Aerobic and anaerobic reduction of nitrated pyrenes in vitro

Nitrated pyrenes are mutagenic and tumorigenic environmental pollutants that are activated to DNA-binding derivatives via nitroreduction. We have investigated the enzymatic nitroreduction of 1-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene to determine if differences in the extent of nitroreduction may help explain differences in their biological potencies. Each nitrated pyrene was incubated aerobically and anaerobically with 105,000 X g supernatant (S105) from Salmonella typhimurium TA98 and the nitroreductase-deficient strain, TA98NR, and with cytosol and microsomes from rat and human liver. Under anaerobic conditions, 1-nitropyrene and 1,3-dinitropyrene were reduced by TA98 S105 to a lesser extent than 1,6- and 1,8-dinitropyrene. The extent of 1,6- and 1,8-dinitropyrene metabolism was not altered relative to TA98 when using TA98NR S105, but the nitroreduction of 1-nitropyrene and 1,3-dinitropyrene was decreased. Both rat and human liver cytosol and microsomes reduced 1,6- and 1,8-dinitropyrene to greater extents than 1-nitropyrene and 1,3-dinitropyrene. Under aerobic conditions rat and human liver cytosols were similar to TA98 S105 in that aminopyrene decreased while nitrosopyrene formation increased. By comparison, oxygen decreased the microsomal formation of both nitrosopyrenes and aminopyrenes. The reduction of succinoylated cytochrome c was measured during the hepatic metabolism of nitro- and nitrosopyrenes under aerobic conditions. The data indicated that reduced nitro- and nitrosopyrene intermediates were directly reducing succinoylated cytochrome c and that the assay could be used as a measure of aerobic nitroreduction. These studies demonstrate that 1,6- and 1,8-dinitropyrene are reduced to a greater extent than 1-nitropyrene and 1,3-dinitropyrene, which corresponds to their relative biological potencies as mutagens and carcinogens. Furthermore, although more extensive nitroreduction is detected under anaerobic conditions, the nitroreduction that occurs aerobically may be important for the mutagenic and tumorigenic properties of these compounds.     

Properties of cytosolic components activating rat hepatic 5'' [corrected]-deiodination in the presence of NADPH.

The effects of cytosol, NADPH and reduced glutathione (GSH) on the activity of 5'-deiodinase were studied by using washed hepatic microsomes from normal fed rats. Cytosol alone had little stimulatory effect on the activation of microsomal 5'-deiodinase. NADPH had no stimulatory effect on the microsomal 5'-deiodinase unless cytosol was added. 5'-deiodinase activity was greatly enhanced by the simultaneous addition of NADPH and cytosol (P less than 0.001); this was significantly higher than that with either NADPH or cytosol alone (P less than 0.001). GSH was active in stimulating the enzyme activity in the absence of cytosol, but the activity of 5'-deiodinase with 62 microM-NADPH in the presence of cytosol was significantly higher than that with 250 microM-GSH in the presence of the same concentration of cytosol (P less than 0.001). The properties of the cytosolic components essential for the NADPH-dependent activation of microsomal 5'-deiodinase independent of a glutathione/glutathione reductase system were further assessed using Sephadex G-50 column chromatography to yield three cytosolic fractions (A, B and C), wherein A represents pooled fractions near the void volume, B pooled fractions of intermediate Mr (approx. 13 000), and C of low Mr (approx. 300) containing glutathione. In the presence of NADPH (1 mM), the 5'-deiodination rate by hepatic washed microsomes is greatly increased if both A and B are added and is a function of the concentrations of A, B, washed microsomes and NADPH. A is heat-labile, whereas B is heat-stable and non-dialysable. These observations provide the first evidence of an NADPH-dependent cytosolic reductase system not involving glutathione which stimulates microsomal 5'-deiodinase of normal rat liver. The present data are consistent with a deiodination mechanism involving mediation by a reductase (other than glutathione reductase) in fraction A of an NADPH-dependent reduction of a hydrogen acceptor in fraction B, followed by reduction of oxidized microsomal deiodinase by the reduced acceptor (component in fraction B).     

Pulmonary phosphatidic acid phosphohydrolase: further studies on the activities in rat lung responsible for the hydrolysis of membrane-bound and aqueously dispersed phosphatidate

Rat lung cytosol and microsomal fractions both contain phosphohydrolase activity towards membrane-bound phosphatidic acid (PAmb) and aqueously dispersed phosphatidic acid (PAaq) which cannot be explained through contamination with the other fraction. The phosphohydrolase activities with PAaq demonstrated Km and Vmax values which were more than an order of magnitude greater than those observed with PAmb and with vesicles prepared from the lipids extracted from [32P]PA-labelled microsomes. The PAaq-dependent activities in both fractions were stimulated by preparing mixed liposomes with phosphatidylcholine. The PAmb-dependent activities in rat lung microsomes and cytosol were markedly stimulated by high concentrations of Triton X-100 and Nonidet P-40. The PAmb- and PAaq-dependent activities in the microsomes were stimulated by deoxycholate. Although no difference was observed in the inhibition profiles of the PAmb- and PAaq-dependent activities of the cytosol in the presence of various mercurials, the PAmb-dependent activity in the microsomes was somewhat more susceptible than the PAaq-dependent activity. The PAmb-dependent activities in both fractions were more susceptible to inhibition by iodoacetamide. These results support the view that separate rat lung enzymes were involved in the hydrolysis of PAmb and PAaq. The relative abilities of rat lung cytosol and microsomes to hydrolyse PA endogenously generated on the microsomes were compared using relative concentrations of cytosol corresponding to the levels in intact rat lung. During the initial period (5-10 min) the cytosol phosphohydrolase activity was more effective than the microsomal activity. At later stages (10-20 min), the rates were comparable.     

Methyl linoleate ozonide as a substrate for rat glutathione S-transferases: reaction pathway and isoenzyme selectivity

The 9,10-mono-ozonide of methyl linoleate was shown to be a substrate for rat hepatic cytosolic, rat lung cytosolic and rat hepatic microsomal glutathione S-transferases (GST). The activities of lung cytosol and liver microsomes with methyl linoleate ozonide (MLO) were found to be high relative to the activity demonstrated by liver cytosol, as compared with their respective activities towards 1-chloro-2,4-dinitrobenzene (CDNB). Only a slight catalytic activity towards the ozonide was noticed for rat lung microsomes. Isoenzyme 2-2 exhibited the highest specific activity (208 nmol/min/mg) when isoenzymes 1-1, 1-2, 2-2, 3-3, 3-4, 4-4 and 7-7 were compared. This isoenzyme accounts for approx. 25% of cytosolic GST protein in rat lung, while in rat liver it represents approx. 9%. This may partly explain the high activity towards the ozonide noticed for rat lung cytosol. No stable conjugates were formed as products of the reaction of MLO with glutathione; although two glutathione-conjugates were noticed on TLC, they were only formed as intermediate compounds. Coupling of an aldehyde dehydrogenase assay or a glutathione reductase assay to the GST-catalyzed conjugation, demonstrated that oxidized glutathione and aldehydes are formed as the major products in the reaction. To further confirm the formation of aldehydes, the products of the GST-catalyzed reaction were incubated with 2,4-dinitrophenylhydrazine, which resulted in hydrazone formation. In conclusion, the activity of the GST towards the ozonide of methyl linoleate is similar to their peroxidase activity with lipid hydroperoxides as substrates.     

Type I nitroreductases in soil enterobacteria reduce TNT (2,4,6,-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)

Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also documented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression of the E. cloacae type I nitroreductase gene was induced in an Escherichia coli strain carrying a plasmid, a simultaneous increase in TNT and RDX nitroreductase activities was observed. In addition, neither TNT nor RDX nitroreductase activity was detected in nitrofurazone-resistant mutants of M. morganii. We conclude that a type I nitroreductase present in these two enteric bacteria was responsible for the nitroreduction of both types of explosive.     

Comparison of the sex and subcellular distributions, catalytic and immunochemical reactivities of hepatic epoxide hydrolases in seven mammalian species

Sex and species differences in hepatic epoxide hydrolase activities towards cis- and trans-stilbene oxide were examined in common laboratory animals, as well as in monkey and man. In general trans-stilbene oxide was found to be a good substrate for epoxide hydrolase activity in cytosolic fractions, whereas the cis isomer was selectively hydrated by the microsomal fraction (with the exception of man, where the cytosol also hydrated this isomer efficiently). The specific cytosolic epoxide hydrolase activity was highest in mouse, followed by hamster and rabbit. Epoxide hydrolase activity in the crude 'mitochondrial' fraction towards trans-stilbene oxide was also highest in mouse and low in all other species examined. Microsomal epoxide hydrolase activity was highest in monkey, followed by guinea pig, human and rabbit, which all had similar activities. Sex differences were generally small, but where significant, male animals had higher catalytic activities than females of the same species in most cases. Antibodies raised against microsomal epoxide hydrolase purified from rat liver reacted with microsomes from all species investigated, indicating structural conservation of this protein. Antibodies directed towards cytosolic epoxide hydrolase purified from mouse liver reacted only with liver cytosol from mouse and hamster and with the 'mitochondrial' fraction from mouse in immunodiffusion experiments. Immunoblotting also revealed reaction with rat liver cytosol. The cytosolic and 'mitochondrial' epoxide hydrolases in all three mouse strains and in both sexes for each strain were immunochemically identical. The anomalies in human liver epoxide hydrolase activities observed here indicate that no single common laboratory animal is a good model for man with regard to these activities.     

NADPH-cytochrome c reductases of Trypanosoma cruzi

NADPH-dependent reduction of cytochrome c is catalyzed both by microsomes and the cytosolic fraction isolated from Trypanosoma cruzi homogenates. About one-third of the activity is microsomal and two-thirds is cytosolic. The microsomal activity is increased by Lubrol and sodium cholate, but pretreatment with phenobarbital has negligible effect. On the other hand, detergents do not affect the cytosolic activity but it is increased by phenobarbital. From these observations, it is concluded that the NADPH-dependent reduction of cytochrome c by microsomes and the cytosol corresponds to two distinct enzymes. The cytosolic enzyme has been purified to a single SDS-PAGE band of about 53,000 da and partially characterized.     

Vitamin K dependent carboxylase: subcellular location of the carboxylase and enzymes involved in vitamin K metabolism in rat liver

Vitamin K dependent carboxylation of an exogenous peptide substrate and endogenous protein substrates, vitamin K epoxidation, and reduction of vitamin K epoxide were measured in subcellular fractions from rat liver. The rough microsomal fraction was highly enriched in all four activities; lower levels were found in smooth microsomes. Mitochondria, nuclei, and cytosol had negligible activities. The addition of 0.2% Triton X-100 to intact microsomes resulted in a 10-20-fold stimulation in carboxylation of a peptide substrate. This marked latency suggests that the active site of the carboxylase may be accessible only from the lumen of the microsomal membrane. A lumen-facing orientation of the carboxylase was also supported by its inaccessibility to trypsin in intact microsomes contrasted with marked inhibition by trypsin in detergent-permeabilized microsomes. Vitamin K epoxidase and epoxide reductase activities were also inhibited by trypsin much more effectively in permeabilized than in intact microsomes, although some degree of exposure at the cytosolic surface was also indicated. These data suggest that carboxylation is an early event in prothrombin synthesis occurring primarily on the lumen side of the rough endoplasmic reticulum membrane. The location of the vitamin K epoxidation-reduction cycle enzymes is consistent with their possible role in the carboxylation reaction.     

Effect of enzyme inducers on metabolism of 1-nitropyrene in human hepatoma cell line HepG2.

We measured the response of HepG2 cells to the classic cytochrome (cyt.) P-450 inducers 3-methylcholanthrene (3-MC) and phenobarbital (PB), by evaluating oxidative and/or reductive metabolism of the nitroarenes, 1-NP and 1,6-dinitropyrene (1,6-DNP), in control and induced cells. In HepG2 cells, 3-MC induces ring-hydroxylation of 1-NP, whereas PB stimulates its nitroreduction. PB induces NADPH-cyt. c reductase, but does not affect other cytosolic and microsomal enzymes which contribute to 1-NP nitroreduction in these cells. However, PB-inducible nitroreductase activity seems to be associated primarily with cyt. P-450 isoenzymatic form(s), as indicated by the requirement for NADPH and the response to specific inhibitors such as alpha-naphthoflavone and CO.     

Regulation of lysophosphatidylcholine-metabolizing enzymes in isolated myocardial cells from rat heart

Enzymatic pathways involved in the metabolism of lysophosphatidylcholine were investigated in rat heart myocardial cells. Acyl CoA-dependent acyltransferase activity was localized in microsomes, and was much greater than lysophospholipase activity in either cytosolic or microsomal fractions. The cytosolic lysophospholipase was more sensitive to inhibition by palmitylcarnitine in comparison to free fatty acids. In contrast, free fatty acids (oleate and palmitate) produced a greater inhibition of the microsomal acyltransferase and lysophospholipase than did palmitylcarnitine. A reduction in the assay pH to 6.5 resulted in an increase in microsomal acyltransferase and cytosolic lysophospholipase activities, but brought about a marked reduction in the microsomal lysophospholipase activity. At pH 6.5, the percentage inhibition of the microsomal acyltransferase by palmitylcarnitine was reduced, whereas the inhibition by palmitic acid was enhanced. The inhibition of the microsomal lysophospholipase by both palmitylcarnitine and palmitic acid was reduced at pH 6.5. With respect to myocardial ischemia, the inhibition of microsomal acyltransferase by free fatty acids and the reduction in microsomal lysophospholipase activity due to acidosis may contribute to the elevation of cellular lysophosphoglycerides which are arrhythmogenic.     

Retinol forms retinoic acid via retinal.

Hepatic cytosol from normal deermice having cytosolic alcohol dehydrogenase (ADH+) also displays retinol dehydrogenase activity and converts retinol to retinoic acid, whereas cytosol from ADH- deermice lacks these enzyme activities and does not produce retinoic acid. Furthermore, microsomes from either strain do not convert retinol to retinoic acid. However, when cytosol from ADH- animals is added to the microsomes, retinoic acid is produced. The obligatory role of retinal as an intermediary step in retinoic acid formation is further shown by isotopic dilution of retinoic acid formed from labeled retinol upon addition of unlabeled retinal. Microsomal retinol dehydrogenase also catalyzes the reduction of retinal to retinol, thereby explaining the decrease in retinoic acid production from retinol in liver cytosol of ADH+ deermice when microsomes are added. Thus, the results of this study indicate that retinal is an obligatory intermediate in the hepatic production of retinoic acid from retinol and that cytosolic and microsomal retinol dehydrogenases play a key role in this process.     

Pulmonary phosphatidic acid phosphatase. Properties of membrane-bound phosphatidate-dependent phosphatidic acid phosphatase in rat lung.

1. The membrane-bound phosphatidate-dependent phosphatidic acid phosphatase activity of rat lung has been investigated in cytosol and microsomal fractions using as a substrate [32P]phosphatidate bound to heat inactivated rat liver microsomes. Both activities demonstrated broad pH optima with a maximum of 7.4--8 for the cytosol and a maximum of 6.5--7.5 with microsomal preparations. 2. At low concentrations (0--5 mM) Mg2+ produced a slight stimulation of the cytosol activity but at higher concentrations an inhibition was observed. Low concentrations (1.0--2.0 mM) of EDTA abolished the cytosol activity and reduced the microsomal activity to half. In both cases, the addition of Mg2+ in the presence of EDTA resulted in an activity which was more than 2-fold greater than that observed in the absence of chelator or divalent cation. 3. The cytosol activity was relatively resistant to the addition of ionic and nonionic detergents. In general, the addition of a number of phosphate esters increased rather than decreased the release of 32Pi, indicating a relative specificity for phosphate groups associated with a hydrophobic environment. The addition of aqueous dispersions of phosphatidate, lysophosphatidic acid or phosphatidylglycerophosphate markedly reduced the hydrolysis of membrane-bound [32P]phosphatidate. The cytosol activity was slightly inhibited by the addition of phosphatidylcholine. 4. In an attempt to estimate the relative contributions of the cytosol and microsomal activities in vivo, these activities were assayed using [32P]phosphatidate endogenously generated on rat lung microsomes. With the 32P-labelled microsomes, the hydrolysis remained linear over the 45 min of the experiment. Addition of high speed supernatant produced a rapid release of 32Pi during the first 10 min followed by a more gradual release similar to that oberved with the microsomes alone. The cytosol activity remained greater than the microsomal activity at all times studied. 5. When [14C]phosphatidate-labelled microsomes were incubated in the presence of nonradioactive CDPcholine, the addition of cytosol markedly stimulated the incorporation of radioactivity into phosphatidylcholine. This observation suggests that the phosphatidic acid phosphatase activity associated with the cytosol has a role in phosphatidylcholine (and presumably surfactant) biosynthesis in rat lung.     

Microsomes convert retinol and retinal into retinoic acid and interfere in the conversions catalyzed by cytosol

Rat liver microsomes converted retinol into retinal and retinoic acid. The production of retinal was observed over a range of substrate concentrations (10-100 microM), but retinoic acid was detected only at retinol concentrations of 50 microM or higher. At 50 microM retinol, the rate of microsomal retinal production was 2-fold greater than that of cytosol, but the rate of retinoic acid synthesis was 4-fold less than that of cytosol. Retinal was also converted into retinoic acid by rat liver microsomes, but at a rate 2-5% of that catalyzed by cytosol. Microsomes also interfered with the conversion of retinol and retinal into retinoic acid by rat liver cytosol. A 50% decrease in the cytosolic rates of retinoic acid production from retinol or retinal was caused by microsomal to cytosolic protein ratios of 0.1 and 0.5, respectively. Under the incubation conditions, which included NAD in the medium, addition of microsomes to cytosol did not affect the elimination half-life of retinol or retinoic acid, but did decrease the elimination half-life of retinal by 2-fold. These data show that retinal synthesis from retinol does not necessarily reflect retinoic acid synthesis and suggest that liver microsomes sequester free retinol and convert it into retinal primarily for elimination, rather than to serve as substrate for cytosolic retinoic acid synthesis.     

Characterization of estrogen and antiestrogen binding to the cytosol and microsomes of breast tumors

The binding of [3H]estradiol and [3H]hydroxytamoxifen to the cytosol and microsomal fractions of several human breast tumors was investigated. By washing microsomal membranes with a KCl-free or a KCl-containing medium we could distinguish between intrinsic, extrinsic and contaminant estradiol binding sites in these membranes. We observed that treatment of the microsomes with low salt medium removes about 80% of the total estradiol binding sites, whereas 20% are not extractable. The concentration of unextractable [3H]estradiol binding sites in the microsomes varies in proportion to the level of cytosolic estrogen receptors (ER). About 10% of the total extranuclear specific estrogen binding sites was consistently found tightly associated to the microsomal fraction, which displays an affinity for estradiol (Kd = 0.1-0.6 nM) similar to that of the cytosolic ER. The displacement of [3H]estradiol with unlabeled hormone or with the antiestrogens, nafoxidine, enclomiphene and tamoxifen (TAM) exhibits identical IC50 values either in the cytosol or in the microsomal membranes. On the other hand, the microsomal fraction of breast tumors also binds [3H]hydroxyTAM, but with higher capacity and lower affinity than those of the cytosolic fraction. Furthermore, we did not observe correlation between the concentrations of ER and of antiestrogen binding sites (AEBS) in the tumors. These results indicate that microsomal membranes of human breast tumors contain estrogen binding sites which may be related to the cytosol ER recycling and that specific AEBS are predominantly localized in this membrane system. Furthermore, it is shown that the magnitude of estradiol binding to microsomes depends on the ER positive degree of the tumors, whereas the magnitude of the antiestrogen binding to the microsomes is independent of the ER status of the tumors.     

Possible pathways of lipid peroxide reduction in the liver and their intracellular localization

Lipid peroxidase activity in rat liver was studied. Rat liver cytosolic fraction was found to be capable of reducing lipid hydroperoxides. On the contrary, no lipid hydroperoxide reduction was observed in microsomes. It was found that at least two proteins in rat liver cytosol are capable of reducing phospholipid hydroperoxides. One of them is precipitated by 33-55% at (NH4)2SO4 saturation and requires reduced glutathione (GSH) as a hydrogen donor, while the other one is precipitated by 55-80% at (NH4)2SO4 saturation and reduces phospholipid hydroperoxides in the presence of a unidentified low molecular weight cytosolic factor, but not GSH or NADPH.     

Human liver cytosolic epoxide hydrolases

Human liver epoxide hydrolases were characterized by several criteria and a cytosolic cis-stilbene oxide hydrolase (cEHCSO) was purified to apparent homogeneity. Styrene oxide and five phenylmethyloxiranes were tested as substrates for human liver epoxide hydrolases. With microsomes activity was highest with trans-2-methylstyrene oxide, followed by styrene 7,8-oxide, cis-2-methylstyrene oxide, cis-1,2-dimethylstyrene oxide, trans-1,2-dimethylstyrene oxide and 2,2-dimethylstyrene oxide. With cytosol the same order was obtained for the first three substrates, whereas activity with 2,2-dimethylstyrene oxide was higher than with cis-1,2-dimethylstyrene oxide and no hydrolysis occurred with trans-1,2-dimethylstyrene oxide. Generally, activities were lower with cytosol than with microsomes. The isoelectric point for both microsomal styrene 7,8-oxide and cis-stilbene oxide hydrolyzing activity was 7.0, whereas cEHCSO had an isoelectric point of 9.2 and cytosolic trans-stilbene oxide hydrolase (cEHTSO) of 5.7. The cytosolic epoxide hydrolases could be separated by anion-exchange chromatography and gel filtration. The latter technique revealed a higher molecular mass for cEHCSO than for cEHTSO. Both cytosolic epoxide hydrolases showed higher activities at pH 7.4 than at pH 9.0, whereas the opposite was true for microsomal epoxide hydrolase. The effects of ethanol, methanol, tetrahydrofuran, acetonitrile, acetone and dimethylsulfoxide on microsomal epoxide hydrolase depended on the substrate tested, whereas both cytosolic enzymes were not at all, or only slightly, affected by these solvents. Effects of different enzyme modulators on microsomal epoxide hydrolase also depended on the substrates used. Trichloropropene oxide and styrene 7,8-oxide strongly inhibited cEHCSO whereas cEHTSO was moderately affected by these compounds. Immunochemical investigations revealed a close relationship between cEHCSO and rat liver microsomal, but not cytosolic, epoxide hydrolase. Interestingly, cEHTSO has no immunological relationship to rat microsomal, nor to rat cytosolic epoxide hydrolase. cEHTSO from human liver differed also from its counterpart in the rat in that it was only moderately affected by tetrahydrofuran, acetonitrile and trichloropropene oxide. Five steps were necessary to purify cEHCSO. The enzyme has a molecular mass (49 kDa) identical to that of rat liver microsomal epoxide hydrolase.     

Characterization of enzymes participating in carbonyl reduction of 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human placenta

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) has been identified as one of the strongest nitrosamine carcinogens in tobacco products in all species tested. Carbonyl reduction to 4-methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) followed by glucuronosylation is considered to be the main detoxification pathway in humans. In previous investigations, we have identified a microsomal NNK carbonyl reductase as being identical to 11ß-hydroxysteroid dehydrogenase 1, a member of the short-chain dehydrogenase/reductase (SDR) superfamily. Recently, we provided evidence that carbonyl reduction of NNK does also take place in cytosol from mouse and human liver and lung. In human liver cytosol, carbonyl reductase, a SDR enzyme, and AKR1C1, AKR1C2 and AKR1C4 from the aldo-keto reductase (AKR) superfamily were demonstrated to be responsible for NNK reduction. Since NNK and/or its metabolites can diffuse through the placenta and reach fetal tissues, we now investigated NNK carbonyl reduction in the cytosolic fraction of human placenta in addition to that in microsomes. Concluding from the sensitivity to menadione, ethacrynic acid, rutin and quercitrin as specific inhibitors, mainly carbonyl reductase (EC 1.1.1.184) seems to perform this reaction in human placenta cytosol. The presence of carbonyl reductase was confirmed by RT-PCR. This is the first report to provide evidence that NNAL formation in placenta is mediated by carbonyl reductase.     

Characterization of enzymes participating in carbonyl reduction of 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human placenta

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) has been identified as one of the strongest nitrosamine carcinogens in tobacco products in all species tested. Carbonyl reduction to 4-methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) followed by glucuronosylation is considered to be the main detoxification pathway in humans. In previous investigations, we have identified a microsomal NNK carbonyl reductase as being identical to 11beta-hydroxysteroid dehydrogenase 1, a member of the short-chain dehydrogenase/reductase (SDR) superfamily. Recently, we provided evidence that carbonyl reduction of NNK does also take place in cytosol from mouse and human liver and lung. In human liver cytosol, carbonyl reductase, a SDR enzyme, and AKR1C1, AKR1C2 and AKR1C4 from the aldo-keto reductase (AKR) superfamily were demonstrated to be responsible for NNK reduction. Since NNK and/or its metabolites can diffuse through the placenta and reach fetal tissues, we now investigated NNK carbonyl reduction in the cytosolic fraction of human placenta in addition to that in microsomes. Concluding from the sensitivity to menadione, ethacrynic acid, rutin and quercitrin as specific inhibitors, mainly carbonyl reductase (EC 1.1.1.184) seems to perform this reaction in human placenta cytosol. The presence of carbonyl reductase was confirmed by RT-PCR. This is the first report to provide evidence that NNAL formation in placenta is mediated by carbonyl reductase.     

NAD (P) H-dependent reduction of nicotinamide N-oxide by an unique enzyme system consisting of liver microsomal NADPH-cytochrome C reductase and cytosolic aldehyde oxidase

NAD (P) H-dependent reduction of nicotinamide N-oxide was investigated with rabbit liver preparations. Microsomes, microsomal NADPH-cytochrome c reductase or cytosolic aldehyde oxidase alone exhibited no nicotinamide N-oxide reductase activity in the presence of NADPH or NADH. However, when the microsomal preparations were combined with the cytosolic enzyme, a significant N-oxide reductase activity was observed in the presence of the reduced pyridine nucleotide. The activity was enhanced by FAD or methyl viologen. Cytosol alone supplemented with NADPH or NADH exhibited only a slight, but when combined with microsomes, a significant N-oxide reductase activity. Based on these facts, we propose a new electron transfer system consisting of NADPH-cytochrome c reductase and aldehyde oxidase, which exhibits nicotinamide N-oxide reductase activity in the presence of the reduced pyridine nucleotide.     

Acute Effects of Soman, Sarin, and Tabun on Microsomal and Cytosolic Components of the Calmodulin System in Rat Striatum

Two hours after administration of Soman (120 micrograms/kg, s.c.), Sarin (150 micrograms/kg, s.c.), or Tabun (240 micrograms/kg, s.c.), microsomes and cytosol were prepared from rat striata. Microsomal and cytosolic calmodulin (CaM) levels, microsomal adenylate and guanylate cyclase activities, protein kinase activities, and Ca2+ + Mg2+-ATPase activities were determined while cytosolic phosphodiesterase (PDE) activities were determined. CaM levels in both cell fractions were significantly increased by Soman and Sarin. Cyclic AMP-PDE and adenylate cyclase activities were decreased by Soman and Sarin. All three agents decreased activities of cyclic GMP-PDE and guanylate cyclase. Sarin and Tabun administration caused significant increases in microsomal protein kinase activity and none of the agents affected activity of divalent cation ATPases. The intensity of effects of the three organophosphates roughly paralleled their observed neurotoxic potencies. The results indicate that components of the CaM system are implicated as either causative or adaptive changes induced by these agents.