Effects of different levels of dietary iron on pregnancy superimposed upon growth in the rat

The effects of feeding four levels of dietary iron, 10, 50, 250 and 1.250 mg/kg were studied during pregnancy in growing and adult rats. Hematological measurements, plasma iron and total iron binding capacity, and iron content in liver, spleen and tibia were compared relative to pregnancy, diet and growth. Iron content in fetuses and fetal livers were compared relative to diet and growth of the dams. All parameters were lowest in rats fed the 10 mg Fe/kg diet. The highest level of iron fed, 1,250 mg/kg, resulted in increased iron content in liver, spleen and tibia of all treatment groups but did not alter hematological values or fetal iron content. Pregnant rats fed any of the four levels of iron had significantly lower Hb, Ht, total and storage iron concentration and ferritin and hemosiderin iron in liver than nonpregnant rats fed the same levels. The level of dietary iron needed by growing pregnant rats for maximal iron content in fetuses and fetal livers was between 50 and 250 mg Fe/kg which was fivefold that needed to obtain maximal hemoglobin concentration in dams. However, adult pregnant rats only needed between 10 to 50 mg Fe/kg for both maximal iron stores in fetal tissues and maximal hemoglobin concentration.     

Identification and characterization of human cytochrome P450 isoforms interacting with pimozide

Using human liver microsomes (HLMs) and recombinant human cytochrome P450 (CYP450) isoforms, we identified the major route of pimozide metabolism, the CYP450 isoforms involved, and documented the inhibitory effect of pimozide on CYP450 isoforms. Pimozide was predominantly N-dealkylated to 1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one (DHPBI). The formation rate of DHPBI showed biphasic kinetics in HLMs, which suggests the participation of at least two activities. These were characterized as high-affinity (K(m1) and Vmax1) and low-affinity (K(m2) and Vmax2) components. The ratio of Vmax1 (14 pmol/min/mg protein)/K(m1) (0.73 microM) was 5.2 times higher than the ratio of Vmax2 (244 pmol/min/mg protein)/K(m2) (34 microM). K(m2) was 91 times higher than K(m1). The formation rate of DHPBI from 25 microM pimozide in nine human livers correlated significantly with the catalytic activity of CYP3A (Spearman r = 0.79, P = .028), but not with other isoforms. Potent inhibition of DHPBI formation from 10 microM pimozide was observed with ketoconazole (88%), troleandomycin (79%), furafylline (48%) and a combination of furafylline and ketoconazole (96%). Recombinant human CYP3A4 catalyzed DHPBI formation from 10 microM pimozide at the highest rate (V = 2.2 +/- 0.89 pmol/min/pmol P450) followed by CYP1A2 (V = 0.23 +/- 0.08 pmol/min/pmol P450), but other isoforms tested did not. The K(m) values derived with recombinant CYP3A4 and CYP1A2 were 5.7 microM and 36.1 microM, respectively. Pimozide itself was a potent inhibitor of CYP2D6 in HLMs when preincubated for 15 min (Ki = 0.75 +/- 0.98 microM) and a moderate inhibitor of CYP3A (Ki = 76.7 +/- 34.5 microM), with no significant effect on other isoforms tested. Our results suggest that pimozide metabolism is catalyzed mainly by CYP3A, but CYP1A2 also contributes. Pimozide metabolism is likely to be subject to interindividual variability in CYP3A and CYP1A2 expression and to drug interactions involving these isoforms. Pimozide itself may inhibit the metabolism of drugs that are substrates of CYP2D6.     

Vasopressin reduces taurochenodeoxycholate-induced hepatotoxicity by lowering the hepatocyte taurochenodeoxycholate content

BACKGROUND/AIMS: Vasopressin has been reported to reduce bile flow, but its effects on bile acid secretion and bile acid-related hepatotoxicity are still unclear. We therefore investigated the influence of vasopressin on the hepatotoxicity and biliary excretion of taurochenodeoxycholic acid in primary cultured rat hepatocytes and isolated perfused rat liver models. METHODS/RESULTS: 1) Addition of vasopressin to hepatocyte cultures significantly decreased lactate dehydrogenase release as compared to cultures exposed to 1 mM taurochenodeoxycholic acid alone, and also reduced intracellular taurochenodeoxycholic acid content from 19.3 +/- 2.2 to 13.0 +/- 1.6 nmol/mg protein. After 30 min of preincubation with 1 mM taurochenodeoxycholic acid, rinsing and reculture of hepatocytes in bile acid-free medium resulted in gradual decrease in the intracellular level of the bile acid, and addition of vasopressin (10(-9) M) to the reculture medium accelerated this process. 2) Superimposition of vasopressin (330 pmol/l) for 10 min on taurochenodeoxycholic acid infusion (1.0 mumol/min: 25 mumol/l) caused a rapid increase in bile flow and biliary excretion of taurochenodeoxycholic acid (697 +/- 42 vs 584 +/- 27 nmol/10 min per g liver) from perfused rat livers, and significantly reduced lactate dehydrogenase release. 3) Superimposition of the PKC blocker H-7 (5 mumol/l) on taurochenodeoxycholic acid infusion (1.0 mumol/min: 25 mumol/l) caused a gradual increase in bile flow and biliary excretion of taurochenodeoxycholic acid. Furthermore, an additional infusion of vasopressin (100 pmol/l) for 10 min in the presence of H-7 produced a greater increase in bile flow and biliary excretion of taurochenodeoxycholic acid as compared with H-7 alone (754 +/- 71 vs. 657 +/- 26 nmol/g liver). 4) Continuous infusion of vasopressin (330 pmol/l) significantly increased the late peak (10-50 min) of horseradish peroxidase excretion from perfused livers (from 8.48 +/- 1.02 to 21.7 +/- 6.02 ng/g liver). CONCLUSIONS: These findings suggest that vasopressin exerts a protective effect against taurochenodeoxycholic acid-induced hepatotoxicity by stimulating the secretion of this bile acid via intracellular vesicular transport systems.     

Glucuronidation of retinoids by rat recombinant UDP: glucuronosyltransferase 1.1 (bilirubin UGT)

Rat liver recombinant BR1UGT1.1 was found to have significant activity toward retinoid substrates. UGT1.1 glucuronidation activity was 91 +/- 18 pmol/mg x min for atRA and 113 +/- 19 pmol/mg x min for 5,6-epoxy-atRA. The apparent K(M) and V(max) of atRA acid glucuronidation by UGT1.1 were 59.1 +/- 5.4 microM and 158 +/- 43 pmol/mg x min, respectively. SDS-PAGE and Western blot analysis of UGT1.1-transfected HK293 membrane proteins photolabeled with [11,12-3H]atRA revealed a protein of approximately 56 kDa that was labeled by [3H]atRA, detected by anti-pNP UGT antibody and not present in membranes from nontransfected HK293 cells. Liver microsomes from Gunn rats, which lack UGT1.1, had significant activity toward atRA (111 +/- 28 pmol/mg x min).     

Isoform specificity of trimethylamine N-oxygenation by human flavin-containing monooxygenase (FMO) and P450 enzymes: selective catalysis by FMO3

In the present study, we expressed human flavin-containing monooxygenase 1 (FMO1), FMO3, FMO4t (truncated), and FMO5 in the baculovirus expression vector system at levels of 0.6 to 2.4 nmol FMO/mg of membrane protein. These four isoforms, as well as purified rabbit FMO2, and eleven heterologously expressed human P450 isoforms were examined for their capacity to metabolize trimethylamine (TMA) to its N-oxide (TMAO), using a new, specific HPLC method with radiochemical detection. Human FMO3 was by far the most active isoform, exhibiting a turnover number of 30 nmol TMAO/nmol FMO3/min at pH 7.4 and 0.5 mM TMA. None of the other monooxygenases formed TMAO at rates greater than 1 nmol/nmol FMO/min under these conditions. Human fetal liver, adult liver, kidney and intestine microsomes were screened for TMA oxidation, and only human adult liver microsomes provided substantial TMAO-formation (range 2.9 to 9.1 nmol TMAO/mg protein/min, N = 5). Kinetic studies of TMAO formation by recombinant human FMO3, employing three different analytical methods, resulted in a Km of 28 +/- 1 microM and a Vmax of 36.3 +/- 5.7 nmol TMAO/nmol FMO3/min. The Km determined in human liver microsomes ranged from 13.0 to 54.8 microM. Therefore, at physiological pH, human FMO3 is a very specific and efficient TMA N-oxygenase, and is likely responsible for the metabolic clearance of TMA in vivo in humans. In addition, this specificity provides a good in vitro probe for the determination of FMO3-mediated activity in human tissues, by analyzing TMAO formation at pH 7.4 with TMA concentrations not higher than 0.5 mM.     

Busulfan-glutathione conjugation catalyzed by human liver cytosolic glutathione S-transferases

We have examined the catalytic activity of glutathione S-transferases (GST) in the conjugation of busulfan with glutathione (GSH) in human liver cytosol, purified human liver GST, and cDNA-expressed GST-alpha 1-1. Human liver microsomes and cytosol were incubated with 40 microM busulfan and 1 mM GSH. Cytosol catalyzed the formation of the GSH-busulfan tetrahydrothiophenium ion (THT+) in a concentration-dependent manner, whereas microsomes lacked activity. The total and spontaneous rates of THT+ formation increased with pH (pH range, 6.50-7.75), with the maximum difference at pH 7.4. Due to the limited aqueous solubility of busulfan, a K(m) for busulfan was not determined. The intrinsic clearance (Vmax/K(m)) of busulfan conjugation was 0.167 microliter/min/mg with 50-1200 microM busulfan and 1 mM GSH. GSH Vmax and K(m) for busulfan conjugation were 30.6 pmol/min/mg and 312 microM, respectively. Ethacrynic acid (0.03-15 microM) inhibited cytosolic busulfan-conjugating activity with 40 microM busulfan and 1 mM GSH. Enzyme-mediated THT+ formation was decreased 97% by 15 microM ethacrynic acid with no effect on the spontaneous reaction. In incubations with affinity-purified liver GST and GST-alpha 1-1, the intrinsic clearance for busulfan conjugation was 0.87 and 2.92 microliters/min/mg, respectively. Busulfan is a GST substrate with a high K(m) relative to concentrations achieved clinically (1-8 microM).     

Incorporation in vivo of 1-14C-palmitic acid into placental and fetal liver lipids of the rabbit

The incorporation of free fatty acid into the placental and fetal liver lipids of rabbits was studied after fetal injections of albumin-bound 1-14C-palmitic acid. The fetuses were killed either 5--10 or 10--20 min after the injection. The placentas and livers were extracted for lipids and the specific activities of triglycerides (TG), phospholipids (PL), free fatty acids (FFA), monoglycerides (MG) and diglycerides (DG) measured. The lipids of the liver and placenta took up 17.0 and 3.6% of the dose, respectively, and of that liver TG accounted for 74% and the placental TG 34% of the label in each tissue. Most of the remaining counts were in the PL fraction with the rest more or less evely distributed between the FFA, DG and MG fractions. No activity was recorded in the cholesterol esters. The placental TG, PL, DG and MG specific activities reached the same level as that of the placental FFA, while in the liver these esters had higher specific activities (than the liver FFA). The liver TG, DG and PL had higher specific activities when compared with those of the placenta. The specific activity of the placental FFA was lower at 10--20 min than at 5--10 min; the opposite was seen for the placental TG. No time-related changes were seen in the liver lipids. It is concluded that (i) both placenta and fetal liver incorporate FFA into glycerides and PL; (ii) the liver incorporates FFA more rapidly and to a greater extent than the placenta; (iii) most of the FFA is incorporated into TG and to a lesser extent (PL; (iv) in both organs hydrolysis of PL or TG occurs. These results are discussed with reference to placental transport of FFA and fetal fat metabolism.     

Evaluation of the xenobiotic biotransformation capability of six rodent hepatoma cell lines in comparison with rat hepatocytes

Phase I and II activities were examined in six rodent hepatoma cell lines and compared with those of cultured rat hepatocytes both in basal conditions and after exposure to 5 microM methylcholanthrene, 2 mM phenobarbital, and 15 microM beta-naphtoflavone. The metabolic profile of testosterone was also studied. The highest aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase activities were found in MH1C1 cells. Comparable values for 7-ethoxyresorufin O-deethylase activity, ranging from 21.6 to 42.9 pmol/mg x min, were observed in the hepatocytes and hepatoma cells, except the HTC cells. In contrast, only Fao cells showed 7-pentoxyresorufin O-depentylase activity at levels similar to those of hepatocytes (6.2 +/- 1.0 and 7.4 +/- 1.2 pmol/mg x min, respectively). Rat hepatocytes actively hydroxylated p-nitrophenol, but this activity was not measurable in hepatoma cells. Glutathione transferase activity was maintained in all the hepatoma cell lines at similar levels to those found in hepatocytes (684 +/- 56 nmol/mg x min). The seven hydroxylated metabolites of testosterone produced by cultured hepatocytes were negligible in hepatoma cells. Exposure of cells to inducers revealed that aryl hydrocarbon hydroxylase activity was mainly increased after treatment with 3-methylcholanthrene and beta-naphtoflavone, and the highest values were found in rat hepatocytes followed by MH1C1 and Fao cells. 3-Methylcholanthrene and naphtoflavone treatment also resulted in a marked increase in 7-ethoxyresorufin O-deethylase activity in hepatocytes as well as in H4IIC3, McA-Rh7777, MH1C1, and Fao cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatic resection in the fetal rabbit. Histologic comparison of tissue regeneration in the fetus versus the adult

Fetal tissues present peculiar features of repair after injury. Although the development of fetal hepatocytes have already been studied in vitro and in transplant models, an in vivo study of fetal liver regeneration is still missed in the literature, to the best of our knowledge. Eight time-dated pregnant California rabbits (23, 24, 25, 30 days of gestational age) and 2 adult male California rabbits were anesthetized following a standardized i.v. protocol (ketamine 50 mg/kg; xilazine 5 mg/kg; propiopromazine 0.75 mg/kg; spontaneous breathing; no anesthetic gas). All the pregnant does underwent a midline laparotomy and a minimal hysterotomy to approach a fetus per each animal. In 2 cases, 1 fetus was delivered and prior to sacrifice the fetal liver was sampled in toto (30 days of gestational age). These pregnancies were allowed to continue to term and were uneventful with a full-term spontaneous delivery of the remaining fetuses. In the other 6 pregnancies, after the hysterotomy, the fetal abdomen was entered through a right-sided longitudinal incision and the liver was partially resected by thermocauterization. Fetal abdomen was closed in 1 layer (non absorbable suture 7-0). The fetus was then returned in the uterus and, after amniotic fluid restoration with warmed saline, the hysterotomy was sutured in double layer (polyglycolic 5-0). Maternal abdomen was closed in 1 layer (polyglycolic 4-0) and the skin in a continuous overlying fashion (silk 3-0). The abdominal cavity of the 2 adult male rabbits was entered through a right subcostal incision. Partial liver resection was performed, and abdominal and skin closure followed the same techniques used for the pregnant does. The treated livers were then sampled in toto at 24, 48, 72 hrs and 4 days after surgery from the fetuses, and at 7 days from the adult rabbits. Histological stains were: H & E; Van Gieson; Masson; Alcian Bleu; PAS. Fetal histology showed a low inflammatory reaction poor in PMN cells with minimal deposition of collagen and a high amount of glycogen in the hepatocytes. The inflammatory response to resection was much more evident in the adult samples as much as the abundant intra and extra-cellular deposition of collagen associated to a minor amount of intracellular glycogen. The peculiar features of liver regeneration in the fetus, deserve further experimental studies.     

Neurochemical and electrophysiological evidence for the existence of a functional gamma-hydroxybutyrate system in NCB-20 neurons

Clonal neurohybridoma NCB-20 cells express a valproate-insensitive succinic semialdehyde reductase activity that transforms succinic semialdehyde into gamma-hydroxybutyrate. This activity (1.14+/-0.16 nmol/min/mg protein) was similar to the lowest activity existing in adult rat brain. [3H]gamma-Hydroxybutyrate labels a homogeneous population of sites on NCB-20 cell membranes (Kd=250+/-44.4nM, Bmax=180+/-16.2fmol/mg protein) that apparently represents specific gamma-hydroxybutyrate binding sites characterized previously on brain cell membranes. Finally, an Na+-dependent uptake of [3H]gamma-hydroxybutyrate was expressed in NCB-20 cells with a Km of 35+21.1 microM and a Vmax of 80+/-14.2 pmol/min/mg protein. A three-day treatment with 1 mM dibutyryl-cyclic-AMP induced a three-fold increase in the cellular succinic semialdehyde reductase activity. In parallel, a K+-evoked release of [3H]gamma-hydroxybutyrate occurred. This release was Ca2+ dependent and was not present in undifferentiated cells. Cyclic-AMP treatment induced a decrease of [3H]gamma-hydroxybutyrate binding sites, which could be due to spontaneous gamma-hydroxybutyrate release. Patch-clamp experiments carried out on differentiated NCB-20 cells revealed the presence of Ca2+ conductances which were partially inhibited by 50 microM gamma-hydroxybutyrate. This gamma-hydroxybutyrate-induced effect was blocked by the gamma-hydroxybutyrate receptor antagonist NCS-382, but not by the GABA(B) antagonist CGP-55845. These results demonstrate the presence of an active gamma-hydroxybutyratergic system in NCB-20 cells which possesses the ability to release gamma-hydroxybutyrate. These cells express specific gamma-hydroxybutyrate receptors which modulate Ca2+ currents independently of GABA(B) receptors.     

Gene expression of alpha1-6 fucosyltransferase in human hepatoma tissues: a possible implication for increased fucosylation of alpha-fetoprotein

The 1-6 fucosylated -fetoprotein (AFP) present in serum of patients with hepatocellular carcinoma (HCC) has been employed for the differential clinical diagnosis of HCC from chronic liver diseases. The molecular mechanism by which this alteration occurs, however, remains largely unknown. To address this issue, we purified GDP-L-Fuc:N-acetyl-beta-D-glucosaminide 1-6 fucosyltransferase (1-6 FucT), an enzyme involved in the 1-6 fucosylation of N-glycans from porcine brain, as well as from a human gastric cancer cell line, and cloned their genes. In this study, levels of 1-6 FucT mRNA expression and the activity of this enzyme for 12 human HCC tissues were examined and compared with that in surrounding tissues and normal livers. The mean +/- SD for 1-6 FucT activity was 78 +/- 41 pmol/h/mg in normal control liver, 202 +/- 127 pmol/h/mg in adjacent uninvolved liver tissues (chronic hepatitis: 181 +/- 106 pmol/h/mg; liver cirrhosis: 233 +/- 164 pmol/h/mg), and 195 +/- 72 pmol/h/mg in HCC tissues. The mRNA expression of 1-6 FucT was also enhanced in proportion to enzymatic activity except for a few cases, suggesting that 1-6 FucT expression is increased in chronic liver diseases, especially liver cirrhosis. Transfection of 1-6 FucT gene into cultured rat hepatocytes markedly increased 1-6 FucT activity and led to an increase in lens culinaris agglutinin (LCA) binding proteins in both cell lysates and condition media. When the 1-6 FucT gene was transfected into a human HCC cell line, Hep3B, which originally showed low levels of 1-6 FucT expression, 1-6-fucosylated AFP was dramatically increased in the condition media. Collectively, these results suggest that the enhancement of 1-6 FucT expression increased the fucosylation of several proteins, including AFP, and that the level of 1-6-fucosylated AFP in patients with HCC was in part caused by up-regulation of the 1-6 FucT gene expression.     

Glutamate dehydrogenase, alanine aminotransferase, thymidine kinase, and arginase in fetal and adult human and rat liver

In fetal livers of both man and rat thymidine kinase activity was 12 times higher than in the adult, glutamate dehydrogenase and arginase were present at 20-50% of their adult values, whereas alanine aminotransferase activity was only an insignificant fraction of that in the adult. Although the developmental changes for the four enzymes were quantitatively similar in both species, qualitatively there were some significant differences. In adult human liver, glutamate dehydrogenase activity was distributed almost equally between the cytosol and particles; the concentration of only the soluble enzyme increased after birth. In rat liver, glutamate dehydrogenase remained exclusively a particulate enzyme. The soluble hepatic alanine aminotransferase activity rose in both species after birth (from less than 2 U/g to 41-57 U/g, respectively). Thymidine kinase was wholly soluble in the fetal livers; only in adult human liver was additional activity (at least 50% of the total) found in the particles. Arginase isozymes, identical and apparently the same single isozyme in fetal and adult rat liver, show an ontogenetic change in man. A shift from a single form, common to human fetal liver and fetal kidney, to at least two variants in adult human liver, indicates another complexity of the fully differentiated liver in man.     

Pharmacological characterization of a specific binding site for angiotensin IV in cultured porcine aortic endothelial cells

This study demonstrated the existence of a specific binding site for angiotensin IV in porcine aortic endothelial cells. Non-equilibrium kinetic analyses at 37 degrees C allowed the calculation of a kinetic Kd of 0.44 nM. Pseudo-equilibrium saturation binding studies at 37 degrees C for 90 min indicated the presence of a single high-affinity site (Kd = 3.87 +/- 0.60 nM), saturable and abundant (Bmax = 9.64 +/- 1.44 pmol/mg protein). Competitive binding studies demonstrated the following rank order of effectiveness: angiotensin IV > angiotensin III > angiotensin II > angiotensin I > angiotensin II-(1-7), while 2-n-butyl-4-chloro-5-hydroxymethyl-1 [(2'-(1H-tetrazol-5-yl) biphenyl-4-yl) methyl] imidazol (DuP 753: losartan), 1-(4-amino-3-methyl-phenyl) methyl-5-diphenylisoethyl-4,5,6,7-tetrahydro-1H-imidazo [4,5-C] pyridine-6-carboxylic acid (PD 123177) or nicotinic acid-Tyr-(N alpha -benzyl-oxycarbonyl-Arg) Lys-His-Pro-Ile-OH (CGP 42112A) were inactive at the concentration of 100 microM. This binding site is, therefore, distinct from angiotensin II receptors, AT1 and AT2. Addition of the divalent cations Mg2+, Mn2+ or Ca2+ to the incubation buffer resulted in 90-95% inhibition of the [125I]angiotensin IV-specific binding to porcine aortic endothelial cells. Furthermore, the chelator, EGTA, at 5 mM increased the number of binding sites (Bmax = 17.8 +/- 2.5 pmol/mg protein), with no change in affinity (Kd = 5.7 +/- 1.3 nM). Exposure of porcine aortic endothelial cell membranes to the non-hydrolyzable GTP analog, GTP gamma S, had no effect on [125I]angiotensin IV binding. The presence of a high concentration of binding sites for angiotensin IV in porcine aortic endothelial cells suggests that this peptide may play an important role in the modulation of the cardiovascular system.     

Relative quantities of catalytically active CYP 2C9 and 2C19 in human liver microsomes: application of the relative activity factor approach

The relative catalytic activities of CYP2C9 and CYP2C19 in human liver microsomes has been determined using the approach of relative activity factors (RAFs). Tolbutamide methylhydroxylation and S-mephenytoin 4'-hydroxylation were used as measures of CYP2C9 and CYP2C19 activity, respectively. The kinetics of these reactions were studied in human liver microsomes, in microsomes from human lymphoblastoid cells, and in insect cells expressing CYP2C9 and CYP2C19. RAFs were calculated as the ratio of Vmax (reaction velocity at saturating substrate concentrations) in human liver microsomes of the isoform-specific index reaction divided by the Vmax of the reaction catalyzed by the cDNA expressed isoform. RAFs were also determined for SUPERMIX, a commercially available mixture of cDNA expressed human drug metabolizing CYPs formulated to achieve a balance of enzyme activities similar to that found in human liver microsomes. Lymphoblast RAF2C9 in human liver microsomes ranged from 54 to 145 pmol CYP/mg protein (mean value: 87), while a value of 251 pmol CYP/mg protein was obtained for SUPERMIX. Insect cell RAF2C9 in human liver microsomes ranged from 1.6 to 143 pmol CYP/mg protein (mean value: 49), while a value of 201 pmol CYP/mg protein was obtained for SUPERMIX. Both lymphoblast and insect cell RAF2C19 in human liver microsomes ranged from 4 to 45 pmol CYP/mg protein (mean values: 29 and 28, respectively), while a value of 29 pmol CYP/mg protein was obtained for SUPERMIX. The nature of the cDNA expression system used had no effect on the kinetic parameters of CYP2C9 as a tolbutamide methylhydroxylase, or of CYP2C19 as a S-mephenytoin hydroxylase. However insect cell expressed CYP2C19 (which includes oxidoreductase) had substantially greater activity as a tolbutamide methylhydroxylase when compared to lymphoblast expressed CYP2C19. The ratio of mean lymphoblast-determined RAF2C9 to RAF2C19 in human livers was 3.0 (range 1.6-17.9; n = 10), while this ratio for SUPERMIX was 8.6. The ratio of mean insect cell-determined RAF2C9 to RAF2C19 in human livers was 1.7 (range 0.04-16.2; n = 10), while this ratio for SUPERMIX was 7.0. Neither ratio is in agreement with the 20:1 ratio of immunoquantified levels of CYP2C9 and 2C19 in human liver microsomes reported in previous studies. SUPERMIX may contain catalytically active CYP2C9 in levels higher than those in human liver microsomes.     

Experimental immunization with anti-rheumatic bacterial extract OM-89 induces T cell responses to heat shock protein (hsp)60 and hsp70; modulation of peripheral immunological tolerance as its possible mode of action in the treatment of rheumatoid arthritis (RA)

High intracellular 1,2,-sn-diacylglycerol (DAG) usually activates protein kinase C (PKC). In choline-deficient Fischer 344 rats, we previously showed that fatty liver was associated with elevated hepatic DAG and sustained activation of PKC. Steatosis is a sequelae of many liver toxins, and we wanted to determine whether fatty liver is always associated with accumulation of DAG with activation of PKC. Obese Zucker rats had 11-fold more triacylglycerol in their livers and 2-fold more DAG in their hepatic plasma membrane than did lean control Zucker rats. However, this increased diacylglycerol was not associated with translocation or activation of PKC in hepatic plasma membrane (activity in obese rats was 897 pmol/mg protein X min(-1) vs. 780 pmol/mg protein X min(-1) in lean rats). No differences in PKC isoform expression were detected between obese and lean rats. In additional studies, we found that choline deficiency in the Zucker rat did not result in activation of PKC in liver, unlike our earlier observations in the choline deficient Fischer rat. This dissociation between fatty liver, DAG accumulation and PKC activation in Zucker rats supports previous reports of abnormalities in PKC signaling in this strain of rats.     

Expression in human prostate of drug- and carcinogen-metabolizing enzymes: association with prostate cancer risk

The role of two common polymorphisms of enzymes involved in the metabolism of drugs and carcinogens was studied in relation to prostate cancer. The gene encoding one of these enzymes (NAT2) is located in an area where frequent allelic loss occurs in prostate cancer. Mutations at the genes CYP2D6 and NAT2 were analysed by allele-specific polymerase chain reaction and restriction mapping in DNA from 94 subjects with prostate cancer and 160 male healthy control subjects. Eleven prostate specimens were analysed for genotype and enzymatic activities NAT2, CYP2D6 and CYP3A by using the enzyme-specific substrates sulphamethazine and dextromethorphan. Enzyme activities with substrate specificities corresponding to NAT2, CYP2D6 and CYP3A are present in human prostate tissue, with mean +/-s.d. activities of 4.8+/-4.4 pmol min(-1) mg(-1) protein, 156+/-91 and 112+/-72 nmol min(-1) mg(-1) protein respectively. The Km values for the prostate CYP2D6 and CYP3A enzyme activities corresponded to that of liver CYP2D6 and CYP3A activities, and the CYP2D6 enzyme activity is related to the CYP2D6 genotype. The N-acetyltransferase, in contrast, had a higher Km than NAT2 and was independent of the NAT2 genotype. The CYP2D6 and CYP3A enzymes, and an N-acetyltransferase activity that is independent of the regulation of the NAT2 gene, are expressed in human prostate tissue. The presence of carcinogen-metabolizing enzymes in human prostate with a high interindividual variability may be involved in the regulation of local levels of carcinogens and mutagens and may underlie interindividual differences in cancer susceptibility.     

Development of a homologous radioimmunoassay for mouse growth hormone receptor

A RIA for mouse GH receptor (mGHR) was developed. A synthetic peptide corresponding to the carboxyl-terminal 14 amino acids of the mGHR (GHR-2 peptide) was used as the antigen for antiserum production. The synthetic peptide was also used as the standard and radioligand in the RIA. The ability of the antiserum to recognize the mGHR was demonstrated by quantitating receptor concentrations in liver and mammary gland from virgin and 15-day-pregnant mice. Serial dilutions of these samples yielded displacement curves parallel to the synthetic peptide. No significant cross-reactivity was seen with serum from virgin or 15-day-pregnant mice, mGH, recombinant mGH-binding protein (mGHBP), a synthetic peptide identical to the hydrophilic tail of mGHBP, or a 14-amino acid synthetic peptide corresponding to amino acids 338-351 of mGHR (GHR-1 peptide). The concentration range of the mGHR RIA was 0.5-200 nM, and the intra- and interassay coefficients of variation were 6.5% and 6.1%, respectively. The concentration of liver GHR increased significantly during pregnancy compared with that in virgin mice, from 0.246 +/- 0.045 pmol/mg protein (mean +/- SEM; n = 5) in the virgin animals to 1.015 +/- 0.159 pmol/mg protein (n = 5) in pregnant mice. In contrast, the mGHR concentration in the mammary gland decreased significantly during pregnancy from 0.606 +/- 0.201 pmol/mg protein (mean +/- SEM; n = 5) to 0.299 +/- 0.027 pmol/mg protein (n = 5). Comparison of the total number of binding sites in livers from virgin and pregnant mice using the GH RRA and the combined results of the mGHR and mGHBP RIAs showed that the two methods gave almost identical results for livers from virgin animals, or 0.363 +/- 0.063 pmol/mg protein (mean +/- SEM; n = 3) and 0.371 +/- 0.008 pmol/mg protein (n = 3) for the GH RRA and the mGHR plus mGHBP RIAs, respectively. However, in livers from pregnant animals, the combined results from the mGHR and mGHBP RIAs were approximately 1.8 times higher than those obtained by the GH RRA, or 6.732 +/- 0.612 pmol/mg protein (mean +/- SEM; n = 3) and 3.693 +/- 0.67 pmol/mg protein (n = 3) for the mGHR plus the mGHBP RIAs and the GH RRA, respectively. The increase in the total GH binding capacity in livers from pregnant mice compared with those from virgin animals was largely due to an increase in the GHBP content. The increase in GHR was only 2.4-fold, or from 0.153 +/- 0.01 pmol/mg protein (mean +/- SEM; n = 3) in virgin mice to 0.364 +/- 0.03 pmol/mg protein (n = 3) in the 15-day-pregnant mice, whereas GHBP increased almost 30-fold during pregnancy, or from 0.218 +/- 0.003 pmol/mg protein (mean +/- SEM; n = 3) in virgin animals to 6.369 +/- 0.607 pmol/mg protein (n = 3) in pregnant mice.     

Plasma levels of parathyroid hormone, vitamin D, calcitonin, and calcium in association with endurance exercise

Nine male marathon runners were investigated during habitual training (week 0), after 3 weeks of training break (week 3), and after 2 weeks (week 5) and 4 weeks (week 7) of retraining. Maximal oxygen uptake, body fat (BF), and plasma levels of 25(OH)D3, 1,25(OH)2D3, parathyroid hormone (PTH), calcitonin (CT), albumin, and albumin-corrected calcium were determined throughout weeks 0-7. The maximal oxygen uptake decreased after training break and increased during retraining (P = 0.002). BF did not change significantly. Plasma 1,25(OH)2D3 was elevated after training break and decreased after 2 and 4 weeks of retraining [week 0: 44.0 +/- 3.7 (SEM) pg x 1(-1); week 3: 52.4 +/- 6.0 pg x 1(-1); week 5: 42.0 +/- 2.8 pg x 1(-1); week 7: 36.9 +/- 2.3 pg x 1(-1); P = 0.03]. Plasma 25(OH)D3 did not change significantly. Plasma PTH increased throughout the training break and retraining (week 0: 1.36 +/- 0.25 pmol x 1(-1); week 3: 2.02 +/- 0.43 pmol x 1(-1); week 5: 2.23 +/- 0.60 pmol x 1(-1); week 7: 2.63 +/- 0.34 pmol x 1(-1); P = 0.03). Albumin-corrected calcium values were transiently decreased during retraining (week 3: 2.77 +/- 0.08 mM; week 5: 2.47 +/- 0.05 mM; week 7: 2.66 +/- 0.07 mM; P = 0.01). Plasma CT did not change during training break, but was transiently decreased during retraining (week 0: 9.97 +/- 0.39 pmol x 1(-1); week 3: 9.91 +/- 0.37 pmol x 1(-1); week 5: 8.19 +/- 0.50 pmol x 1(-1); week 7: 9.02 +/- 0.45 pmol x 1(-1); P = 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Female exposure to high G: chronic adaptations of cardiovascular functions

The substrate structure-activity relationships described for the major human drug-metabolizing cytochrome P450 (P450 or CYP) enzymes suggest that the H1 receptor antagonist terfenadine could interact with CYP2D6 either as a substrate or as an inhibitor, in addition to its known ability to act as a substrate for CYP3A4. Based on this substrate structure-activity relationship, computer modeling studies were undertaken to explore the likely interactions of terfenadine with CYP2D6. An overlay of terfenadine and dextromethorphan, a known substrate of CYP2D6, showed that it was possible to superimpose the site of hydroxylation (t-butyl group) and the nitrogen atom of terfenadine with similar regions in dextromethorphan. These observations were substantiated by the ease of docking of terfenadine into a protein model of CYP2D6. Experimentally, terfenadine inhibited CYP2D6 activity in human liver microsomes with an IC50 of 14-27 microM, depending on the CYP2D6 substrate used. The inhibition of CYP2D6 was further defined by determining the Ki for terfenadine against bufuralol 1'-hydroxylase activity in four human livers. Terfenadine inhibited bufuralol 1'-hydroxylase activity with a Ki of approximately 3.6 microM. The formation of the hydroxylated metabolite (hydroxyterfenadine) in microsomes prepared from human liver and specific P450 cDNA-transfected B lymphoblastoid cells indicated that only CYP2D6 and CYP3A4 were involved in this transformation. As expected, the rate of formation was greatest with CYP3A4 (Vmax = 1257 pmol/min/nmol of P450), with CYP2D6 forming the metabolite at a 6-fold lower rate (Vmax = 206 pmol/min/nmol of P450). The two enzymes had similar KM values (9 and 13 microM, respectively). These data indicate that, as predicted from modeling studies, terfenadine has the structural features necessary for interaction with CYP2D6.     

Studies on transmembrane and paracellular phenomena in the foot of the slug agriolimax reticulatus (Mü)

Employing livers from rats fed on a protein-free diet for two weeks, the effects of protein deficiency on both biosynthesis and degradation of rRNA were investigated and the following results were obtained. 1. Protein deficiency led to a decrease of total liver RNA content per DNA to about 80% of that in normal rat liver. 2. From the kinetics of rRNA labelling with [14C]orotic acid in vivo, the half-lives of cytoplasmic rRNA's of normal and protein-deficient rat livers were determined to be 6.2 and 5.1 days, respectively. Furthermore, considering the pool size of rRNA in rat liver, the turnover rate of cytoplasmic rRNA was calculated to be 0.212 pmole/min/mg of nuclear DNA in normal rats and 0.240 pmole/min/mg of nuclear DNA in protein-deficient rats. 3. From the electrophoretic patterns of nucleolar RNA's of both groups of rat livers labeled with [14C]orotic acid, the time courses of the specific activities of nucleolar 45S, 32S, and 28S rRNA's were analysed and the half-life of each nucleolar RNA in both groups of rat livers was determined. Nucleolar 45S, 32S, and 28S RNA's had half-lives of 6.0, 15.9, and 26.5 min in normal rats, respectively, and 5.5, 19.4, and 22.9 min in protein-deficient rats, respectively Considering the pool size of each nucleolar RNA obtained from the leectrophoretic pattern, the turnover rates of 45S, 32S, and 28S RNA's were calculated to be the same, i.e., o.189 pmoles/min/mg of nuclear DNA, in normal rat liver and 0.372, 0.372, and 0.358 pmoles/min/mg of nuclear DNA in protein-deficient rat liver, respectively. 4. These results indicate that protein deficiency increased both the rate of degradation of cytoplasmic rRNA and that of nucleolar rRNA synthesis in rat liver. While in normal rat liver the rates of rRNA synthesis and degradation were rather similar, the rate of rRNA synthesis in protein-deficient rats was about 1.5 times higher than that of its degradation. Therefore, the decrease of total liver RNA content in protein deficiency might be accounted for by stimulated degradation of rRNA in the nucleus. 5. The activities of RNase in nuclear fractions of both groups of rat livers were compared. Both activities of nuclear acid RNase and especially that of the free form of alkaline RNase in protein-deficient rat liver were higher than those in normal rat liver.