Rocket fuel for the quantification of S-nitrosothiols. Highly specific reduction of S-nitrosothiols to thiols by methylhydrazine.

Reduction of S-nitrosothiols to the corresponding thiol function is the key step in analyzing S-nitrosocysteinyl residues in proteins. Though it has been shown to give low yields, ascorbate-dependent reduction is commonly performed in the frequently used biotin-switch technique. We demonstrate that the compound methylhydrazine can act as a specific and efficient reducing agent for S-nitrosothiols. The corresponding thiol function is exclusively generated from low molecular weight and proteinaceous S-nitrosothiols while methylhydrazine failed to reduce disulfides. It was possible to optimize the experimental conditions so that thiol autoxidation is excluded, and high reaction yields (>90%) are obtained for the thiol function. The biotin-switch technique performed with methylhydrazine-dependent reduction shows remarkably improved sensitivity compared to the ascorbate-dependent procedure.

Inhibition of carnitine acetyltransferase by mildronate, a regulator of energy metabolism.

Carnitine acetyltransferase (CrAT; EC 2.3.1.7) catalyzes the reversible transfer of acetyl groups between acetyl-coenzyme A (acetyl-CoA) and L-carnitine; it also regulates the cellular pool of CoA and the availability of activated acetyl groups. In this study, biochemical measurements, saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, and molecular docking were applied to give insights into the CrAT binding of a synthetic inhibitor, the cardioprotective drug mildronate (3-(2,2,2-trimethylhydrazinium)-propionate). The obtained results show that mildronate inhibits CrAT in a competitive manner through binding to the carnitine binding site, not the acetyl-CoA binding site. The bound conformation of mildronate closely resembles that of carnitine except for the orientation of the trimethylammonium group, which in the mildronate molecule is exposed to the solvent. The dissociation constant of the mildronate CrAT complex is approximately 0.1 mM, and the K(i) is 1.6 mM. The results suggest that the cardioprotective effect of mildronate might be partially mediated by CrAT inhibition and concomitant regulation of cellular energy metabolism pathways.

The carnitine transporter SLC22A5 is not a general drug transporter, but it efficiently translocates mildronate.

In addition to its function as carnitine transporter, novel organic cation transporter type 2 (OCTN2; human gene symbol SLC22A5) is widely recognized as a transporter of drugs. This notion is based on several reports of direct measurement of drug accumulation. However, a rigorous, comparative, and comprehensive analysis of transport efficiency of OCTN2 has not been available so far. In the present study, OCTN2 orthologs from human, rat, and chicken were expressed in 293 cells using an inducible expression system. Uptake of trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (ASP(+)), cephaloridine, ergothioneine, gabapentin, mildronate, pyrilamine, quinidine, spironolactone, tetraethylammonium, verapamil, and vigabatrin was determined by liquid chromatography/mass spectrometry. For reference, uptake of carnitine was measured in parallel. Our results indicate that OCTN2-mediated uptake of drugs was not significantly different from zero or, with tetraethylammonium and ergothioneine, was minute relative to carnitine. The carnitine congener mildronate, by contrast, was transported very efficiently. Thus, OCTN2 is not a general drug transporter but a highly specific carrier for carnitine and closely related molecules. Transport parameters (cellular accumulation, transporter affinity, sodium dependence) were similar for mildronate and carnitine. Efficiency of transport of mildronate was even higher than that of carnitine. Hence, our results establish that OCTN2 is a key target of the cardioprotective agent mildronate because it controls, as integral protein of the plasma membrane, cellular entry of mildronate and enables efficient access to intracellular targets. The highest levels of human OCTN2 mRNA were detected by real-time reverse transcription-polymerase chain reaction in kidney, ileum, breast, small intestine, skeletal muscle, and ovary but also in some heart and central nervous system tissues.

Interaction of mildronate with the mitochondrial carnitine/acylcarnitine transport protein.

The interaction of mildronate [3-(2,2,2-trimethylhydrazine) propionate] with the purified mitochondrial carnitine/acylcarnitine transporter reconstituted in liposomes has been studied. Mildronate, externally added to the proteoliposomes, strongly inhibited the carnitine/carnitine antiport catalyzed by the reconstituted transporter with an IC(50) of 560 muM. A kinetic analysis revealed that the inhibition is completely competitive, that is, mildronate interacts with the substrate-binding site. The half-saturation constant of the transporter for external mildronate (K(i)) is 530 muM. Carnitine/mildronate antiport has been measured as [(3)H]carnitine uptake into proteoliposomes containing internal mildronate or as [(3)H]carnitine efflux from proteoliposomes in the presence of external mildronate, indicating that mildronate is transported by the carnitine/acylcarnitine transporter and that the inhibition observed was due to the transport of mildronate in the place of carnitine. The intraliposomal half-saturation constant for mildronate transport (K(m)) has been determined. Its value, 18 mM, is much higher than the external half-saturation constant (K(i)) in agreement with the asymmetric properties of the transporter. In vivo, the antiport reaction between cytosolic (administered) mildronate and matrix carnitine may cause intramitochondrial carnitine depletion. This effect, together with the inhibition of the physiological transport, will lead to impairment of fatty acid utilization.

Pharmacokinetics and biological fate of 3-(2,2, 2-trimethylhydrazinium)propionate dihydrate (MET-88), a novel cardioprotective agent, in rats.

In this study, we examined the disposition, metabolism, and excretion of a novel cardioprotective agent, 3-(2,2, 2-trimethylhydrazinium)propionate dihydrate (MET-88), in rats. The disposition of MET-88 after oral and i.v. administration of 2, 20, and 60 mg/kg indicated that the pharmacokinetics of MET-88 were nonlinear. The profiles of radioactive MET-88 and total radioactivity in plasma were consistent at doses of 20 and 60 mg/kg. However, at 2 mg/kg, the plasma MET-88 levels were obviously lower than the total. The excretion of radioactivity after oral administration of MET-88 indicated that increasing doses led to a shift from exhaled CO(2) to urinary excretion as the major excretion route. Major metabolites in plasma after oral administration of MET-88 were glucose, succinic acid, and 3-hydroxypropionic acid, and in vitro studies revealed that MET-88 was converted to 3-hydroxypropionic acid by gamma-butyrobetaine hydroxylase (EC 1.14. 11.1). An isolated liver perfusion system modified to trap CO(2) gas was used to examine the excretion pathway of MET-88. [(14)C]CO(2) gas was decreased by the addition of iodoacetic acid, DL-fluorocitric acid, or gamma-butyrobetaine to this system, and subsequent thin-layer chromatography analyses of perfusates revealed that MET-88 was first converted to 3-hydroxypropionic acid by gamma-butyrobetaine hydroxylase and then was biosynthesized to glucose and metabolized to CO(2) gas via the glycolytic pathway and tricarboxylic acid cycle.

Batch culture biodegradation of methylhydrazine contaminated NASA wastewater.

The batch culture degradation of NASA wastewater containing mixtures of citric acid, methylhydrazine, and their reaction product was studied. The organic contaminants present in the NASA wastewater were degraded by Achromobacter sp., Rhodococcus B30 and Rhodococcus J10. While the Achromobacter sp. showed a preference for the degradation of the citric acid, the Rhodococcus species were most effective in reducing the methylhydrazine and the reaction product. Removals of more than 50% were observed for citric acid, methylhydrazine and the reaction product when the NASA wastewater was inoculated with the microbes in batch cultures. Simulation and chemical characterization of citric acid and hydrazine mixtures show that the interaction is partly of a chemical nature and leads to the formation of a conjugated UV/Visible absorbing compound. An 'azo' carbonyl derivative of the citric acid, consistent with the spectral data obtained from the investigation, has been proposed as the possible product.

Purification and characterization of the rat liver gamma-butyrobetaine hydroxylase.

The biosynthesis of carnitine from lysine and methionine involves five enzymatic reactions. Gamma-butyrobetaine hydroxylase (BBH; EC 1.14.11.1) is the last enzyme of this pathway. It catalyzes the reaction of hydroxylation of gamma-butyrobetaine to carnitine. This enzyme had never been purified to homogeneity from rat tissue. This paper describes the purification and characterization of the rat liver BBH. This protein has been purified some 413 fold by ion exchange, affinity and gel-filtration chromatographies and appears as a dimere of 43,000 Daltons subunits by PAGE. The affinity chromatography column used in the purification process utilizes 3-(2,2,2-trimethylhydrazinium)propionate (THP), a BBH inhibitor, as the ligand. Polyclonal antibodies were raised against the liver enzyme. They were able to precipitate BBH activity in either a crude liver extract or a purified fraction of the enzyme. Furthermore, it crossreacts with a 43 kDa protein in the liver. No evidence for extra hepatic enzyme was found.

[Comparative study of the antioxidant activity of gamma-butyrobetaine and its natural and synthetic derivatives in vitro]. / Sravnitel'naia otsenka antioksidantnol aktivnosti gamma-butirobetaina i ego prirodnykh i sinteticheskikh proizvodnykh in vitro.

Experiments in vitro with the use of a rat brain homogenate demonstrated antioxidant activity of methyl ether (3(2,2,2-trimethylhydrazinium) propionate possessing a positive charge at the quaternary nitrogen atom. The antioxidant activity was due to intensified neutralization of superoxide anion radicals and, correspondingly, inhibited peroxidation of endogenous brain lipids. The other compounds under study, namely, gamma-butyrobetaine, its methyl ether, and 3(2,2,2-trimethylhydrazine) propionate did not exhibit antioxidant properties.

Magnetic polyethyleneimine (PEI) microcapsules as retrievable traps for carcinogen electrophiles formed in the gastrointestinal tract.

Semi-permeable magnetic microcapsules containing polyethyleneimine (PEI) have been developed as retrievable carcinogen traps. In vitro, the soluble core PEI and membrane both bound reactive substances of limited aqueous stability, such as from [14C]N-methyl-N-nitrosourea ([14C]NMU), and aqueous stable dyes of molecular weight up to 1000. The core/membrane location ratio of binding was dependent upon membrane characteristics of the microcapsule batch used. Microcapsules administered intragastrically to rats bound up to 0.006% of [14C]dimethylhydrazine ([14C]DMH) and 1.4% of [14C]NMU administered i.p. or intrarectally, respectively. Time-dependency of [14C]DMH binding was consistent with labelling of microcapsules within the small intestine. There were no detectable metabolites from [14C]DMH trapped within the colon, whereas binding of [14C]NMU indicated that microcapsules could bind transient species present within the colon in competition with the faecal bulk. These results indicate that this approach could be used to detect highly unstable and possibly genotoxic substances in situ, hitherto unknown, formed within the intestinal lumen.

Organ-specific effects of 1,2-dimethylhydrazine in hamster.

Uptake and metabolism of the carcinogen 1,2-dimethylhydrazine (DMH) were compared in isolated epithelial cells from the colon and the small intestine. A new method was developed to separate colonic epithelial cells into surface columnar cells and crypt cells without the use of any proteolytic enzymes. Colonic columnar cell-enriched fraction exhibited DMH metabolism two to three times higher than that of crypt cells. The carcinogen binding was much lower in the small intestine as compared to the colon. In the small intestine, the crypt cell-enriched fraction showed higher carcinogen binding as compared to villus cells. Pyrazole was found to inhibit DMH binding by isolated small intestinal and colonic epithelial cells. The extent of inhibition was maximum in cells showing the greatest ability to incorporate DMH.

Effect of dietary selenium on biotransformation and excretion of mutagenic metabolites of N-nitrosodimethylamine and 1,1-dimethylhydrazine in the liver perfusion/cell culture system.

The mutagenicity of N-nitrosodimethylamine (NDMA) and 1,1-dimethylhydrazine (UDMH) has been studied in an isolated liver perfusion/cell culture system. The liver donors, male Wistar rats, were either selenium (Se)-deficient or had a physiologically adequate Se status (Se-supplemented). Mutagenicity was measured in perfusate and bile with Chinese hamster V79 cells as the genetic target. Se deficiency increased the mutagenic effect of NDMA in the perfusate, whereas no mutagenicity was detected in the bile of either Se-deficient or Se-supplemented livers. No significant increase in the mutagenicity of UDMH was seen in the perfusate with Se deficiency, but the bile became mutagenic. Se deficiency thus increased the mutagenicity of both NDMA and UDMH: with NDMA, the effect was observed in the perfusate, and with UDMH, in the bile.

Enzymatic activation of the carcinogens 2-phenylethylhydrazine and 1,2-dimethylhydrazine to carbon-centered radicals.

Spin-trapping experiments demonstrate that oxidation of 1,2-dimethylhydrazine and 2-phenylethylhydrazine generates a comparable yield of carbon-centered radicals when catalyzed by horseradish peroxidase - H2O2. Using oxyhemoglobin as the catalyst, 2-phenylethylhydrazine oxidation generates ten times more carbon-centered radicals than 1,2-dimethylhydrazine oxidation. This result is in agreement with oxygen consumption studies from which the apparent KM values of 8.0 mM and 72 mM were calculated for the oxyhemoglobin-catalyzed oxidation of 2-phenylethylhydrazine and 1,2-dimethylhydrazine, respectively. These differences in metabolic activation of mono- and disubstituted hydrazines may be of importance regarding the carcinogenic properties of these derivatives.

Enzymatic activation of the carcinogens 2 phenylethylhydrazine and 1,2 dimethylhydrazine to carbon centered radicals

Spin-trapping experiments demonstrate that oxidation of 1,2-dimethylhydrazine and 2-phenylethylgydrazine generates a comparable yield of carbon-centered radicals when catalyzed by horseradish peroxidase-H2O2. Using oxyhemoglobin as the catalyst, 2-phenylethylgydrazine oxidation generates ten times carbon-centered radicals than 1,2-dimethylhidrazine oxidation. This results is in agreement with oxygen consumption studies from which the apparent KM values of 8.0 mM and 72 mM were calculated for the oxyhemoglobin-catalyzed oxidation of 2-phenylethylhydrazine and 1,2-dimethylhydrazine, respectively. These differences in metabolic activation of mono- and disubstituted hydrazines may be of importance regarding the carcinogenic properties of these derivatives

In vivo formation of sigma-methyl- and sigma-phenyl-ferric complexes of hemoglobin and liver-cytochrome P-450 upon treatment of rats with methyl- and phenylhydrazine.

Ferric sigma-phenyl complexes of hemoglobin and liver cytochrome P-450 are formed in vivo upon administration of C6H5NHNH2 to rats. Small amounts of the sigma-methyl complex of hemoglobin were also detected in vivo upon treatment of rats with CH3NHNH2. At the doses used for CH3NHNH2 (25 and 50 mg/kg) the states and levels of hemoglobin in the blood and spleen, and of cytochrome P-450 in the liver were almost unchanged. On the contrary, C6H5NHNH2 (25-100 mg/kg) led to a decrease of the HbO2 blood level (10-50%), together with an increase in the HbFe(III) level and the appearance of the HbFe(III)-C6H5 complex. The concentration of this complex reaches its maximum value (2 mM) 1 h after C6H5NHNH2 administration (20% of total hemoglobin). At the same time large amounts of HbO2, HbFe(III) and HbFe(III)-C6H5 appeared in the spleen, and remained high up to 24 h after treatment. Treatment of rats with C6H5NHNH2 (25-100 mg/kg) led to a significant decrease in the level of liver cytochrome P-450 (a 70% decrease 2 h after treatment with 100 mg/kg C6H5NHNH2). About 15% of the remaining cytochrome P-450 existed as a cyt.-P-450-Fe(III)-C6H5 complex, a new example of cytochrome P-450-Fe-metabolite complex which is stable in vivo.

Activation of the colon carcinogen 1,2-dimethylhydrazine in a rat colon cell-mediated mutagenesis assay.

Suspensions of rat colon epithelial cells metabolized the potent colon carcinogen, 1,2-[14C]dimethylhydrazine (DMH), into 14C-labeled, alkali-soluble volatile products, presumably CO2. The colon cell suspensions, however, were less effective than rat hepatocyte suspensions. In addition, we used a cell-mediated mutagenesis assay to test rat colon epithelial cells grown from tissue explants for their ability to metabolize DMH into products mutagenic for human P3 teratoma cells. Mutagenesis in the P3 cells was indicated by an acquired resistance to 6-thioguanine. Cocultivation of the colon cells with the P3 cells in the cell-mediated assay resulted in mutagenesis, whereas in the absence of the colon cells, no mutagenesis by DMH was observed. Similar results were obtained in a hepatocyte-mediated mutagenesis assay. Colon cells were also able to activate another carcinogen, benzo(a)pyrene, into products mutagenic for the P3 cells. Individual epithelial clonal populations isolated from the colon cultures grown from tissue explants, however, expressed different capacities to activate DMH and benzo(a)pyrene into mutagens, and a high degree of DMH activation by cells from a colon clone was not necessarily associated with a similar degree of benzo(a)pyrene activation. Our results indicate that the colon itself contains epithelial cell types capable of effectively converting DMH into mutagenic (and presumably carcinogenic) products without necessarily involving intermediary metabolism by hepatocytes as previously thought.

Effect of inhibitors of the FAD-containing monooxygenase system from rat liver microsomes on monomethylhydrazine metabolism and activation to reactive metabolites.

Several inhibitors of the FAD-containing monooxygenase (FAD-MO) system from rat liver microsomes (imipramine, chlorpromazine, mercaptoethylamine, dithiothreitol, naphthylthiourea, phenylthiocarbamide) and one inhibitor of the liver microsomal cytochrome P-450 (P-450)-mediated biotransformations (SKF 525 A), were tested as possible inhibitors of monomethylhydrazine (MMH) biotransformation to CO2 and to reactive metabolites that bind covalently to nucleic acids and proteins. Results confirm previous suggestions that both FAD-MO and P-450 are involved in MMH metabolism to CO2 and suggest a similar participation of both systems for production of reactive metabolites interacting with macromolecules.

[Effect of sex hormones on the 1,2-dimethylhydrazine metabolic activity in the kidneys of CBA-strain mice]. / Vliianie polovykh gormonov na aktivnost' metabolizma 1,2-dimetilgidrazina v pochkakh myshei linii CBA.

1,2-dimethylhydrazine (DMH) metabolizing activity of kidney microsomes was shown to be two times higher in male, than in female CBA mice. Castration decreased DMH metabolizing activity of male kidney microsomes to the females' level. DMH metabolizing activity of castrated males treated with testosterone propionate was identical to that of intact males. The incorporation of 14C-DMH into kidney DNA was also higher in male, than in female CBA mice.

In vitro DNA and nuclear proteins alkylation by 1,2-dimethylhydrazine.

The methylating carcinogen 1,2-dimethylhydrazine (DMH) CAS 540.73.8 is highly organ-specific and, under certain experimental conditions, produces a high incidence of adenocarcinoma in the colon of rodents. We have tried to assess the possibility that part of the organ-specifity in the carcinogenic effect of DMH could be attributed to its metabolism by specific microsomal enzymes. In particular, we compared the in vitro effects of DMH in the presence of either colon or liver microsomes from animals that had been treated with microsomal enzyme inducers. V79 Chinese hamster cells were used as the target to evaluate the damage to the genetic material, as judged by (1) formation of adducts of DNA bases and (2) amino acid modifications in nuclear proteins using [Me-14C]DMH and appropriate analytical detection systems. Our results tend to support the above postulated hypothesis.