of 45.8 kDa and contains 426 amino acid residues. The sequence of cytosolic glutamine transaminase K obtained in the present work has strong similarities to other aminotransferases, including >90% identity with kynurenine pyruvate aminotransferase. Our preparation of purified rat kidney cytosolic glutamine transaminase K possesses some kynurenine pyruvate aminotransferase activity. Thus, rat kidney cytosol possesses at least two distinct enzymes that catalyze glutamine transaminase K/cysteineS-conjugate β-lyase/kynurenine py- ruvate aminotransferase reactions. The present work underscores the difficulties associated with characterizing aminotransferases with overlapping specificities.

Stability study on renal type I mineralocorticoid receptor

The purpose of this work is to review stability and activation properties of type I receptor, in order to explain the reasons for its extreme in vitro instability. We demonstrate that the treatment of rat kidney cytosol with H 2O 2 prevents aldosterone binding, DNA/steroid-receptor complex interactions, and prevents the receptor thermal inactivation. In contrast, exogenous sulfhydryl reducing reagents are necessary to insure maximum binding of mineralocorticoid receptor and DNA/steroid-receptor interaction. However, the presence of β-mercaptoethanol in thermal induced incubations reverts the H 2O 2 protection. We also demonstrate that contaminations with free or sequestered iron are harmful for both, receptor binding capacity (in a reversible form) and for hormone-receptor /DNA binding properties (in a partially reversible form). We propose a sulfhydryl oxidative mechanism for type I mineralocorticoid receptor inactivation in which iron contaminants might accelerate this process by oxidative catalysis. We also demonstrate that when thiol groups are blocked by specific reagents such as N-ethyl-maleimide or dithionitrobenzoic acid, type I sites loose binding capacity, but the protein is protected from oxidation as well as inactivation.

Immunochemical Characterization of the ATP-Stimulated Glucocorticoid-Receptor-Translocation Promoter from Various Orga of Rat1

We previously found a novel endogenous factor in rat liver cytosol, named ATP-stimulated glucocorticoid-receptor-translocation promoter (ASTP), that increased the binding of activated glucocorticoid-receptor to nuclei in the presence of ATP. In this work, we immunized rabbits with the purified ASTP protein and characterized the antibodies with regard to titer, cross-reactivity and specificity. An IgG fraction from sera of the immunized rabbits contained specific antibodies to ASTP. The anti-ASTP IgG could precipitate the ASTP protein without the activity. Immunoblot analysis revealed a major band of 48 kDa in rat liver cytosol that migrated to the same position as the purified ASTP protein by SDS-PAGE, and an additional minor band of about 50 kDa. Monospecific antibodies purified from the IgG fraction using the antigen (the purified 48-kDa ASTP protein) immobilized on a polyvinylidene difluoride membrane also reacted with both the 48-kDa ASTP protein and the 50-kDa protein in rat liver cytosol, suggesting that this 50-kDa protein is immunologically related to the 48-kDa ASTP protein. Densitometric quantification of immunoblots demonstrated that the rat kidney cytosol contained ASTP protein at a concentration of about 20% of that of liver cytosol. Other tissues such as brain, skeletal muscle, heart, and lung, contained neither the ASTP protein nor the activity.

Demonstration of nuclear translocation of the mineralocorticoid receptor (MR) using an anti-MR antibody and confocal laser scanning microscopy.

Using a synthetic peptide that corresponds to a unique region of the N-terminal domain of the human mineralocorticoid receptor (MR), amino acids 87-96, we have generated a polyclonal antibody, human (h) MRsN. This sequence shares no homology with the corresponding sequences of the glucocorticoid receptor or other steroid/thyroid hormone receptor superfamily members. Antibody hMRsN cross-reacts with MR from human, rat, and mouse cells and recognizes denatured MR from either crude preparations or partially purified rat kidney cytosol, rat colon, or recombinant hMR overexpressed in baculovirus-infected Sf9 cells. Immunoprecipitation of the native MR from either partially purified or crude preparations of rat kidney cytosol with hMRsN, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver stain, demonstrated a major protein band with a mol wt of 116 kilodaltons. In addition, using confocal laser scanning microscopy and digital image analysis, the immunocytochemical localization of the recombinant hMR over-expressed in Sf9 cells 24 h post-transfection was determined. In the absence of ligand, the MR was detected solely in the cytoplasm, after a 30-min exposure to 100 nM aldosterone the MR was perinuclear, and after 60 min, the MR was predominantly nuclear. To ascertain that this phenomenon was not unique to insect cells, aldosterone induced MR nuclear translocation in mouse macrophage cells was also demonstrated immunocytochemically, clearly indicating a role for nuclear translocation in MR function.

Immunomodulation and drug acetylation: Influence of the immunomodulator tilorone on hepatic, renal and blood N-acetyltransferase activity and on hepatic cytosolic acetyl coenzyme a content

The biochemical alteration responsible for immunomodulator enhancement of drug acetylation in vivo was probed ex vivo and in vitro in the rat. Rat liver or kidney cytosol, obtained by differential centrifugation, or whole blood served as the source of N-acetyltransferase (NAT). Addition of tilorone (0.5–8.0mM) to incubation mixtures containing procainamide (PA, 0.6mM) and acetyl coenzyme A (AcCoA, 0.42 mM) resulted in the inhibition of N-acetylprocainamide formation, while lower concentrations of tilorone had no effect. Pretreatment of rats with tilorone (50 mg/kg) administered orally 48 hr prior to sacrifice did not alter hepatic apparent K m and V max for NAT toward PA compared to control animals. Utilization of an AcCoA regenerating system in the incubation mixtures also resulted in no significant differences in the apparent Michaelis-Menten parameters obtained. Acetylation activity in kidney and whole blood also was not altered by immunomodulator pretreatment. Hepatic cytosolic AcCoA content was reduced significantly 48 hr after tilorone pretreatment (5.10 ± 2.1 vs 11.97 ± 2.2 nmol/mg protein) (P < 0.05). These data indicate that an increase in NAT content or activity is not the biochemical alteration responsible for immunomodulator enhancement of drug acetylation, and that the required cofactor, cytosolic AcCoA, is decreased by immunomodulator pretreatment.

Differences between aldosterone and its antagonists in binding kinetics and ligand-induced hsp90 release from mineralocorticosteroid receptor

We have previously reported that mineralocorticosteroid receptor (MR) is a 8–9 S heterooligomeric complex that includes the 90 kDa heat shock protein (hsp90). To elucidate how antagonist-receptor complexes are biologically inactive in terms of transcriptional regulation, we analyzed the binding of mineralocorticosteroid agonists and antagonists with MR and the ligand-induced transformation of its heterooligomeric structure. This study was performed in the cytosol of adrenalectomized rat kidney and of COS cells transiently transfected with human MR cDNA. Although aldosterone antagonists (SC9420 and RU26752) bind MR with the same affinity as aldosterone, they dissociate much more rapidly from the 8–9 S form of both rat and human MR than does aldosterone. Using sedimentation gradient analysis, we showed that the interaction between hsp90 and the steroid binding subunit of MR is highly dependent upon the nature of the steroid ligand since the binding of aldosterone antagonists results in an easy release of hsp90. We propose that both rapid dissociation of ligand and weakened hsp90-receptor interaction play a key role in the mechanism of mineralocorticosteroid antagonism. In the COS cell model, cortisol, described as a weak mineralocorticosteroid agonist, dissociates also more rapidly from human MR than does aldosterone. Our results suggest that ligand binding kinetics and ligand dependent modification in receptor structure are important modulators of MR function as a transcriptional regulatory factor.

Catabolism of platelet‐activating factor and its acyl analog

Recent investigations have shown the presence of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine, i.e. the acyl analog of platelet-activating factor (PAF), in unstimulated tissues as well as its formation along with platelet-activating factor upon stimulation of a variety of cells. We demonstrate here that this acyl analog of PAF can be catabolized by purified lysophospholipases I and II from bovine liver with near stoichiometric formation of 2-acetyl-sn-glycero-3-phosphocholine. Lysophospholipase II also deacetylated PAF to lysoPAF and evidence is presented to show that this is an intrinsic activity of this enzyme. This suggested that some lysophospholipases may contribute to intracellular inactivation of PAF by deacetylation. Anion-exchange chromatography of rat liver cytosol confirmed this possibility. However, similar experiments with rat kidney cytosol and rat and human platelet cytosol clearly separated lysophospholipase activities without PAF acetylhydrolase activity from specific PAF acetylhydrolases not having lysophospholipase activity. Thus, lysophospholipases are clearly involved in the metabolism of the acyl analog of PAF and in some tissues, such as liver, may even contribute to abolishing the biological activity of PAF through deacetylation.

Primary structure and cellular distribution of two fatty acid-binding proteins in adult rat kidneys.

Fatty acid-binding proteins (FABPs) were purified from the kidneys of female and male rats and characterized by primary structure and histological distribution in the kidney. Two FABPs (14 and 15.5 kDa) were found in male rat kidney cytosol whereas only 14-kDa FABP could be recognized in female rat kidneys throughout the purification steps. The amino acid sequence of the 14-kDa FABP was identical to that of rat heart FABP deduced from the cDNA sequence (Heuckeroth, R. O., Birkenmeier, E. H., Levin, M. S., and Gordon, J. I. (1987) J. Biol. Chem. 262, 9709-9717). Structural analysis of the male-specific 15.5-kDa FABP identified this second FABP as a proteolytically modified form of alpha 2u-globulin, an 18.7-kDa major urinary protein of adult male rats (Unterman, R. D., Lynch, K. R., Nakhasi, H. L., dolan, K. P., Hamilton, J. W., Cohn, D. V., and Feigelson, P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3478-3482) which shares a common ancestry with a number of hydrophobic ligand-binding proteins such as serum retinol-binding proteins. Immunohistochemical investigation disclosed that heart-type FABP (14-kDa FABP) is localized in the cytoplasm of the epithelia of the distal tubules in both male and female rat kidneys whereas 15.5-kDa FABP immunostaining was observed predominantly in the endosomes or lysosomes of proximal tubules in male rat kidneys. These results suggest strongly the functional divergence of two FABPs in the rat kidney.

Design of a gas chromatograph with parallel radioactivity and mass spectrometric detection

A Perkin Elmer 3920 gas chromatograph, equipped with a versatile inlet system (i.e. an injector/trap), was interfaced to a radioactivity detector and a mass-selective detector (H/P 5970B) to identify 14C-labeled compounds. The use of a pre-trap as a demountable, programmable-temperature injector, in conjunction with the injector/trap, allowed the introduction of 0.5-ml samples of rat kidney cytosol extracts to 0.32 mm I.D. capillary columns. The instrumentation greatly facilitated the identification of the major radiolabeled metabolite of d-limonene associated with the male rat-specific protein alpha 2u-globulin as 1,2-cis-d-limonene oxide.

Separation of different forms of [formula omitted] synthetase by fast protein liquid chromatography

A method is described by which different forms of S- adenosylmethionine synthetase are separated. The method makes use of fast protein liquid chromatography on an anion exchange hydrophilic polyether resin. Different forms of S- adenosylmethionine synthetase from rat and human liver and kidney and rat zajdela hepatoma cells can be separated within 15 min. From a mixture of rat liver and kidney cytosols all three forms α, β and γ can be separated. The time needed to separate the different forms of S- adenosylmethionine synthetase is reduced from 3 h with conventional gel filtration methods, to 15 min using this HPLC anion-exchange method. Also the amount of tissue needed to detect the different forms is reduced from 125 mg to 12.5 mg of fresh rat liver tissue. These advantages make this newly developed method applicable when large numbers of samples have to be analyzed or when only small amounts of tissue are available.

Mineralocorticoid and Renal Receptor Binding Activity of 21-Deoxyaldosterone

Since several aldosterone metabolites are known to be active, we have assessed the mineralocorticoid biological and renal receptor binding activities of the aldosterone metabolites, 21-deoxyaldosterone (21-deoxy-Aldo), 21-deoxytetrahydroaldosterone (21-deoxy-THAldo), and 3 alpha, 5 beta-tetrahydroaldosterone (THAldo). We synthesized these steroids by bioreduction of aldosterone with intestinal bacteria. Mineralocorticoid agonist activity of 21-deoxy-Aldo, 21-deoxy-THAldo and THAldo, determined by bioassay using adrenalectomized rats, was 1-5%, less than 0.01%, and 0.1-0.5% that of aldosterone, respectively. 21-Deoxy-Aldo showed no antagonist activity. The relative affinity in competing with [3H]aldosterone for binding to mineralocorticoid receptors in adrenalectomized rat kidney cytosols was 94%, less than 0.01%, and less than 0.01% that of aldosterone. The relative binding affinity for rat renal glucocorticoid receptors was 23%, less than 0.01%, and less than 0.01% that of dexamethasone, and for corticosteroid-binding globulin 17%, less than 0.01%, and less than 0.01% that of cortisol. These results show that the naturally occurring steroid, 21-deoxy-Aldo, possesses mineralocorticoid agonist activity which is equivalent to that of 11-deoxycorticosterone, and has substantial affinity for rat renal mineralocorticoid and glucocorticoid receptors. The results also implicate the pathophysiological role of 21-deoxy-Aldo as a potential mineralocorticoid in 21-hydroxylase deficiency, where urinary excretion of this steroid is invariably elevated.

The monocytic differentiation of HL60 induced by rat kidney NADPH-linked high-Km aldehyde reductase protein

Abstract The human promyelocytic leukemia cell line HL60 differentiates to monocyte/macrophage cells when incubated with NADPH-linked high-Km aldehyde reductase (EC 1.1.1.2) purified from the cytosol of rat kidney. Differentiation was assessed by cell growth, morphology, adhesiveness, nitro blue tetrazolium reduction, and nonspecific esterase activity. The extent of differentiation induced by the reductase and measured at 4 days by nitro blue tetrazolium reduction is dose-dependent with an ED50 (dose required for half-maximal effect) of 71 nM. In the presence of 10 nM retinoic acid the ED50 for reductase is reduced to 18 nM and an isobologram analysis of this effect indicates that the combination is synergistic. Inactivation of the enzymatic activity is not associated with a decrease in differentiation-induced activity. These results suggest that the structure of the enzyme protein and not its enzymatic activity is involved with induction of differentiation. This view is supported by the demonstration that aldehyde reductase binds specifically to HL60 cells with a KD of 70 nM and that there are 13,000 binding sites/cell. Thus, the extent of differentiation induced by various concentrations of aldehyde reductase are directly related to the expected level of receptor occupancy.

Bioactivation mechanism of l-thiomorpholine-3-carboxylic acid

l-Thiomorpholine-3-carboxylic acid ( l-TMC) is a cyclized analog of S-(2-chloroethyl)- l-cysteine, which is cytotoxic in vitro and nephrotoxic in vivo. To determine whether l-TMC may play a role in S-(2-chloroethyl)- l-cysteine-induced toxicity, the cytotoxicity of l-TMC was studied in isolated rat kidney cells. l-TMC produced time- and concentration-dependent cytotoxicity. Probenecid, an inhibitor of the renal anion transport system, and l-α-hydroxyisocaproic acid, a substrate for l-amino acid oxidase, inhibited l-TMC-induced cytotoxicity. Rat kidney cytosol catalyzed the metabolism of l-TMC to a product absorbing at 300 nm. The increase in absorbance at 300 nm was accompanied by an increase in oxygen consumption and was inhibited by l-α-hydroxyisocaproic acid; moreover, the absorbance of the metabolite was quenched by addition of potassium cyanide or sodium borohydride, which indicated the formation of an imine. When l-TMC was incubated with rat kidney cytosol and sodium borodeuteride was added at the end of the incubation period, analysis by gas chromatography/mass spectrometry of the tert-butyldimethylsilyl ester of l-TMC showed the formation of [ 2H]TMC, indicating the intermediate formation of the imine 5,6-dihydro-2 H-1,4-thiazine-3-carboxylic acid; chemically synthesized TMC imine showed similar behavior. The enzyme responsible for the metabolism of l-TMC was purified from rat kidney and was identified as l-amino acid oxidase. These observations indicate a role for l-amino acid oxidase in the bioactivation and cytotoxicity of l-TMC.

Effects of the new nitrosourea derivative, fotemustine, on the glutathione reductase activity in rat tissues in vivo and in isolated rat hepatocytes

Fotemustine, a new clinically active nitrosourea, is demonstrated herein to be a poor inhibitor of glutathione reductase activity from rat liver, lung and kidney cytosols. In order to show that an intracellular step of activation does not lead to a toxic intermediary metabolite, rat hepatocytes were incubated with fotemustine. Their glutathione-related pathways were checked and shown not to be altered, while under similar experimental conditions BCNU was shown to be dramatically harmful. Furthermore, association of fotemustine with a H 2O 2 production leading drug, diquat, was shown to be inefficient—while BCNU is efficient—in potentiating the diquat toxicity. Considering the role of glutathione level in the detoxification of mutagens and carcinogens, the advantage of foemustine over BCNU in therapeutic use seems substantiated.