Rat Liver Cytosol Research Articles

Disulfide S-Monoxides Convert Xanthine Dehydrogenase into Oxidase in Rat Liver Cytosol More Potently Than Their Respective Disulfides

Xanthine oxidase (XO)/xanthine dehydrogenase (XD) oxidizes oxypurines to uric acid, with only the XO form producing reactive oxygen species. In the present study, the effects of cystamine S-monoxide and cystine S-monoxide (disulfide S-monoxides) on the conversion of XD to XO in rat liver were examined. A partially purified enzyme fraction from the rat liver was incubated with xanthine in the presence or absence of NAD+, and the uric acid formed was measured by HPLC. Under basal conditions, XO activity represented about 15% of the total XO plus XD activity. Cystamine S-monoxide and cystine S-monoxide converted XD into XO in a dose-dependent manner, and the concentrations required to increase XO activity by 50% were approximately 1 and 2 microM, respectively. Their respective thiols (cysteamine and cysteine) and disulfides (cystamine and cystine) up to 10 microM showed weak or no effects on the activities of XO and XD and their conversion. Experiments utilizing a sulfhydryl reducing reagent (dithiothreitol) and sulfhydryl modifiers (4,4'-dithiodipyridine and 1-fluoro-2,4-dinitrobenzene) indicated that disulfide S-monoxides-induced conversion of XD to XO occurs via disulfide bridge formation in XD, but not the modification of sulfhydryl groups. These results suggest that disulfide S-monoxides have the potential to increase the generation of reactive oxygen species through the conversion of XD to XO in liver.

NADPH‐dependent coenzyme Q reductase is the main enzyme responsible for the reduction of non‐mitochondrial CoQ in cells

We purified an NADPH-dependent coenzyme Q reductase (NADPH-CoQ reductase) in rat liver cytosol and compared its enzymatic properties with those of the other CoQ10 reductases such as NADPH: quinone acceptor oxidoreductase 1 (NQO1), lipoamide dehydrogenase, thioredoxine reductase and glutathione reductase. NADPH-CoQ reductase was the only enzyme that preferred NADPH to NADH as an electron donor and was also different from the other CoQ10 reductases in the sensitivities to its inhibitors and stimulators. Especially, Zn2+ was the most powerful inhibitor for NADPH-CoQ reductase, but CoQ10 reduction by the other CoQ10 reductases could not be inhibited by Zn2+. Furthermore, the reduction of the CoQ9 incorporated into HeLa cells was also inhibited by Zn2+ in the presence of pyrithione, a zinc ionophore. Moreover, NQO1 gene silencing in HeLa cells by transfection of a small interfering RNA resulted in lowering of both the NQO1 protein level and the NQO1 activity by about 75%. However, this transfection did not affect the NADPH-CoQ reductase activity and the reduction of CoQ9 incorporated into the cells. These results suggest that the NADPH-CoQ reductase located in cytosol may be the main enzyme responsible for the reduction of non-mitochondrial CoQ in cells.

Interaction of the Electrophilic Ketoprofenyl-Glucuronide and Ketoprofenyl-Coenzyme A Conjugates with Cytosolic Glutathione S-Transferases

Carboxylic acid-containing drugs are metabolized mainly through the formation of glucuronide and coenzyme A esters. These conjugates have been suspected to be responsible for the toxicity of several nonsteroidal anti-inflammatory drugs because of the reactivity of the electrophilic ester bond. In the present study we investigated the reactivity of ketoprofenyl-acylglucuronide (KPF-OG) and ketoprofenyl-acyl-coenzyme A (KPF-SCoA) toward cytosolic rat liver glutathione S-transferases (GST). We observed that KPF-SCoA, but not KPF-OG inhibited the conjugation of 1-chloro-2,4-dinitrobenzene and 4-nitroquinoline N-oxide catalyzed by both purified cytosolic rat liver GST and GST from FAO and H5-6 rat hepatoma cell lines. Photoaffinity labeling with KPF-SCoA suggested that the binding of this metabolite may overlap the binding site of 4-methylumbelliferone sulfate. Furthermore, high-performance liquid chromatography and mass spectrometry analysis showed that both hydrolysis and transacylation reactions were observed in the presence of GST and glutathione. The formation of ketoprofenyl-S-acyl-glutathione could be kinetically characterized (apparent K(m) = 196.0 +/- 70.6 microM). It is concluded that KPF-SCoA is both a GST inhibitor and a substrate of a GST-dependent transacylation reaction. The reactivity and inhibitory potency of thioester CoA derivatives toward GST may have potential implications on the reported in vivo toxicity of some carboxylic acid-containing drugs.

Abstract 390: VLDL Exits From The Endoplasmic Reticulum In A Specialized VLDL-transporting-vesicle (VTV) In Rat Primary Hepatocytes.

Transport of very low-density lipoprotein (VLDL) from its site of synthesis, the endoplasmic reticulum (ER), to the Golgi is required for its eventual secretion from hepatocytes. This step represents a potential therapeutic target in controlling VLDL export and thus control of its metabolic derivative, LDL, the major carrier of cholesterol and determinant of atherosclerosis. The present study was designed to understand how VLDL exits from the ER at the molecular level. We developed an in vitro ER-budding assay in which rat liver ER (500 ug) was pre-loaded with 14 C-triacylglycerol (TAG) to mark VLDL and 3 H-proteins to mark newly synthesized proteins. The ER was incubated with rat liver cytosol (1 mg), GTP, and ATP at 37 o C for 30 min. The reaction mix was fractionated on a continuous sucrose gradient and the distribution of 14 C-TAG and 3 H-protein across the gradient was determined. 14 C-TAG was found in the light density region of the gradient, the expected place for TAG-rich VLDL carrying vesicles whereas 3 H-proteins appeared in the mid portion, the expected place in the gradient for protein vesicles. We examined the distribution of apolipoprotein B100 (apoB100), a marker for VLDL and albumin (a typical liver secretory protein) across the same gradient by Western blotting. As expected, apoB100 was distributed in light fractions whereas albumin was mainly in the mid portion of the gradient. These data show that VLDL and albumin are transported in vesicles of differing density. We hypothesize that a specialized vesicle is utilized for VLDL transport, which we name the VLDL-transporting-vesicle (VTV). Our results show that the release of VTV from rat liver ER requires cytosol, GTP, Sar1 (a GTPase), ATP, and incubation at 37 o C. VTV was sealed as judged by apoB100 signal post proteinase K treatment. VTVs concentrate ApoB100, Sar1, and exclude ER-resident protein calnexin. VTV fuses with liver cis -Golgi and delivers its cargo, VLDL, to the Golgi lumen. 2D-gels and electron microscopy data reveal that VTVs are different in their protein composition and are larger in size when compared to albumin carrying vesicles. In conclusion, we have identified and characterized a new ER-derived vesicle, VTV, which transports nascent VLDL from the ER to the Golgi in primary hepatocytes.

In Vitro Metabolism of 8-2 Fluorotelomer Alcohol: Interspecies Comparisons and Metabolic Pathway Refinement

The detection of perfluorinated organic compounds in the environment has generated interest in their biological fate. 8-2 Fluorotelomer alcohol (8-2 FTOH, C(7)F(15)CF(2)CH(2)CH(2)OH), a raw material used in the manufacture of fluorotelomer-based products, has been identified in the environment and has been implicated as a potential source for perfluorooctanoic acid (PFOA) in the environment. In this study, the in vitro metabolism of [3-(14)C] 8-2 FTOH and selected acid metabolites by rat, mouse, trout, and human hepatocytes and by rat, mouse, and human liver microsomes and cytosol were investigated. Clearance rates of 8-2 FTOH in hepatocytes indicated rat > mouse > human >/= trout. A number of metabolites not previously reported were identified, adding further understanding to the pathway for 8-2 FTOH metabolism. Neither perfluorooctanoate nor perfluorononanoate was detected from incubations with human microsomes. To further elucidate the steps in the metabolic pathway, hepatocytes were incubated with 8-2 fluorotelomer acid, 8-2 fluorotelomer unsaturated acid, 7-3 acid, 7-3 unsaturated acid, and 7-2 secondary fluorotelomer alcohol. Shorter chain perfluorinated acids were only observed in hepatocyte and microsome incubations of the 8-2 acids but not from the 7-3 acids. Overall, the results indicate that 8-2 FTOH is extensively metabolized in rats and mice and to a lesser extent in humans and trout. Metabolism of 8-2 FTOH to perfluorinated acids was extremely small and likely mediated by enzymes in the microsomal fraction. These results suggest that human exposure to 8-2 FTOH is not expected to be a significant source of PFOA or any other perfluorocarboxylic acids.

BENZO(A)PYRENE METABOLISM: ROLE OF BIOALKYLATION

Previous studies of the metabolic activation of chemical carcinogens including benzo(a)pyrene have demonstrated that selected aromatic hydrocarbons can undergo a series of biological substitution reactions in tissue cytosol preparations when in the presence of the methyl donor S-adenosyl-L-methionine. Although identification of the methylated metabolites of carcinogens had been tentatively established using liquid chromatographic techniques, confirmation of the identity of these methylated derivatives has remained elusive. In the present study, the carcinogen benzo(a)pyrene was found to undergo methylation reactions in the presence of rat liver cytosol preparations to yield products that were identified as methylated, formyl and hydroxyalkyl derivatives of the parent carcinogen. Identification and confirmation of the identity of the 6-methylbenzo(a)pyrene metabolite was confirmed by both UV detection, gas chromatographic and mass spectral identification, and NMR analysis of metabolic fractions obtained from incubations of benzo(a)pyrene. The results conclusively demonstrate that the carcinogen benzo(a)pyrene undergoes biological substitution reactions to yield 6-methylbenzo(a)pyrene in the presence of cytosolic enzymes. The presence of 6-methylbenzo(a)pyrene as a metabolite of benzo(a)pyrene illustrates and confirms the presence of centers of reactivity in unsubstituted aromatic hydrocarbons. These centers of reactivity serve as sites for biochemical substitution reactions, yielding methyl substituted hydrocarbons. Furthermore, these methylated metabolites of polycyclic aromatic hydrocarbons are further metabolically activated to hydroxyalkyl and formyl derivatives which can be subsequently activated to electrophilic esters leading to DNA and protein reactive species.

BIOALKYLATION OF BENZ(A)ANTHRACENE: IMPLICATIONS FOR CARCINOGENESIS

Previous studies on the metabolic activation of unsubstituted polycyclic aromatic hydrocarbons have largely focused on the formation of oxidation products as a result of reaction of the parent hydrocarbon with microsomal enzymes. These products of reaction have included both epoxide as well as a diol epoxide metabolites. In addition, formation of quinones as well as hydroxyl substituted derivatives of various polycyclic aromatic hydrocarbons have been investigated as metabolic endpoints. Several studies have taken place illustrating the formation of these metabolites, as well as the formation of biologically relevant adducts in a variety of animal models as well as cell culture preparations. Although these metabolic products have been widely characterized and studied in a variety of systems, there have been limited studies characterizing the formation of methyl substituted derivatives of unsubstituted aromatic hydrocarbons, and the subsequent formation of hydroxyalkyl derivatives of various compounds. The present study investigates the formation of both alkyl and hydroxyalkyl substituted derivatives of the unsubstituted polycyclic aromatic hydrocarbon benz(a)anthracene in preparations of rat liver cytosol fortified with S-adenosyl-L-methionine. The results demonstrate that methylated derivatives are formed as metabolic products, and that the identity of these products are both the 7-methybenz(a)anthracene and 7,12-dimethylbenz(a)anthracene. These results confirm that aromatic hydrocarbons undergo biological substitution reactions at specific centers of reactivity of the unsubstituted molecule yielding bioalkylated derivatives.

The acyl-CoA thioesterase I is regulated by PPARα and HNF4α via a distal response element in the promoter

The cytosolic acyl-coenzyme A thioesterase I (Acot1) is an enzyme that hydrolyzes long-chain acyl-CoAs of C(12)-C(20)-CoA in chain length to the free fatty acid and CoA. Acot1 was shown previously to be strongly upregulated at the mRNA and protein level in rodents by fibrates. In this study, we show that Acot1 mRNA levels were increased by 90-fold in liver by treatment with Wy-14,643 and that Acot1 mRNA was also increased by 15-fold in the liver of hepatocyte nuclear factor 4alpha (HNF4alpha) knockout animals. Our study identified a direct repeat 1 (DR1) located in the Acot1 gene promoter in mouse, which binds the peroxisome proliferator-activated receptor alpha (PPARalpha) and HNF4alpha. Chromatin immunoprecipitation (ChIP) assay showed that the identified DR1 bound PPARalpha/retinoid X receptor alpha (RXRalpha) and HNF4alpha, whereas the binding in ChIP was abrogated in the PPARalpha and HNF4alpha knockout mouse models. Reporter gene assays showed activation of the Acot1 promoter in cells by the PPARalpha agonist Wy-14,643 after cotransfection with PPARalpha/RXRalpha. However, transfection with a plasmid containing HNF4alpha also resulted in an increase in promoter activity. Together, these data show that Acot1 is under regulation by an interplay between HNF4alpha and PPARalpha.

Metabolism in Vitro and in Vivo of the DNA Base Adduct, M1G

Oxidative damage is considered a major contributing factor to genetic diseases including cancer. Our laboratory is evaluating endogenously formed DNA adducts as genomic biomarkers of oxidative injury. Recent efforts have focused on investigating the metabolic stability of adducts in vitro and in vivo. Here, we demonstrate that the base adduct, M1G, undergoes oxidative metabolism in vitro in rat liver cytosol (RLC, Km = 105 microM and vmax/Km = 0.005 min-1 mg-1) and in vivo when administered intravenously to male Sprague Dawley rats. LC-MS analysis revealed two metabolites containing successive additions of 16 amu. One- and two-dimensional NMR experiments showed that oxidation occurred first at the 6-position of the pyrimido ring, forming 6-oxo-M1G, and then at the 2-position of the imidazole ring, yielding 2,6-dioxo-M1G. Authentic 6-oxo-M1G was chemically synthesized and observed to undergo metabolism to 2,6-dioxo-M1G in RLC (Km = 210 microM and vmax/Km = 0.005 min-1 mg-1). Allopurinol partially inhibited M1G metabolism (75%) and completely inhibited 6-oxo-M1G metabolism in RLC. These inhibition studies suggest that xanthine oxidase is the principal enzyme acting on M1G in RLC and the only enzyme that converts 6-oxo-M1G to 2,6-dioxo-M1G. Both M1G and 6-oxo-M1G are better substrates (5-fold) for oxidative metabolism in RLC than the deoxynucleoside, M1dG. Alternative repair pathways or biological processing of M1dG makes the fate of M1G of interest as a potential marker of oxidative damage in vivo.

A Proteomic Analysis of Bromobenzene Reactive Metabolite Targets in Rat Liver Cytosol in Vivo

Metabolic activation and protein covalent binding are early and apparently obligatory events in the cytotoxicity of many simple organic chemicals including drugs and natural products. Although much has been learned about the chemistry of reactive metabolite formation and reactivity toward protein nucleophiles, progress in identifying specific protein targets for reactive metabolites of various protoxins has been much slower. We previously reported nine microsomal and three cytosolic proteins as targets for reactive metabolites of bromobenzene in rat liver. These results, and contemporary work by others, indicate that protein covalent binding is not totally random in cells. Moreover, as protein targets for other protoxins were identified, little commonality of target proteins became apparent. In the present work, we used two-dimensional gel electrophoresis to separate liver cytosolic proteins from rats treated with 14C-bromobenzene; 110 of the 836 observed spots contained measurable radioactivity that varied over a 600-fold range of adduct density. Of these 110 spots, in-gel digestion coupled with mass spectrometry identified apparently single proteins in 57 spots. A few other spots clearly contained more than one identifiable protein, and in several cases, the same protein was identified in several spots having different apparent molecular masses and/or pI. Altogether, 33 unique new protein targets for bromobenzene metabolites were identified and compared to those known for acetaminophen, naphthalene, butylated hydroxytoluene, benzene, thiobenzamide, and halothane via a target protein database available at http://tpdb.medchem.ku.edu:8080/protein_database/. With increasing numbers of target proteins becoming known, more commonality in targeting by reactive metabolites from diverse chemical agents may be seen. Such commonality may help to separate toxicologically significant covalent binding events from a background of covalent binding that is toxicologically inconsequential.

SEC-ICP-MS and ESI-MS as tools to study the interaction between cisplatin and cytosolic biomolecules

Most of cisplatin’s citotoxic properties are due to the interaction of the drug with DNA. However, other biological molecules present in the cell cytosol, such as MT (metallothionein) and GSH (glutathione), are potential targets for cisplatin and have been related to its side-effects or with the cellular resistance mechanisms to the drug. Experiments simulating physiological conditions have been performed to study the specific cisplatin metabolites which interact with GSH and MT and to characterize the different drug–biomolecule adducts over time. A combination of size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) and electrospray ionization-mass spectrometry (ESI-MS) techniques has been used to provide sensible multi-elemental detection and structural information of the species of interest. Time dependent transformation of 10 μM cisplatin at neutral pH (7.4) produces different concentrations of the mono-aquo and oligomeric derivatives, as could be confirmed by ESI-MS. No di-aquo derivative was seen to be produced under these conditions at any of the incubation times used. Cisplatin and the oligomeric derivative were incubated with GSH and MT at different drug:biomolecule ratios. Adducts from cisplatin–GSH (1:500) and from cisplatin–MT (1:10) incubations were characterized by SEC-ICP-MS. While both GSH and MT reacted with cisplatin producing different compounds, only GSH reacted with the oligomeric derivative of cisplatin. SEC-ICP-MS experiments showed that, under neutral pH conditions, Cd atoms remained bound to the cisplatin:MT adducts, but Zn atoms were lost. Results were compared with those obtained by in vitro and in vivo experiments with rat kidney, liver and inner ear cytosols.

Identification of 1-furan-2-yl-3-pyridin-2-yl-propenone, an anti-inflammatory agent, and its metabolites in rat liver subcellular fractions

1-Furan-2-yl-3-pyridin-2-yl-propenone (FPP-3) has been characterized to have an anti-inflammatory activity through the inhibition of the production of nitric oxide and tumor necrosis factor-alpha. In the present studies, the phase 1 metabolism of FPP-3 was investigated in rat liver microsomes and cytosols. When FPP-3 was incubated with rat liver microsomes and cytosols in the presence of NADPH, 2 major peaks were detected on a liquid chromatography/electrospray ionization-mass spectrometry. Two metabolites (i.e., M1 and M2) were characterized as reduced forms on propenone: M1 (1-furan-2-yl-3-pyridin-2-yl-propan-1-one) was the initial metabolite and M2 (1-furan-2-yl-3-pyridin-2-yl-propan-l-ol) was a secondary alcohol believed to be formed from M1.

Site-Specific Arylation of Rat Glutathione S-Transferase A1 and A2 by Bromobenzene Metabolites in Vivo

The hepatotoxicity of bromobenzene (BB) derives from its reactive metabolites (epoxides and quinones), which arylate cellular proteins. Application of proteomic methods to liver proteins from rats treated with a hepatotoxic dose of [14C]-BB has identified more than 40 target proteins, but no adducted peptides have yet been observed. Because such proteins are known to contain bromophenyl- and bromodihydroxyphenyl derivatives of cysteine, histidine, and lysine, the failure to observe modified peptides has been attributed to the low level of total covalent binding and to the "dilution" effect of multiple metabolites reacting at multiple sites on multiple proteins. In this work glutathione S-transferase (GST), a well-known and abundant BB-target protein, was isolated from liver cytosol of rats treated with 14C-BB by use of a glutathione (GSH)-agarose affinity column and further resolved by reverse-phase high-performance liquid chromatography (HPLC) into subunits M1, M2, A1, A2 and A3. The subunits were identified by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whole-molecule mass spectrometry, and peptide mass mapping and found to contain radioactivity corresponding to 0.01-0.05 adduct per molecule of protein. Examination of tryptic digests of these subunits by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization mass spectrometry (ESI-MS) failed to reveal any apparent adducted peptides despite observed sequence coverages up to 87%. However, use of HPLC-linear ion-trap quadrupole Fourier transform mass spectrometry (LTQ-FTMS) to search for predicted modified tryptic peptides revealed peaks corresponding, with a high degree of mass accuracy, to a bromobenzoquinone adduct of peptide 89-119 in both GSTA1 and A2. The identity of these adducts and their location at Cys-111 was confirmed by tandem mass spectrometry (MS-MS). No evidence for the presence of any putative BB-adducts in GST M1, M2, or A3 was obtained. This work highlights the challenges involved in the unambiguous identification of reactive metabolite adducts formed in vivo.

Lysosome membrane lipid microdomains: novel regulators of chaperone-mediated autophagy

Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins in lysosomes. The limiting step of this type of autophagy is the binding of substrates to the lysosome-associated membrane protein type 2A (LAMP-2A). In this work, we identify a dynamic subcompartmentalization of LAMP-2A in the lysosomal membrane, which underlies the molecular basis for the regulation of LAMP-2A function in CMA. A percentage of LAMP-2A localizes in discrete lysosomal membrane regions during resting conditions, but it exits these regions during CMA activation. Disruption of these regions by cholesterol-depleting agents or expression of a mutant LAMP-2A excluded from these regions enhances CMA activity, whereas loading of lysosomes with cholesterol significantly reduces CMA. Organization of LAMP-2A into multimeric complexes, required for translocation of substrates into lysosomes via CMA, only occurs outside the lipid-enriched membrane microdomains, whereas the LAMP-2A located within these regions is susceptible to proteolytic cleavage and degradation. Our results support that changes in the dynamic distribution of LAMP-2A into and out of discrete microdomains of the lysosomal membrane contribute to regulate CMA.

Characterization of Alternative Cytosolic Forms and Cellular Targets of Mouse Mitochondrial Thioredoxin Reductase

Thioredoxin reductase (TR) and thioredoxin (Trx) define a major cellular redox system that maintains cysteine residues in numerous proteins in the reduced state. Both cytosolic (TR1 and Trx1) and mitochondrial (TR3 and Trx2) enzymes are essential in mammals, but the function of the mitochondrial system is less understood. In this study, we characterized subcellular localization of three TR3 forms that are generated by alternative first exon splicing and that differ in their N-terminal sequences. Only one of these forms resides in mitochondria, whereas the two other isoforms are cytosolic. Consistent with this finding, TR3 did not have catalytic preferences for mitochondrial Trx2 versus cytosolic Trx1, both of which could serve as TR3 substrates. Similarly, TR1 was equally active with Trx1, Trx2, or a bacterial Trx. We generated recombinant selenoprotein forms of TR1 and TR3 and found that these enzymes were inhibited by zinc, but not by calcium or cobalt ions. We further developed a proteomic method for identification of targets of TRs in mammalian cells utilizing affinity columns containing recombinant TR3 forms differing in C-terminal sequences. Using this procedure, we found that Trx1 was the major target of TR3 in both rat and mouse liver cytosol. The truncated form of TR3 lacking selenocysteine was particularly efficient in binding Trx1, consistent with the previously observed role of truncated TR1 in apoptosis. Overall, these data establish that the function of TR3 is not limited to its role in Trx2 reduction.

Limited Reactivity of Formyl Chloride with Glutathione and Relevance to Metabolism and Toxicity of Dichloromethane

Formyl chloride has been indirectly implicated as an intermediate in the oxidation of CH(2)Cl(2) and proposed to be a product of the oxidation of some other compounds. Formyl chloride was synthesized and added to aqueous solutions, with CO formed as a product. The presence of glutathione (GSH) did not reduce the yield of CO at any of the pH values tested. At pH >or= 9, a small amount of S-formyl GSH was detected (<or =3% of CO formed) and identified by comparison with synthetic material using mass spectrometry. Incubations of CH(2)Cl(2) with human liver microsomes at neutral pH produced mainly CO and only trace protein adducts (detected from (14)CH(2)Cl(2)) and no S-formyl GSH at the level of detection. Neither rat liver cytosol nor purified GSH transferase (rat) 5-5 or (human) T1-1 significantly enhanced the level of S-formyl GSH recovered in incubations with either NADPH-fortified microsomes and CH(2)Cl(2) or synthetic formyl chloride. The oxidation pathway is the principal route of metabolism of CH(2)Cl(2) at low doses in vivo, and previous literature assumes that the formyl chloride product is reactive with GSH and protein. We provide evidence that formyl chloride can react with GSH but that, in contrast to suggestions in the literature, the extent is very limited because of the known high rate of rearrangement to CO in aqueous solution. The very limited reaction of formyl chloride with nucleophiles is consistent with the low toxicity of CH(2)Cl(2), and formyl chloride binding cannot be used as an explanation for discrepancies in pharmacokinetic models [Gargas, M. L., Clewell, H. J., III, and Andersen, M. E. (1986) Toxicol. Appl. Pharmacol. 82, 211-223; Clewell, H. J., III (1995) Toxicology 102, 83-94].

Molecular Cloning of a Novel Type of Rat Cytoplasmic 17β-Hydroxysteroid Dehydrogenase Distinct from the Type 5 Isozyme

Rat liver contains two cytosolic enzymes (TBER1 and TBER2) that reduce 6-tert-butyl-2,3-epoxy-5-cyclohexene-1,4-dione into its 4R- and 4S-hydroxy metabolites. In this study, we cloned the cDNA for TBER1 and examined endogenous substrates using the homogenous recombinant enzyme. The cDNA encoded a protein composed of 323 amino acids belonging to the aldo-keto reductase family. The recombinant TBER1 efficiently oxidized 17beta-hydroxysteroids and xenobiotic alicyclic alcohols using NAD+ as the preferred coenzyme at pH 7.4, and showed low activity towards 20alpha- and 3alpha-hydroxysteroids, and 9-hydroxyprostaglandins. The enzyme was potently inhibited by diethylstilbestrol, hexestrol and zearalenone. The coenzyme specificity, broad substrate specificity and inhibitor sensitivity of the enzyme differed from those of rat NADPH-dependent 17beta-hydroxysteroid dehydrogenase type 5, which was cloned from the liver and characterized using the recombinant enzyme. The mRNA for TBER1 was highly expressed in rat liver, gastrointestinal tract and ovary, in contrast to specific expression of 17beta-hydroxysteroid dehydrogenase type 5 mRNA in the liver and kidney. Thus, TBER1 represents a novel type of 17beta-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution. In addition, TBER2 was identified as 3alpha-hydroxysteroid dehydrogenase on chromatographic analysis of the enzyme activities in rat liver cytosol and characterization of the recombinant 3alpha-hydroxysteroid dehydrogenase.

In vivo oxidative metabolism of a major peroxidation-derived DNA adduct, M1dG.

3-(2-Deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M1dG) is a DNA adduct arising from the reaction of 2-deoxyguanosine with the lipid peroxidation product, malondialdehyde, or the DNA peroxidation product, base propenal. M1dG is mutagenic in bacteria and mammalian cells and is present in the genomic DNA of healthy human beings. It is also detectable, albeit at low levels, in the urine of healthy individuals, which may make it a useful biomarker of DNA damage linked to oxidative stress. We investigated the possibility that the low urinary levels of M1dG reflect metabolic conversion to derivatives. M1dG was rapidly removed from plasma (t(1/2) = 10 min) after i.v. administration to rats. A single urinary metabolite was detected that was identified as 6-oxo-M1dG by MS, NMR spectroscopy, and independent chemical synthesis. 6-Oxo-M1dG was generated in vitro by incubation of M1dG with rat liver cytosols, and studies with inhibitors suggested that xanthine oxidase and aldehyde oxidase are involved in the oxidative metabolism. M1dG also was metabolized by three separate human liver cytosol preparations, indicating 6-oxo-M1dG is a likely metabolite in humans. This represents a report of the oxidative metabolism of an endogenous DNA adduct and raises the possibility that other endogenous DNA adducts are metabolized by oxidative pathways. 6-Oxo-M1dG may be a useful biomarker of endogenous DNA damage associated with inflammation, oxidative stress, and certain types of cancer chemotherapy.

Immobilization stress increases endogenous monoamine oxidase (MAO) inhibitor in rat liver

Although the physiological role of endogenous monoamine oxidase (MAO) inhibitor still remains unclear, the present study examined whether or not immobilization stress (IMMO) induce MAO inhibitor. An endogenous inhibitor of MAO was separated by gel filtration from 105,000 g supernate in rat liver cytosol following IMMO. The molecular weight of this inhibitor was estimated to be 500–600 by gel filtration. This inhibitor was proved to be heat-stable resistant to protease treatment. IMMO for 2 h significantly decreased MAO. These results suggest that this inhibitor is induced by IMMO. MAO activity in rat liver might be regulated by the level of this inhibitor.

Proteomic identification of glucocorticoid receptor interacting proteins

The glucocorticoid receptor (GR) acts as a ligand dependent transcription factor but can also cross talk with other signaling pathways via protein-protein interactions. In this paper we describe methods to study novel cytosolic GR interacting proteins, using mAb based immunoaffinity chromatography of GR from rat liver cytosol. Co-purifying proteins were identified by 2-DE in combination with MALDI-TOF-MS. Non-liganded/non-activated and in vitro liganded/activated GR, respectively, co-purifies with specific sets of proteins. Of these 34 were conclusively identified, seven have previously been reported to be part of the GR-complex, revealing 27 new possible interacting candidates for the GR-complex. Of the novel GR interacting proteins the major vault protein, TATA binding interacting protein 49a and glycoprotein PP63 were of special interest. Furthermore, using 2-D DIGE we show that the set of proteins interacting with non-liganded GR is distinctly different in protein amount compared to the proteins found with liganded/activated GR. This suggests the presence of different GR complexes in the cell, which was further substantiated by the finding of several separate GR native protein complexes, "GR-receptosomes", using blue native gel electrophoresis. Our findings suggest the existence of several new mechanisms for GR signaling and regulation.