Selenium regulates gene expression for estrogen sulfotransferase and alpha 2U-globulin in rat liver

Dietary intake of the essential trace element selenium (Se) regulates expression of genes for selenoproteins and certain non-Se-containing proteins. However, these proteins do not account for all of Se's biological effects. The objective of this work was to identify additional genes whose expression is regulated by Se. Identification of these genes may reveal new functions for Se or define mechanisms for its biological effects. Weanling male Sprague-Dawley rats were fed a Torula yeast-based Se-deficient basal diet or the same diet supplemented with 0.5 mg Se/kg diet as sodium selenite for 13 weeks. Total RNA was used as template for RNA fingerprinting. Two differentially expressed cDNA fragments were identified and cloned. The first had 99% nucleotide identity with rat liver estrogen sulfotransferase (EST) isoform-6. The second had 99% nucleotide sequence identity with rat liver alpha 2u-globulin. The mRNA levels for both were markedly reduced in Se deficiency. Laser densitometry showed that EST mRNA in Se deficiency was 7.3% of that in Se-adequate rat liver. The level of alpha 2u-globulin mRNA in Se-deficient rat liver was only 12.6% of that in Se-adequate rat liver. These results indicate that dietary Se may play a role in steroid hormone metabolism in rat liver.     

Hydroxysteroid sulfotransferase activity in the rat brain and liver as a function of age and sex

The high concentrations of dehydroepiandrosterone sulfate and pregnenolone sulfate in the mammalian brain, despite the blood-brain barrier's impermeability to these compounds, and the apparent independence of these concentrations from those in plasma prompted us to investigate whether enzymatic sulfation of dehydroepiandrosterone was detectable in the rat brain. Low hydroxysteroid sulfotransferase activities were detectable in in vitro incubations of homogenates from all rat brain regions except the cerebellum, being highest in the hypothalamus and pons. This activity was not ascribable to enzyme in brain capillary blood. The activity was mainly cytosolic, although there was also significant activity in the partially purified nuclear fraction. The enzyme had different properties from those of hepatic isozymes, with a pH optimum of 6.5 and a high Km of approximately 2 mM for dehydroepiandrosterone. The enzyme was also active with pregnenolone as substrate. Activities in the brain were approximately 300-fold lower than in the liver but, as in the liver, these were higher in females than in males. The variations in brain activity as a function of age did not parallel those in the liver. Relatively high activities were found in the fetal brain and declined at birth, while activities were insignificant in the fetal liver and rose following birth. There was a major peak in activity in pubertal female brains, but this peak was less important, and later, in males. No evidence was found to indicate that the low brain enzyme activities and high Km were attributable either to the presence of an inhibitor or to the steroid sulfation actually being a secondary activity of another brain sulfotransferase. We discuss whether the sulfotransferase activities found are adequate to synthesize the dehydroepiandrosterone and pregnenolone sulfate found in brain.     

Hexokinase isoenzymes in the rat placenta

The phosphorylation of glucose to glucose-6-phosphate, the first enzymatic step for glucose utilization is catalysed by a family of four hexokinase isoenzymes (HKI-IV) which display a tissue-specific distribution. The expression of HK isoenzymes was investigated in the rat placenta. High levels of HKI and HKII mRNA were found in the junctional and the labyrinthine zones. HKIII mRNA was present at low levels in the junctional zone and glucokinase (HKIV) mRNA was not detected, indicating that HKI and HKII are the two major placental HK isoenzymes. HKII activity was increased in placenta of insulinopenic diabetic rats. This regulation is likely to support the increase in glucose utilization and storage characteristics of the enlarged placentae of diabetic rats.     

Heterogeneous expression of P450 2C12 mRNA in female rat liver

The heterogeneous expression of P450 2C12 mRNA was studied in the rat liver. Liver was sampled from female rat, approximately 40 mg portions being removed from 18 different locations. Total RNA was isolated and subjected to northern blot analysis. The membrane was hybridized with a (32)P-labeled rat P450 2C12 gene-specific oligonucleotide probe. After the hybridized P450 2C12 gene-specific probe had been washed out, the membrane was hybridized again with a rat oligonucleotide probe for the detection of actin mRNA expression. In comparison with actin mRNA expression, P450 2C12 mRNA was not evenly expressed throughout the 18 different locations in the liver. The present results show the heterogeneous expression of P450 2C12 mRNA in the rat liver.     

Molecular cloning of three sulfotransferase cDNAs from mouse liver

Sulfate conjugation plays an important role in the biotransformation of not only xenobiotics but also many endogenous substances. Sulfotransferases, the enzymes that are responsible for this process, exist as a superfamily of genes. It has long been recognized that significant species differences exist among drug and carcinogen metabolizing enzymes such as cytochrome P450. Species differences in both regulation and catalytic activities of sulfotransferases may also exist. To investigate this, we conducted cDNA cloning and cDNA expression studies of sulfotransferase in the mouse. Three sulfotransferase cDNA clones were isolated from a female B6CBA mouse liver. Two of the clones, mSTa1 and mSTa2, were highly homologous to each other. Alignment of mSTa1 and mSTa2 cDNAs' nucleotide sequences with those of other sulfotransferase cDNAs revealed the greatest sequence identity with the rat STsmp cDNA. This analysis suggests that mSTa1, mSTa2 and rSTsmp cDNAs are derived from orthologous genes belonging to the alcohol/hydroxysteroid sulfotransferase gene family. The third clone, mSTp1 showed high identity to rSTp, hSTp1, hSTp3, and rSTp1C1, suggesting that mSTp1 belongs to the phenol family.     

Heterogeneous distribution of the sodium-dependent alanine transport activity in the rat hepatocyte plasma membrane

The localization of the sodium-dependent alanine uptake activity in rat liver cells was studied. Fractions representative of the canalicular, the contiguous (lateral) and the blood-sinusoidal surface of the hepatocyte were isolated by means of centrifugal fractionation and density gradient centrifugation. The distribution of various marker-enzyme activities in conjunction with the occurrence of alanine transport activity was studied both in fractions obtained after zonal density gradient centrifugation, and in the subcellular fractions mentioned above. It is concluded that the sodium-dependent alanine transport activity is primarily located in the blood-sinusoidal plasma membrane of the hepatocyte.     

Changes in cardiac output distribution after liver dearterialization in the rat

The changes in cardiac output distribution in rats have been studied after hepatic artery ligation and additional ligament division. These procedures resulted initially in a marked decrease in the arterial blood supply to the liver and this effect was pronounced still four weeks after the operation. Evidence was found that the decrease in liver blood supply induced by the procedure was in part counterbalanced by arteriovenous shunting in the preportal region. Furthermore, a temporary redistribution of cardiac output with reduced fractions to colon and spleen was found two weeks after these operations.     

Size distribution of rat liver nuclear RNA containing mRNA sequences

Total rat liver poly(A)-containing polysomal mRNA was size-fractionated on polyacrylamide gels in 98% formamide. Complementary DNA (cDNA) was prepared from the 8--14-S mRNA fraction and separated into sequences representing abundant and non-abundant mRNAs. The cDNA complementary to the abundant small mRNA of the rat liver cell (approximately 20 species) was hybridized to nuclear RNA of different lengths to determine the size distribution of nuclear RNA molecules which contain these messenger sequences. It was found that: 1. All abundant 8--14-S poly(A)-containing mRNAs have larger nuclear precursor molecules; 20% of the different messenger sequences are found in nuclear RNA of several times their cytoplasmic length. 2. 70% of the mass of the examined nuclear messenger sequences is in RNA molecules of a size similar to their polysomal mRNA; 30% are in larger than 18-S RNA and 2% are between 37 S and 44 S. 3. The majority of small messenger-containing RNA molecules in the RNA prepared from isolated nuclei are of true nuclear origin, since their frequency distribution differs significantly from that of the polysomal 8--14-S mRNA.     

Bacterial expression and characterization of a cDNA for human liver estrogen sulfotransferase

A distinct human estrogen sulfotransferase (hEST-1) cDNA has been isolated from a human liver lambda Zap cDNA library using a PCR procedure. The enzymatically active protein has been expressed in two bacterial expression systems and the kinetic and immunologic properties of the enzyme have been characterized. The full-length cDNA for hEST-1 is 994 base pairs in length and encodes a 294 amino acid protein with a calculated molecular mass of 35,123 Da. Purified hEST-1 migrated with an apparent molecular mass of 35,000 Da during SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of hEST-1 expressed in E. coli with a rabbit anti-hEST-1 antibody yields a band of approximately 35,000 Da. The anti-hEST-1 antibody also detects a single band in human liver and jejunum cytosol which migrates with the same molecular mass as expressed hEST-1. There was also no cross-reactivity of hEST-1 with rabbit anti-hP-PST or rabbit anti-hDHEA-ST antibodies upon immunoblot analysis. hEST-1 was expressed in bacteria and purified to homogeneity. Expressed hEST-1 activity has a significantly greater affinity for estrogen sulfation than that found for the other human STs which conjugate estrogens. hEST-1 maximally sulfates beta-estradiol and estrone at concentrations of 20 nM. hEST-1 also sulfates dehydroepiandrosterone, pregnenolone, ethinylestradiol, and 1-naphthol, at significantly higher concentrations; however, cortisol, testosterone and dopamine are not sulfated. The results presented in this paper describe the expression and characterization of a human EST distinct from other human STs which sulfate estrogens. The high affinity of hEST-1 for estrogens indicates that this ST may be important in both the metabolism of estrogens and in the regulation of their activities.     

Metabolic effects and distribution space of flufenamic acid in the isolated perfused rat liver

Most HIV prevention programs for women target individual risk behaviors while the influence of larger contextual factors, such as city of residence, are often neglected. This preliminary study compares women drug users from two different cities in the largely rural state of Kentucky on HIV risk behaviors. The women are from Lexington, a medium sized metropolitan area, and from Louisville, a large metropolitan area. Comparisons between the women from the two cities indicate that there are many similarities in their risk behaviors, but also some important differences. The women from Lexington (the smaller city), are more likely to be at risk for becoming infected with HIV due to their drug use, while the women from Louisville (the larger city) are more likely to be at risk because of their sex exchange practices and economic situation. The implications for prevention are discussed.     

The effects of vasoconstrictors on distribution of ischemic tissue flow in isolated perfused rat liver

Discusses plans of marriage among students and the problems of student families. Comprehensive sociohygienic study of the health status and communal conditions was carried out among the students of the Russian Peoples' Friendship University, including married students with children. This social group is characterized by a peculiar life style, intensive intellectual and social activity; moreover, it is the most favorable period for marriage and childbirth. Special attention is paid to difficulties (material, communal, educational, etc.) experienced by the female students becoming mothers in the course of studies. Specific medicodemographic tendencies brought the authors to a conclusion that development and introduction of programs of medicosocial support for student families, specifically, mothers, is needed, which should be aimed at the soonest possible solution of the acute problems of maintaining the health of mothers and their children.     

Estrogen sulfotransferase expression in the human liver: marked interindividual variation and lack of gender specificity

Estrogen sulfotransferase (EST) catalyzes the specific sulfonation of estrogen at the 3'-hydroxyl position using 3'-phosphoadenosine-5'-phosphosulfate as an activated sulfate donor. Sulfonation renders the hormone biologically inactive as well as changing its half-life within the human body. Studies in the rat and mouse have suggested that expression of EST in the liver is age- and sex-dependent, being prominent only in sexually mature young males. Although a human EST cDNA has previously been cloned, the characteristics of hepatic EST expression in human subjects remain to be defined. In this study, we have investigated and compared the expression of EST in 10 human liver samples by using an EST-specific antibody and performing enzyme activity assays. We found a marked interindividual variation (up to 25-fold) in the hepatic expression of EST. However, EST protein level in the human liver is correlated neither with gender nor with age. Interestingly, paired-group analysis revealed a statistically significant difference in the hepatic expression of EST protein and activity between alcohol users and nonusers. We conclude that, unlike what is observed in the rodent liver, EST expression in the human liver is not sex-limited. Thus hepatic EST may play a role in estrogen metabolism and homeostasis in both genders of human subjects. The marked individual variation suggests that EST gene expression is subject to sensitive control by genetic or environmental factors. The potential correlation between alcohol consumption and hepatic EST expression deserves further evaluation.     

Separation and quantification of corticosteroid-induced, bone and liver alkaline phosphatase isoenzymes in canine serum

Quantifying alkaline phosphatase (ALP) isoenzymes in canine serum would provide a useful index in a clinical laboratory. To achieve this goal, we tested a semi-automatic assay combining wheat germ lectin (WGL) precipitation and chemical inhibition of isoenzymes of the TNS gene with levamisole to quantify bone ALP (BALP) and corticosteroid-induced ALP (CALP), respectively. The liver ALP (LALP) isoenzyme was then calculated from the equation: TALP = BALP + LALP + CALP BALP, LALP and CALP standards from serum of puppies, bile-duct ligated dogs and dogs on 4.4 mg/kg/day prednisolone for 30 days, respectively, were used. The suitability of standard sera was tested by affinity electrophoresis. Levamisole (4.2 mM) inhibits 98% of BALP and LALP but only 42% CALP. Multiplying measured CALP by 1.8 gives the total CALP value in serum. WGL precipitated 92.3% BALP, 23.3% LALP and 26.8% heated CALP standards. These values were used to adjust precipitated ALP to obtain the exact levels of BALP. WGL was then tested on pooled serum standards in which the relative proportions of all the ALPs were known and controlled. BALP was adequately quantified except when LALP and CALP levels were extremely high. The assay was also applicable under conditions resulting in high ALP. Therefore, combining WGL and levamisole inhibition provides an adequate separation and quantification of canine ALP isoenzymes. The method has great potential for diagnostic use and should be tested further for routine implementation.     

Subcellular distribution of curium in beagle liver

1. A lamellar body-enriched fraction was prepared from rabbit lung and characterized by electron microscopy, surface activity studies, phospholipid assay and marker enzymes. 2. Both phospholipases A1 and A2 were found to be present in lamellar bodies. After these had been ruptured both enzymes were found to be principally in the soluble phase. 3. The possible roles for phospholipases in lamellar body development and in the respiratory distress syndrome of the newborn are discussed.     

Crude liver membrane fractions and extracellular matrix components as substrata regulate differentially the preservation and inducibility of cytochrome P-450 isoenzymes in cultured rat hepatocytes

The influence of cell-substrata interactions on the preservation of basal or in-vivo-induced microsomal cytochrome P-450 isoenzyme contents in cultured rat hepatocytes and on the adaptive responses after exposure to phenobarbital or 3-methylcholanthrene in vitro, was investigated. Hepatocytes from untreated or phenobarbital-treated rats were cultured in serum-free, aprotinin-supplemented culture medium in 96-well microtiter plates coated with collagen type I (COL), laminin, fibronectin or crude liver membrane fractions/collagen type I (CMF/COL). Basal cell functions were characterized by measuring the total protein content and lactate dehydrogenase release. The relative contributions of CYP1A1/2, CYP2B1/2, CYP2C6, CYP2C11, CYP3A and CYP4A isoenzymes were determined with ELISA using monoclonal antibodies raised against purified cytochromes P-450 from rat liver microsomes. The characterization of the CMF revealed that contaminations with mitochondria, nuclei and lysosomes are relatively low. Among these, membranes derived from the endoplasmic reticulum appeared to be the major organelle contaminant of the CMF. The matrix components laminin, fibronectin and collagen type IV were found in appreciable amounts. Hepatocytes from untreated rats, cultured for up to nine days on CMF/COL-coated plates, retained their relative cytochrome P-450 contents at 1.5-3-fold higher levels when compared to cells cultured on COL, fibronectin or laminin. Similarly, hepatocytes from phenobarbital-treated rats preserved the contents of barbiturate-inducible CYP2B1/2 and CYP3A proteins best when cultured on CMF/COL. After exposure of hepatocytes cultured on CMF/COL to phenobarbital from days 3-6, CYP3A proteins were enhanced more than twofold and CYP2B1/2, depending on the exposure level, increased 1.3-6-fold. After exposure to 3-methylcholanthrene, a threefold increase of CYP1A proteins was found in CMF/COL and laminin cultures. These results indicate that CMF/COL, as a substratum in rat hepatocyte cultures, regulates gene expression of cytochromes P-450 isoenzymes for up to 9 days and provides a matrix which enables the cells to respond qualitatively similar to the response observed in different zones of the liver. This activity cannot be replaced by single-matrix components.