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.

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.

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.

Metabolism and activation of 1,1-dimethylhydrazine and methylhydrazine, two products of nitrosodimethylamine reductive biotransformation, in rats.

Nitrosodimethylamine (DMN) and two of its metabolites, methylhydrazine (MH) and 1,1-dimethylhydrazine (UDMH), were metabolized to CO2 by liver slices obtained from Sprague-Dawley rats. Under the conditions used, DMN and MH produced reactive metabolites that bound covalently to nucleic acids, but UDMH did not. Rat liver microsomes or 9,000 X g supernatants were able to transform DMN, MH, and UDMH to CH2O. In the cases of MH and UDMH, enzymatic and nonenzymatic pathways of CH2O formation were observed in both liver microsomes and 9,000 X g supernatants. DMN, MH, and UDMH led to covalent binding (CB) to proteins in incubation mixtures containing either microsomes or 9,000 X g supernatants. In the case of DMN, the process was enzymatic and required NADPH in both cellular fractions. In the case of MH, the process was enzymatic in microsomes and required NADPH and O2. With UDMH or MH and 9,000 X g supernatants, nonenzymatic interactions resulting in CB to proteins dominated. All these results suggest that part of the CO2 produced during DMN metabolism might be derived from UDMH and MH. Similarly, a significant part of the CB of DMN metabolites to proteins in incubation mixtures containing microsomes or 9,000 X g supernatants might be derived from enzymatic and nonenzymatic reactions of UDMH or MH. Also, a minor part of the CB of DMN-reactive metabolites to nucleic acids might have resulted from MH's further biotransformation to reactive metabolites. Overall, biotransformation of DMN and MH might not be a detoxication process, as previously thought, but one related to some of the DMN toxic effects.

Bile acids: effects on absorption of 1,2-dimethylhydrazine and 7,12-dimethylbenz[a]anthracene in the colon of the rat and guinea pig.

The specific effects of bile acids as cocarcinogens were investigated. Absorptions of [14C]7,12-dimethylbenz[a]anthracene, [14C]dimethylhydrazine ([14C]DMH), and [3H]inulin from loops of colons from outbred Sprague-Dawley rats and Hartley guinea pigs were determined. In each animal absorption of one carcinogen and inulin was studied in one control loop and in an experimental loop containing either deoxycholic acid (DOC) or chenodeoxycholic acid (CDOC). DOC had a more pronounced effect on increasing loss of carcinogen from the intestinal lumen than did CDOC. This role of bile acids was consistent with their known effect of increasing intestinal permeability. Less carcinogen remained in the colon mucosa when DOC was present in the intestinal lumen. Although [14C]DMH was absorbed more rapidly from the intestinal lumina of guinea pigs than from those of rats, the rat accumulated more of the carcinogen in the intestinal mucosa and liver.

Effect of dietary protein concentration on yield of mutagenic metabolites from 1,2-dimethylhydrazine in mice.

The effects of varying dietary protein concentrations on the metabolism of 1,2-dimethylhydrazine (DMH) to mutagenic products by male C57BL/6 X C3H F mice were assayed by in vivo and in vitro methods. DMH and its metabolite, azoxymethane (AOM), did not increase the mutation frequency of Salmonella typhimurium (strain G-46) in vitro alone or in the presence of mouse liver homogenates capable of activating the promutagen dimethylnitrosamine. Methylazoxymethanol (MAM), another metabolite of DMH, was mutagenic in vitro without activation. S.c. administration of DMH, AOM, or MAM at dosages ranging from 0.2 to 0.8 mmol/kg of body weight caused dose-dependent increases in mutations of S. typhimurium in the host-mediated assay, and molar potencies increased progressively from DMH to AOM to MAM. S.c. or i.p. injections of AOM increased host-mediated mutagenesis within 20 min, while increases in mutagenesis by DMH required at least 1 hr. When [14C]DMH was administered, [14C]azomethane was expired immediately, while 14CO2 began to appear 1 hr after DMH administration. The percentage of administered [14C]DMH expired as azomethane varied inversely with dietary protein concentration, while AOM-induced host-mediated mutagenesis was directly proportional to dietary protein (p less than 0.01). The percentage of DMH converted to mutagenic end products was limited by losses of the volatile metabolite azomethane, especially in protein-deficient mice. Greater expiration of azomethane and decreased conversion of AOM to MAM, both seen with restriction of dietary protein, were associated with a smaller body burden of DMH metabolites.

Cell specificity in hepatocarcinogenesis: preferential accumulation of O6-methylguanine in target cell DNA during continuous exposure to rats to 1,2-dimethylhydrazine.

Concentrations of the major methylated DNA purines were determined in two liver cell cell populations of Fischer 344 rats during administration of 30 ppm 1,2-dimethylhydrazine (SDMH) in the drinking water for periods of up to 4 weeks. Quantitation of 7-methylguanine and O6-methylguanine (O6MG) was achieved by highly sensitive optical methods following separation of DNA bases by high-performance liquid chromatography. Overall alkylation as indicated by the concentration of 7-methylguanine was near maximum in both hepatocytes and liver nonparenchymal cells by the third day of SDMH administration. Similar amounts of 7-methylguanine were present in the two liver cell populations at seven of nine time points during 4 weeks of exposure. In contrast, dramatic differences in the cumulative concentrations of O6MG were seen in the two cell populations. Nonparenchymal cells accumulated O6MG during the first 8 days of exposure and had up to a 30-fold greater concentration of this product than did the corresponding hepatocytes. In the hepatocytes, a rapid decline in O6MG concentration was observed between 1 and 3 days of exposure to SDMH. Thereafter, only low levels of this promutagenic lesion were present in hepatocytes. Exposure of rats to the same regimen of SDMH for up to 10 months caused angiosarcomas in the livers of over 90% of the animals, while hepatocellular carcinomas occurred in only 40%. Thus, a strong correlation exists between the inability to repair O6MG and cell specificity for carcinogenesis.

In vivo kinetics of O6-methylguanine and 7-methylguanine formation and persistence in DNA of rats treated with symmetrical dimethylhydrazine.

The rates of appearance and removal of 7-methylguanine and O6-methylguanine in DNA from rat liver, kidney, and colon were determined at various intervals up to 120 hr after i.p. administration of 10.2, 40.7, 81.5, or 163 mg 1,2-dimethylhydrazine (SDMH) per kg body weight (one-sixteenth, one-fourth, one-half, or one 50% lethal dose) using high-pressure liquid chromatography and fluorescence spectrophotometry. In most cases, increasing doses of SDMH slowed the rate of methylation of DNA, especially of the liver; colon DNA was methylated at a faster rate than was liver DNA, and kidney DNA was methylated at the slowest rate following SDMH administration. Removal of O6-methylguanine was slow (half-life, 37 to 50 hr) when this base was present in liver DNA at concentrations above 400 mumol/mol guanine; as the concentration fell below 300 mumol O6-methylguanine per mol guanine, the removal rate more than doubled (half-life, 16 to 19 hr). Some evidence was obtained to suggest that in the first 12 hr after maximum DNA methylation following SDMH administration, a rapid time-dependent removal of 7-methylguanine from liver and kidney but not colon DNA occurred. In these instances, then, the rates of formation and removal of aberrant methylated bases did not follow first-order kinetics.

Aberrant and nonrandom methylation of chromosomal DNA-binding proteins of colonic epithelial cells by 1,2-dimethylhydrazine.

The alkylating carcinogen 1,2-dimethylhydrazine (DMH) induces colonic tumors with high incidence. The sites of in vivo modification of target macromolecules were studied using [methyl-3H]DMH. Carcinogen binding to subcellular fractions of colonic epithelial cells was analyzed at time intervals ranging from 10 min to 36 hr, with particular emphasis on the alkylation of nuclear constituents. DMH modifies not only nucleic acids but also histones and other DNA-binding proteins in the target cells. Separation of the 3H-methylated amino acids showed aberrant methylation patterns after exposure to [3H]DMH, as compared with methylations observed with [methyl-3H]methionine as a natural methyl group donor. DMH-modified histone H1 contained methylated lysin, arginine, and histidine residues not normally found, and other histones had abnormal methylarginine contents. High-mobility-group proteins and other nuclear proteins contained methyllysine residues not normally detected. Proteins known to be associated with template-active and more accessible DNA sequences, such as the high-mobility group proteins and the multiacetylated forms of histones H3 and H4, were preferentially damaged after exposure to [3H]DmH. The result suggests that carcinogen-induced chromosomal damage is not random and may selectively affect proteins in the actively transcribing or replicating genes in the target cells. That damage affects the amino acids most likely to be involved in DNA binding.

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.

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.

Fatty acid stimulated N-demethylation of 1,2-dimethylhydrazine and tetramethylhydrazine by rat colonic mucosa.

A fatty acid stimulated, NADPH-independent pathway for the N-demethylation of 1,1-dimethylhydrazine (1,1-DMH) with the generation of HCHO was demonstrated in 10,000 g soluble fractions of colonic mucosal homogenates. Tetramethylhydrazine and, to a lesser extent, aminopyrine, but not 1,2-DMH or methylhydrazine, were also substrates for this reaction. Isolated superficial colonic epithelial cells metabolized 1,1-DMH at a faster rate than proliferative epithelial cells. Indomethacin, an inhibitor of cyclooxygenase activity, and 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of both cyclooxygenase and lipoxygenase activities, suppressed HCHO production from 1,1-DMH by 50 and 80%. However, in the presence of indomethacin or ETYA, arachidonate hydroperoxide stimulated HCHO formation. This suggested a peroxidative mechanism for 1,1-DMH metabolism, related in part to prostaglandin synthesis. A possible role for lipoxygenase activity in mediating 1,1-DMH metabolism was suggested by the ability of linoleate, which did not increase prostaglandin synthesis, to stimulate 1,1-DMH metabolism and by the fact that ETYA was more effective than indomethacin as an inhibitor of 1,1-DMH metabolism. The fatty acid stimulated pathway for N-demethylation was clearly distinct from the mixed function oxidase activities. NADPH did not stimulate 1,1-DMH metabolism to HCHO. 7,8-Benzoflavone or SKF-525A, inhibitors of cytochrome P-450, and methimazole, an inhibitor of N-demethylation catalyzed by the hepatic microsomal FAD-containing monooxygenase, did not suppress HCHO formation. To the extent that 1,1-DMH and tetramethylhydrazine reach the colon unchanged, the results suggest that fatty acid stimulated cooxidation pathways in colonic mucosa may contribute to the metabolism of these agents. Metabolism by superficial cells which are destined to slough may be an important defense mechanism against the toxic and carcinogenic actions of these hydrazines in colon.

The effect of mixed-function oxidase and amine oxidase inhibitors on the activation of dialkylnitrosamines and 1,2-dimethylhydrazine to bacterial mutagens in mice.

The effect of the mixed-function oxidase inhibitor phenylimidazole (PI) and the amine oxidase inhibitors iproniazid (IPRO) and aminoacetonitrile (AAN) on the mutagenic activity of various carcinogens was determined in intrasanguineous host-mediated assays, using mice as hosts and E. coli 343/113 as an indicator of mutagenic activity. The carcinogenic compounds dimethyl-, diethyl-, methylethyl-, and diethanolnitrosamine (DMNA, DENA, MENA, and DELNA respectively) and 1,2-dimethylhydrazine (SDMH) were administered i.p. to mice pretreated or not with one of the inhibitors. After 4 h exposure to each of the carcinogens, E. coli cells recovered from the liver of non-pretreated mice showed considerable induction of VALr mutations; after pretreatment of the hosts with the three inhibitors, significant reduction of the amounts of induced mutants in vivo was observed. Particularly, PI proved a very efficient inhibitor of DENA, MENA, DELNA, and SDMH mutagenicity (93%-97% reduction), suggesting that these carcinogens are mainly activated by cytochrome P-450-dependent enzymes. However, since PI might also inhibit the NAD-mediated activation of DELNA by alcohol dehydrogenase (ADH), the present experiments do not rule out an additional role of ADH in the in vivo mutagenic activation of DELNA. AAN and IPRO were less and much less effective, respectively, in reducing the mutagenic activity of all compounds. Surprisingly, PI showed less inhibition of the mutagenic activity of DMNA (60% reduction), as compared to the other carcinogens; this indicates that metabolic routes other than the cytochrome P-450-dependent enzyme system may be important for the activation of DMNA.

Metabolism of 1,2-dimethylhydrazine in isolated perfused rat liver.

The metabolism of 1,2-dimethylhydrazine (DMH) was investigated in isolated perfused rat liver. The separation of [14C]DMH and its metabolites azomethane (AM), azoxymethane (AOM) and methylazoxymethanol (MAM) was performed by high pressure liquid chromatography (HPLC). The fractions were quantitatively detected by liquid scintillation counting of the radioactivity. It was demonstrated that DMH is highly metabolized by the liver. After 1 h of perfusion, the median amounts of DMH and its metabolites in the medium were: DMH, 16%; AM, 3%; AOM, 42%; MAM, 30% of the given dose. During this time 9.9% and 0.7% of the total radioactivity were eliminated as AM and CO2 in the oxygen stream of the perfusion apparatus; 0.6% were excreted by bile and 12.3% stored in hepatic tissue. The reaction rate (t1/2) of each individual metabolic step was estimated by means of a mathematical kinetic model as follows: DMH leads to AM, 21.8 min; AM leads to AOM, 1.5 min; AOM leads to MAM, 41.5 min; MAM leads to, 611 min. The results are discussed in comparison to in vivo experiments.

Inhibition of the alkylation of nucleic acids and of the metabolism of 1,2-dimethylhydrazine by aminoacetonitrile.

Pretreatment of rats with aminoacetonitrile inhibited the metabolism of [14C]1,2-dimethylhydrazine to 14CO2 and increased the expiration of [14C]-azomethane. Alkylation of nucleic acids following administration of 1,2-dimethylhydrazine was reduced by aminoacetonitrile to 5% of control levels in liver, 11% of control levels in kidney and 43% of control levels in colon.

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.

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.

Changes in tissue and serum activity of cathepsin B-like cysteine proteinase during colorectal carcinogenesis by 1,2-dimethylhydrazine in mice.

The activity of cathepsin B-like cysteine proteinase in the mice colon was studied during carcinogenesis induced by 1,2-dimethylhydrazine dihydrochloride (DMH). Before starting the treatment with DMH, the activity of the observed enzyme was very low in the colorectal area but the activity in the serum was rather high. In the course of carcinogenesis, the local activity markedly increased in the stroma equally as in the cells of developing tumors while the serum activity slightly decreased. Following the total ovariectomy, no significant changes in the local activity or in the serum activity were found. Possible causes of these variations are discussed.

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.