Kidney Cytosol Research Articles

OFFGEL isoelectric focusing and polyacrylamide gel electrophoresis separation of platinum-binding proteins

In this work a 2D electrophoretic separation procedure able to maintain the integrity of platinum–protein bonds has been developed. The method is based on the use of sequential OFFGEL isoelectric focussing (IEF) and PAGE. A systematic study of the reagents used for PAGE, for OFFGEL-IEF separation, and post-separation treatment of gels (such as enzymatic digestion and sample preparation for MS analysis) was tackled regarding their suitability for the identification of platinum binding proteins using standard proteins incubated with cisplatin. The distribution of platinum in high and low molecular weight fractions (separated by cut-off filters) was determined by ICP-MS, which allows evaluating platinum–protein bond stability under the conditions studied. SDS-PAGE in the absence of β-mercaptoethanol or dithiotreitol preserved the platinum–protein bonds. In addition, neither the influence of the electric field during the electrophoretic separation, nor the processes of fixing, staining and destaining of proteins in the gel did result in the loss of platinum from platinum binding proteins. SDS-PAGE under non-reducing conditions provides separation of platinum-binding proteins in very narrow bands with quantitative recoveries. Different amounts of platinum-bound proteins covering the range 0.3–2.0 μg were separated and mineralised for platinum determination, showing good platinum linearity. Limits of detection for a mixture of five standard proteins incubated with cisplatin were between the range of 2.4 and 13.9 pg of platinum, which were satisfactory for their application to biological samples. Regarding OFFGEL-IEF, a denaturing solution without thiourea and without dithiotreitol is recommended. The suitability of the OFFGEL-IEF for the separation of platinum binding proteins of a kidney cytosol was demonstrated.

Metallothionein as Potential Biomarker of Cadmium Exposure in Persian Sturgeon (Acipenser persicus)

Metallothionein (MT) concentration in gills, liver, and kidney tissues of Persian sturgeon (Acipenser persicus) were determined following exposure to sublethal levels of waterborne cadmium (Cd) (50, 400, and 1,000 μg l(-1)) after 1, 2, 4, and 14 days. The increases of MT from background levels were 4.6-, 3-, and 2.8-fold for kidney, liver, and gills, respectively. The results showed that MT level change in the kidney is time and concentration dependent. Also, cortisol measurement revealed elevation at the day 1 of exposure and followed by MT increase in the liver. Cd concentrations in the cytosol of experimental tissues were measured, and the results indicated that Cd levels in the cytosol of liver, kidney, and gills increased 240.71-, 32.05-, and 40.16-fold, respectively, 14 days after exposure to 1,000 μg l(-1) Cd. The accumulation of Cd in cytosol of tissues is in the order of liver > gills > kidney. Pearson correlation coefficients showed that the MT content in kidney is correlated with Cd concentration, the value of which is more than in liver and gills. Thus, kidney can be considered as a tissue indicator in A. persicus for waterborne Cd contamination.

In vitro glutathione conjugation of methyl iodide in rat, rabbit, and human blood and tissues

Methyl iodide (MeI) is an intermediate in the manufacture of some pesticides and pharmaceuticals, and is under review for US registration as a non-ozone depleting alternative for methyl bromide for pre-plant soil fumigation. MeI is primarily metabolized via conjugation with glutathione (GSH), with further metabolism to S-methyl cysteine and methanethiol. To facilitate extrapolations of animal pharmacokinetic data to humans, rate constants for the GSH metabolism of MeI were determined in cytosols prepared from the liver and kidneys of rats, human donors, female rabbits, and rabbit fetuses, from rabbit olfactory and respiratory epithelium, and from rabbit and rat blood using a headspace vial equilibration technique and two-compartment mathematical model. MeI was metabolized in liver and kidney from adults of all three species, but metabolism was not detectable in fetal rabbit kidney. Maximal metabolic rates (Vmax) were similar in liver from rat and human donors (~40 and 47 nmol/min/mg, respectively) whereas the Vmax rates in kidney cytosols varied approximately three-fold between the three species. No difference was observed in the loss of MeI from active and inactive whole blood from either rats or rabbits. The metabolism in olfactory and respiratory epithelial cytosol had Michaelis–Menten constant (Km) values that were several times higher than for any other tissue, suggesting essentially first-order metabolism in the nose. The metabolism of MeI in human liver cytosol prepared from five individual donors indicated two potential populations, one high affinity/low capacity and one with a lower affinity but higher capacity.

Effects of a formula of components from Shengmai Powder on glucocorticoid receptor in rats after thermal injury

To investigate the effects of a formula of components from Shengmai Powder, a compound traditional Chinese herbal medicine, on glucocorticoid receptor (GR) in rats after thermal injury. A total of 32 male SD rats were randomly assigned into normal control group, untreated group, ginsenosides group and components group, with 8 rats in each group. Rats in the normal control group were intragastrically administered with normal saline (NS) at room temperature once daily. Rats in the untreated group were treated with NS before thermal injury, and rats in the components group and ginsenosides group were once daily treated with a mixture of aqueous extracts of Ophiopogonis Japoni, Fructus schizandrae Chinensis and ginsenosides and ginsenosides respectively. Rats were administered for one week. After the last administration, rats in the untreated group and treated groups underwent thermal injury for one hour, and then were sacrificed immediately by decapitation. Blood serum was collected, and the serum corticosterone (CS) and adrenocorticotropic hormone (ACTH) levels were determined with enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) respectively. The liver, lung and kidney homogenates were used to determine the GR binding capacity by radioligand receptor binding assay. The results were analyzed by one point analysis. GR binding capacities in liver, lung and kidney cytosols in the untreated group were obviously lower than those in the normal control group (P<0.01). The GR binding capacities in the liver and lung cytosols in the components group and the ginsenosides group were significantly higher than those in the untreated group (P<0.01), but no significant difference was found in kidney cytosol. Compared with the ginsenosides group, GR binding capacity in liver cytosol in the components group was increased (P<0.01), but there were no noticeable differences when compared with the GR binding capacities in lung and kidney cytosols. Serum CS and ACTH levels of the normal rats were (66+/-16)microg/L and (59+/-18) ng/L respectively. There were significant differences in CS and ACTH levels between the normal control group and the other groups (P<0.01), in which the serum CS and ACTH levels were (113+/-33)microg/L and (125+/-20) ng/L, (123+/-26) microg/L and (110+/-30) ng/L and (118+/-17) microg/L and (115+/-35) ng/L respectively. But there was no significant difference between the untreated group and the other treated groups. The formula of components from Shengmai Powder can enhance the effect of ginsenosides in up-regulating GR in rats after thermal injury.

Transcriptional repression mediated by 45-kDa calcium oxalate monohydrate binding protein

This study was done to investigate the DNA binding ability of a diagnostic biomarker, 45-kDa calcium oxalate monohydrate (COM) binding protein, isolated from human kidney and its effect on transcription. The 45-kDa COM binding protein was isolated and purified from human kidney. The subcellular localization of the protein and the amino acid composition of the protein were analyzed. Oxalate-binding activity in the presence or absence of DNA was determined. The possibility of forming DNA-protein adducts was checked by diethylaminoethyl (DEAE)-Sephadex column chromatography. The effect of the protein on in vitro transcription was also studied. The isolated 45-kDa protein was found to be basic in nature and its AACompIdent analysis showed it to be related to known transcription factors. The protein was found to be present in kidney cytosol and nucleus. The decreased oxalate binding activity in the presence of the DNA, and the shift in the DEAE-Sephadex elution profile established the DNA-binding ability of the protein. The in vitro transcription assay demonstrated the repression effect of the protein on gene expression during hyperoxaluria. Transcriptional repression by the 45-kDa COM binding protein in an in vitro transcription assay system was reduced in the presence of oxalate. Hence, altered expression of certain genes involved in the prognosis of urolithiasis might be mediated by this 45-kDa protein.

14C-labeled Pulegone and Metabolites Binding to α2u-globulin in Kidneys of Male F-344 Rats*

Pulegone is a major constituent of pennyroyal oil and a minor component of peppermint oil. Pulegone is biotransformed to menthofuran and menthones (diastereomeric menthone and isomenthone) in pennyroyal and peppermint as well as in rodents. Pulegone and menthofuran are hepatotoxic to rodents, and menthones are less toxic. The metabolism and disposition of pulegone and menthofuran were previously studied in rodents, and higher concentrations of pulegone- and menthofuran-derived radioactivity were observed in male than female rat kidney. One explanation is the association of pulegone and metabolites with a male rat-specific protein, α2u-globulin. To test this hypothesis, male and female rats were dosed orally with 14C-labeled pulegone (80 mg/kg, 120 μCi/kg) or menthofuran (60 mg/kg, 120 μCi/kg) or menthones (80 mg/kg, 120 μCi/kg) in corn oil, and the kidney cytosol was prepared 24 h after dosing. An equilibrium dialysis experiment showed that in all three studies the radioactivity was associated with kidney cytosol proteins of male but not female rats. The chemicals present in the male rat kidney cytosol after dialysis were extracted with dichloromethane and characterized by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC-MS). All parent compounds were detected, and the metabolites characterized included piperitone from pulegone or menthones treatment, menthones and possibly 8-hydroxymenthones from pulegone treatment, and mintlactones (diastereomeric mintlactone and isomintlactone) and 7a-hydroxymintlactone from menthofuran treatment. Analysis of the male rat kidney cytosol by a gel filtration column demonstrated that the retention was due to reversible binding of these chemicals with the male rat-specific protein α2u-globulin. However, binding of pulegone and/or metabolites to α2u-globulin did not produce accumulation of this protein in the kidney.

Rat NAD +-dependent 3α-hydroxysteroid dehydrogenase (AKR1C17): A member of the aldo-keto reductase family highly expressed in kidney cytosol

Mammalian 3α-hydroxysteroid dehydrogenases (3α-HSDs) have been divided into two types: Cytosolic NADP(H)-dependent 3α-HSDs belonging to the aldo-keto reductase family, and mitochondrial and microsomal NAD +-dependent 3α-HSDs belonging to the short-chain dehydrogenase/reductase family. In this study, we characterized a rat aldo-keto reductase (AKR1C17), whose functions are unknown. The recombinant AKR1C17 efficiently oxidized 3α-hydroxysteroids and bile acids using NAD + as the preferred coenzyme at an optimal pH of 7.4–9.5, and was inhibited by ketamine and organic anions. The mRNA for AKR1C17 was detected specifically in rat kidney, where the enzyme was more highly expressed as a cytosolic protein than NADP(H)-dependent 3α-HSD (AKR1C9). Thus, AKR1C17 represents a novel NAD +-dependent type of cytosolic 3α-HSD with unique inhibitor sensitivity and tissue distribution. In addition, the replacement of Gln270 and Glu276 of AKR1C17 with the corresponding residues of NADP(H)-dependent 3α-HSD resulted in a switch in favor of NADP + specificity, suggesting their key roles in coenzyme specificity.

Wish to die in end-stage ALS

We investigated the metabolism of hexafluoropropene, a nephrotoxic fluoroalkene, in rat liver and kidney subcellular fractions and in rats in vivo. Incubation of hexafluoropropene (1 mM) with microsomes and cytosol in the presence of glutathione (GSH) yielded S-(1,2,3,3,3-pentafluoropropenyl)glutathione (PPFG) and S-(1,1,2,3,3,3-hexafluoropropyl)glutathione (HFPG) as identified by thermospray mass spectrometry and 1H-NMR. In liver microsomes, PFPG formation was predominant (240 nmol/min/mg protein) over HFPG (36 nmol/min/mg), whereas in cytosol, HFPG was the only hexafluoropropene metabolite (136 nmol/min/mg) detectable. In kidney microsomes, GSH-conjugate formation could not be detected; in kidney cytosol, HFPG was exclusively formed (46 nmol/min/mg). Hexafluoropropene inhalation (800 ppm for 1 hr) in rats fitted with a biliary cannula resulted in the biliary elimination of PFPG without detectable formation of HFPG; the exclusively formed urinary metabolite, identified by GC/MS, was N-acetyl-S-(1,1,2,3,3,3-hexafluoropropy)-L-cysteine. The results show that hexafluoropropene is metabolized to two different GSH-conjugates in rat liver and kidney. The data also suggest that hexafluoropropene metabolites formed in the liver and eliminated with bile are not translocated to the kidney and that intrarenal bioactivation by GSH-conjugation may be responsible for hexafluoropropene-induced nephrotoxicity.

Inhibitory effects of diesel exhaust components and flavonoids on 20α-hydroxysteroid dehydrogenase activity in mouse tissues

The inhibitory effects of diesel exhaust components and flavonoids on 20α-hydroxysteroid dehydrogenase (20α-HSD) activity were examined in cytosolic fractions from the liver, kidney and lung of male mice. 9,10-Phenanthrenequinone (9,10-PQ) and 1,2-naphthoquinone (1,2-NQ), which are contained in diesel exhaust particles (DEPs), potently inhibited 20α-HSD activity in liver cytosol. 9,10-PQ also inhibited the enzyme activity in lung cytosol. However, 20α-HSD activity in kidney cytosol was little inhibited by 9,10-PQ or 1,2-NQ. Flavonoids such as quercetin, fisetin and kaempferol exhibited high inhibitory potencies for 20α-HSD activity in liver cytosol, whereas these flavonoids were poor inhibitors for the enzyme activity in kidney cytosol. It is likely that several diesel exhaust components and flavonoids augment the signaling of progesterone in the liver cells, by potently inhibiting 20α-HSD activity in mouse liver cytosol. The possibility that there are distinct enzymes catalyzing 20α-HSD activity in the non-reproductive tissues of male mice is also discussed.

Metal exposure assessment in native fish, Mullus barbatus L., from the Eastern Adriatic Sea

Metallothionein (MT) and metal (Zn, Cu, Fe, Mn, Cd) association in the cytosol of liver, kidney and intestine was studied on native Mullus barbatus specimens. Investigated parameters of all specimens (1 to 8-year-old) were related to the fish length, which reflects fish growth and aging. Cytosolic metal concentrations in liver, kidney and intestine are tissue specific but in general decrease as follows: Fe ≈ Zn > Cu > Mn > Cd. Metallothionein levels are also tissue specific with the highest level in intestine, followed by kidney and liver. Percentage partition of MT and Zn in liver, kidney and intestine cytosol of red mullet is comparable. Associations of MT and metals that induce MT synthesis exist in liver for MT/Zn ( r = 0.28) and MT/Cu ( r = 0.26) and in intestine for MT/Cu ( r = 0.38). Unlike essential metals, there is no significant correlation between MT and the toxic metal Cd, what is ascribed to its very low cytosolic concentration. The most pronounced size-related accumulation is found for the toxic metal Cd in liver cytosol ( r = 0.69), indicating chronic Cd accumulation. Negative correlation between metals (Cd, Zn, Fe, Mn) and the condition factor also indicates chronic metal effects and their metabolic impact.

Cadmium Concentration and Subcellular Distribution in Organs of the Mussel Crenomytilus Grayanus from Upwelling Regions of Okhotsk Sea and Sea of Japan

Cadmium (Cd) accumulation and subcellular distribution in the digestive gland and kidney of mussel Crenomytilus grayanus from naturally Cd elevated areas was studied. Mussels were collected from three sites of Okhotsk Sea and Sea of Japan: control area (site 1), seasonal upwelling region (site 2), and stationary upwelling region (site 3). Mussel from site 3 was shown to accumulate a significantly increased Cd concentration in the digestive gland and an extremely high Cd concentration (1780+/-732 microg g-1 dry weight) in the kidney. Cd was mainly sequestered into the kidney cytosol of the mussels from both upwelling regions and control sites (73% to 96%). However, digestive gland cytosol bound about 55% of Cd for the mussels from sites 1 and 2 and only 22.4% of Cd for the mussel from site 3. In the organs of the mussels from upwelling regions, the most of cytosolic Cd was associated with the protein fraction corresponding to molluscan metallothioneins. MTLP was isolated from kidney cytosol of the mussels from site 3 (powerful upwelling regions) by gel filtration chromatography, whereas in the organs studied of the mussels from other sites, MTLP content was below the detection limit.

Characterization of Phenotypes inGstm1-null Mice by Cytosolic and in Vivo Metabolic Studies Using 1,2-Dichloro-4-Nitrobenzene

Glutathione S-transferase Mu 1 (GSTM1) has been regarded as one of the key enzymes involved in phase II reactions in the liver, because of its high expression level. In this study, we generated mice with disrupted glutathione S-transferase Mu 1 gene (Gstm1-null mice) by gene targeting, and characterized the phenotypes by cytosolic and in vivo studies. The resulting Gstm1-null mice appeared to be normal and were fertile. Expression analyses for the Gstm1-null mice revealed a deletion of Gstm1 mRNA and a small decrease in glutathione S-transferase alpha 3 mRNA. In the enzymatic study, GST activities toward 1,2-dichloro-4-nitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB) in the liver and kidney cytosols were markedly lower in Gstm1-null mice than in the wild-type control. Gstm1-null mice had GST activities of only 6.1 to 21.0% of the wild-type control to DCNB and 26.0 to 78.6% of the wild-type control to CDNB. After a single oral administration of DCNB to Gstm1-null mice, the plasma concentration of DCNB showed larger AUC0-24 (5.1-5.3 times, versus the wild-type control) and higher Cmax (2.1-2.2 times, versus the wild-type control), with a correspondingly lower level of glutathione-related metabolite (AUC0-24, 9.4-17.9%; and Cmax, 9.7-15.6% of the wild-type control). In conclusion, Gstm1-null mice showed markedly low ability for glutathione conjugation to DCNB in the cytosol and in vivo and would be useful as a deficient model of GSTM1 for absorption, distribution, metabolism, and excretion/toxicology studies.

Age- and tissue-dependent metallothionein and cytosolic metal distribution in a native Mediterranean fish, Mullus barbatus, from the Eastern Adriatic Sea

The levels of metallothionein (MT), a biomarker of metal exposure, and of cytosolic metals (Zn, Cu, Cd), known as MT inducers, were investigated as variables of age (1 to 8 years) and tissue mass (liver, kidney, brain) of red mullet ( Mullus barbatus). Within the age from 1 to 8 years the most significant increase is evident for cytosolic Cd in liver (43-fold) and in kidney (5-fold). MT and essential metals are constant with age or slightly increased. Over the growth period, statistically significant MT and metal increase is evident only between 1 and 6–8 years old specimens, while for Cd in liver and kidney cytosol significant increase already exists at 4 years old specimens. Metal distribution in all tissues follows the order: Zn > Cu > Cd, with even 500–800 times lower Cd levels than essential metal levels. Consequently, MTs follow the levels of essential metals, Zn and Cu, indicating MT involvement in homeostasis of essential metals. In contrast to kidney and brain, hepatic MT levels are not age-dependent. Inclusion of hepatic MT measurements and the associated cytosolic metals will be useful in the assessment of long-term metal effects in demersal fish M. barbatus.

Subcellular Distribution of Heavy Metals in Organs of BivalveModiolus Modiolus Living Along a Metal Contamination Gradient

Concentration and distribution of Fe, Zn, Cu, Cd, Mn, Pb, Ni among subcellular fractions (cellular membrane structures and cytosol) and Zn, Cu, Cd among cytoplasmic proteins in the kidney and digestive gland of musselModiolus modiolus living along a polymetallic concentration gradient were studied. It was found in the kidney ofM. modiolus from contaminated sites that the Fe percent increased in the “membrane” fraction, whereas Zn, Pb, Ni and Mn percent increased in the cytosol compared to the kidney of the control mussel. Note kidney cytosol ofM. modiolus from clean and contaminated sites sequestered major parts of Cu and Cd. In the digestive gland ofM. modiolus from contaminated sites Fe, Zn, Cd, Mn, Ni percent increased in the “membrane” fraction, whereas Cu, Pb percent increased in the cytosol compared to digestive gland of control mussel. Gel-filtration chromatography shows kidney ofM. modiolus contains increased metallothionein-like protein levels irrespective of ambient dissolved metal concentrations. It was shown that the metal detoxification system in the kidney and digestive gland ofM. modiolus was efficient under extremely high ambient metal levels. However, under complex environmental contamination in the kidney ofM. modiolus, the metal detoxification capacity of metallothionein-like proteins was damaged.

Characteristics and inhibition by flavonoids of 20α-hydroxysteroid dehydrogenase activity in mouse tissues

Progesterone was stereoselectively reduced to a metabolite 20α-hydroxy-4-pregnen-3-one in the cytosolic fraction from the liver of male mice, indicating that the reduction of progesterone is catalyzed by 20α-hydroxysteroid dehydrogenase (20α-HSD). The cytosolic 20α-HSD activity was observed not only in the liver, but also in the kidney and lung. In liver cytosol, both NADPH and NADH were effective as cofactors for 20α-HSD activity, although NADPH was better than NADH for the enzyme activity. On the other hand, 20α-HSD activity in kidney cytosol required only NADPH as a cofactor. No significant sex-related difference of 20α-HSD activity was observed in liver and kidney cytosols. Flavonoids have been reported to inhibit the biosynthesis and metabolism of steroids. However, little is known about inhibitory effects of flavonoids on 20α-HSD activity. Thus, the effects of 16 flavonoids on 20α-HSD activity were examined, using liver cytosol of male mice. Among flavonoids tested, fisetin, apigenin, naringenin, luteolin, quercetin and kaempferol exhibited high inhibitory potencies for the 20α-HSD activity. We propose the possibility that these flavonoids augment progesterone signaling by inhibiting potently 20α-HSD activity in non-reproductive tissues.

Transamination in the Metabolism of the Nephrotoxicant N-(3,5-Dichlorophenyl)succinimide in Rats

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is nephrotoxic in rats. Due to the involvement of NDPS metabolism in its mechanism of toxicity, the detailed biotransformation of 14C-NDPS in rats was previously evaluated using high-performance liquid chromatography-electrospray ionization-mass spectrometry. In the present report, we describe the identification of two novel amino metabolites of NDPS, which were present in significant amounts in rat kidney tissues. Using liquid chromatography-tandem mass spectrometry and synthetic standards, the two metabolites were identified as N-(3,5-dichlorophenyl)-2-aminosuccinamic acid (2-NDASA) and its N-acetylated derivative (N-acetyl-2-NDASA). The mechanism of formation of 2-NDASA was studied in vitro. Incubations were carried out in rat liver and kidney cytosols using the major oxidative metabolite of NDPS, N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid, as the substrate. Formation of 2-NDASA in vitro was confirmed using mass spectrometry. Inhibitors of alcohol dehydrogenase (4-methylpyrazole) and aldehyde dehydrogenase (disulfiram) reduced 2-NDASA formation by 40 to 50%. Menadione (an inhibitor of aldehyde oxidase) and quercetin (an inhibitor of carbonyl reductase) did not show any effects. (Aminooxy)acetic acid, an inhibitor of pyridoxal 5'-phosphate-containing enzymes such as aminotransferases, almost completely abolished the formation of 2-NDASA. Using liquid chromatography-mass spectrometry, the transamination mechanism was further supported by the incorporation of a 15N-amino group in 2-NDASA when 15N-glutamic acid was included in the incubation mixture. Results from these studies show that transamination is a metabolic pathway in the clearance of NDPS in rats, and that cytosolic dehydrogenases and aminotransferases may be involved in this process.

Subcellular Distribution and Protein Binding of Perfluorooctanoic Acid in Rat Liver and Kidney

Perfluorooctanoic acid (PFOA) is an organic fluorochemical, and its elimination in rats is markedly sex-dependent. Liver and kidney are two primary tissues of distribution of PFOA in rats. In this study, the subcellular distribution of PFOA in male and female rat liver and kidney was examined. The results demonstrated that PFOA content in the liver cytosol of the female rat was significantly higher (49 ± 6% of total radioactive residues, TRR) than in the male liver (26 ± 5% TRR), whereas PFOA distribution in the heavier subcellular fractions, especially the nuclei and cell debris fraction, was marginally higher in male rat liver. In rat kidney, more than 70% of PFOA was distributed in the cytosolic fraction, with no significant difference between sexes. The degree of protein binding of PFOA in rat liver and kidney cytosol was analyzed by two different chromatographic methods. The percentage of protein-bound PFOA in the liver cytosol was found to be approximately 55% in both male and female rats. In contrast, significantly more PFOA was bound to cytosolic proteins in the kidney of male rats (42 ± 6% TRR) than in females (17 ± 5% TRR). Ligand blotting analysis revealed that multiple proteins from the liver cytosol, nuclei, and mitochondria fractions were capable of specific binding to PFOA.

The interactions of androgen receptor with poly(A)-containing RNA and polyribonucleotides.

The androgen receptor from mouse kidney cytosol binds not only to DNA but also to RNA as assayed by competition with DNA-cellulose centrifugation or by direct binding with agarose-polynucleotides. The dihydrotestosterone-receptor complex interacts more strongly to poly(A)-containing mRNA than to native natural DNA; it binds much more tightly to synthetic poly(G) than to natural DNA and other homopolyribonucleotides such as poly(U), poly(A), or poly(C). Poly(U) is moderately effective in competing for the complex, whereas poly(A) and poly(C) re not effective. Competition studies with heteropolyribonucleotides show that polymers containing G are most effective on binding to the androgen receptor, whereas those containing U are moderately effective, and those containing only A and C are least effective. Several analogues of poly(G), such as poly(X), poly(I), and poly(m7G) interact very well with the dihydrotestosterone-receptor with some differences in the bindings. A double-stranded polymer, poly(I) . poly(C) competes poorly for the androgen receptor. The observations that the androgen receptor binds to RNA and interacts with polyribonucleotides selectively suggest that androgen receptor-RNA interaction could play important roles in gene regulation.

Subcellular Distribution and Protein Binding of Perfluorooctanoic Acid in Rat Liver and Kidney

Perfluorooctanoic acid (PFOA) is an organic fluorochemical, and its elimination in rats is markedly sex-dependent. Liver and kidney are two primary tissues of distribution of PFOA in rats. In this study, the subcellular distribution of PFOA in male and female rat liver and kidney was examined. The results demonstrated that PFOA content in the liver cytosol of the female rat was significantly higher (49 ± 6% of total radioactive residues, TRR) than in the male liver (26 ± 5% TRR), whereas PFOA distribution in the heavier subcellular fractions, especially the nuclei and cell debris fraction, was marginally higher in male rat liver. In rat kidney, more than 70% of PFOA was distributed in the cytosolic fraction, with no significant difference between sexes. The degree of protein binding of PFOA in rat liver and kidney cytosol was analyzed by two different chromatographic methods. The percentage of protein-bound PFOA in the liver cytosol was found to be approximately 55% in both male and female rats. In contrast, significantly more PFOA was bound to cytosolic proteins in the kidney of male rats (42 ± 6% TRR) than in females (17 ± 5% TRR). Ligand blotting analysis revealed that multiple proteins from the liver cytosol, nuclei, and mitochondria fractions were capable of specific binding to PFOA.

Strain‐ and sex‐related differences of carbonyl reductase activities in kidney microsomes and cytosol of rats

This study was designed to elucidate strain- and sex-related differences of carbonyl reductase activity in rat kidney by using the oral antidiabetic drug acetohexamide as substrate. The frequency distribution of carbonyl reductase activities in kidney microsomes of male Fischer 344 (Fischer), Sprague-Dawley, Wistar and Wistar-Imamichi (Wistar-IM) rats exhibited a marked strain-related difference. Furthermore, the enzyme activities in kidney microsomes of Fischer, Sprague-Dawley and Wistar rats were male-specific, resulting insignificant sex-related differences in these strains. There was no sex-related difference of carbonyl reductase activity in kidney microsomes of the Wistar-IM strain, which lacked its activity in both sexes. On the other hand, although carbonyl reductase activities were fully detectable in kidney cytosols from all the strains of male and female rats, no strain- or sex-related difference was observed among the cytosolic enzyme activities. These results provide new information for understanding the influence of internal factors on the renal metabolism of ketone-containing xenobiotics.