Accumulation of Aluminium in Rat Liver: Association with Constituents of the Cytosol

Abstract: Aluminium (A1) accumulation occurs in the liver of renal patients and in patients on parenteral nutrition. Human hepatotoxicity is not proven. The role of the liver in storage and biotransformation of A1 and in development of osteo- and neurotoxicity is not clarified as yet. The aim of the present investigation was to study the storage of A1 in total liver and in subcellular liver fractions, and its association with soluble cytosolic molecular species. Therefore, rats were loaded with A1 prior to liver fractionation by ultracentrifugation, and equilibrium gel filtration chromatography of the cytosol, using a previously described method for A1 speciation in serum. A1 accumulated dose-dependently in liver and subcellular liver fractions, the lowest levels occurring in the cytosol. A dose-dependent elevation of A1 in the blood was also observed. Gelfiltration of the cytosol indicated that A1 was associated with a low molecular weight form which was not a citrate complex, and a high molecular weight form, which was larger than transferrin. No induction of and association with metallothionein occurred.     

PACPX--a substituted xanthine--antagonizes both the A1 and A2 subclasses of the P1-purinoceptor: antagonism of the A2 subclass is competitive but antagonism of the A1 subclass is not

1,3-Dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX) was examined for its ability to antagonize adenosine acting on the A1 and A2 subclasses of the P1-purinoceptor. A1-purinoceptors were studied in the isolated, driven left atria of the guinea-pig, and A2-purinoceptors in the isolated, carbachol-contracted taenia coli of the guinea-pig. PACPX antagonized the actions of adenosine in both types of preparation and was a more potent antagonist than 8-phenyltheophylline. The antagonism at the A2-purinoceptor was competitive with a pA2 of 5.95. The antagonism at the A1-purinoceptor was not competitive, although antagonism at the A1-purinoceptor was greater than that at the A2-purinoceptor, based on a comparison of pD2 values. The manner of antagonism of PACPX on the A1-purinoceptors of the heart was different from that found for the A1-receptors in bovine brain, implying that there is a fundamental difference between these central and peripheral A1 subclasses of P1-purinoceptor.     

A novel type of phospholipase A2 inhibitor, thielocin A1 beta, and mechanism of action.

Thielocin A1 beta, a novel phospholipase A2 inhibitor, was isolated from Thielavia terricola RF-143. It inhibited various phospholipase A2s in a dose-dependent manner. Among these, group II phospholipase A2 from rat was most sensitive to thielocin A1 beta (IC50 = 0.0033 microM). The inhibition of phospholipase A2 by thielocin A1 beta was independent of Ca2+ and substrate concentration. In addition, the inhibition of rat group II phospholipase A2 was noncompetitive (Ki = 0.0068 microM) and reversible. Furthermore, thielocin A1 beta quenched the relative fluorescent intensity of Naja naja venom phospholipase A2 and in a dose-dependent manner; 50% quench was noted with a molar ratio of thielocin A1 beta/enzyme of 2.2. These observations indicated that inhibition of phospholipase A2 by thielocin A1 beta may result from direct interaction with the enzyme.     

Processing-induced phase transitions of theophylline--implications on the dissolution of theophylline tablets

Aqueous wet massing of stable anhydrous theophylline (A) with polyvinylpyrrolidone (PVP) resulted in its complete transformation to theophylline monohydrate (M). Drying at 45 degrees C, resulted in the formation of metastable anhydrous theophylline (A*) which then transformed to A. PVP, a known crystallization inhibitor, was effective in inhibiting the A* --> A transition. The higher molecular weight polymer, PVP K90, was more effective in inhibiting the A* --> A transition as compared to PVP K17. The disappearance of M, and the formation of A* and A was simultaneously monitored by XRD. An increase in the drying temperature from 45 to 55 degrees C accelerated the A* --> A transition. In granules prepared by the high-shear process, approximately 50% of theophylline existed as A and the rest as A*. In contrast, the fluid-bed granulation process yielded granules containing only A. Thus, the physical form of theophylline in tablets was influenced by the molecular weight of the binding agent, the granulation method, and the drying temperature. Using A as the starting material, tablets were manufactured by high-shear aqueous wet granulation process and the A* content was quantified. These tablets were stored under various relative humidity (RH) conditions at 25 degrees C for 2 weeks. Storage at RH >or= 33% caused complete A* --> A conversion accompanied by a pronounced decrease in the initial dissolution rate indicating that phase transitions during processing and storage can have a significant influence on product performance.     

Distribution of cyclosporin A between blood cells and plasma of cardiac and renal transplant recipients

The relationship between blood cells and plasma concentrations of cyclosporin A (Cy A) determined by radioimmunoassay, was investigated in 12 heart and 12 kidney transplant recipients. The decision between a linear and nonlinear model was made according to a standardized residuals plot. We observed high blood cells-plasma concentration ratios in the two groups, indicating a high affinity of Cy A for blood cells. The distribution of Cy A between blood cells and plasma was ascribed to a nonlinear saturable model in the two groups. According to our results we have simulated the blood-plasma concentration ratio of Cy A as a function of plasma Cy A concentration and hematocrit.     

Site-dependent contributions of P-glycoprotein and CYP3A to cyclosporin A absorption, and effect of dexamethasone in small intestine of mice

We examined whether the oral bioavailability of cyclosporin A is controlled primarily by P-glycoprotein (P-gp) or CYP3A in the small intestine. In situ loop method was used to evaluate the uptake of cyclosporin A (40nmol) at the upper and lower intestine of wild-type and mdr1a/1b knockout mice treated or not treated with dexamethasone (75mg/kg/day, 7 days, i.p.). Expression of CYP3A mRNA in the control group was higher in the upper than the lower intestine, while that of the multidrug resistance-1a (mdr1a) mRNA was in the opposite order. Dexamethasone administration potently induced CYP3A and mdr1a mRNAs in the lower and upper intestine, respectively. At 45min after cyclosporin A administration into an upper intestinal loop of the control group of wild-type mice, the ratio of residual cyclosporin A to dose did not differ significantly from that of mdr1a/1b knockout mice, whereas in dexamethasone-treated wild-type mice, the residual ratio was increased significantly. The ratio of the cyclosporin A metabolite M17 to cyclosporin A in portal venous blood at an upper intestinal loop of mdr1a/1b knockout mice was much higher than that a lower intestinal loop. The M17/cyclosporin A ratio of portal venous blood at a lower intestinal loop in mdr1a/1b knockout mice was increased significantly by dexamethasone treatment. These results suggest that, under physiological conditions, the oral bioavailability of cyclosporin A is mainly controlled by CYP3A in the upper intestine, rather than liver, but when P-gp is induced by steroid, the intestinal absorption of cyclosporin A may be inhibited.     

五味子甲素对大鼠肝微粒体CYP3A活性的影响

目的：通过体外药物代谢实验探讨五味子甲素对CYP3A活性的影响。方法：以大鼠肝微粒体为载体,选取咪达唑仑（MDZ）作为药物“探针”,建立高效液相色谱（HPLC）检测方法,体外给药测定五味子甲素对MDZ的IC50值以及相关酶动力学参数。结果：孵育体系内源性物质不干扰测定,方法快捷、稳定、灵敏度高。在肝微粒中,五味子甲素对MDZ的IC50浓度为6．26μmol／mL,相关酶动力学参数：Km=15．77μmol／L,Ki=5．50μmol／mL。结论：五味子甲素对大鼠肝微粒体CYP3A活性具有抑制作用,其抑制作用为混合型,即：非竞争与反竞争抑制。     

Structural characterization of a new variant of the CYP2A6 gene (CYP2A6*1B) apparently diagnosed as heterozygotes of CYP2A6*1A and CYP2A6*4C

During the course of investigating the frequency of a CYP2A6 whole deletion-type polymorphism (CYP2A6*4C) in Japanese, an unexpectedly large population of heterozygotes for CYP2A6*4C and the wild-type (CYP2A6*1A) was found. Cloning of a cDNA encoding CYP2A6 from the liver of individuals judged as heterozygotes for CYP2A6*4C and the CYP2A6*1A was carried out to identify the causal allele(s) responsible for a possible overestimation. A clone isolated from the liver cDNA library possessed 58 bp sequences in the 3'-untranslated region, which was replaced with the corresponding region of the CYP2A7 gene. The same gene conversion existed in the genomic DNA, indicating that the replacement was not a cloning artifact. Based on the gene structure of the allele (CYP2A6*1B), this variant was thought to be one of the causal alleles responsible for overestimation of heterozygotes for CYP2A6*4C and CYP2A6* A. To investigate this further, we developed a genotyping method which could distinguish the CYP2A6*A, CYP2A6*1B and CYP2A6*4C alleles from each other. The results clearly showed that CYP2A6*1B was the sole allele responsible for the overestimation. We conclude that the new genotyping method allows determination of six genotypes of the CYP2A6 gene, simultaneously and precisely, in both Oriental and Caucasian populations.     

Kinetics and Mechanism of Isomerization of Cyclosporin A

The kinetics of isomerization of cyclosporin A to isocyclosporin A were studied in various nonaqueous solvents as a function of temperature and added methanesulfonic acid. The rate of isomerization was found to be acid-catalyzed over the acid concentration range studied. The choice of organic solvent significantly altered the rate of isomerization. For a series of alcohols, the rate was enhanced with increasing dielectric constant of the media, however, this correlation did not hold upon introduction of the dipolar aprotic solvent, tetrahydrofuran. Conversion of cyclosporin A to isocyclosporin A in tetrahydrofuran was found to contain diminished side reactions as compared to alcoholic solvents. The rate of conversion of isocyclosporin A to cyclosporin A was determined in aqueous buffers as a function of pH, buffer concentration, and temperature. The rates of conversion were extremely rapid compared to the forward reaction. Based on the pH dependencies of dilute solution reactivities, isocyclosporin A displayed a kinetically generated pK _a value of 6.9 for the secondary amine moiety. From pH 8 to pH 10 the pH–rate profile plot is linear, with a slope approximately equal to unity, indicating apparent hydroxide ion catalysis. The break in pH–rate profile suggests a change in the rate-determining step upon protonation of isocyclosporin A. The rate of isomerization in plasma was comparable with that found in a pH 7.4 buffer solution, indicating that plasma proteins do not significantly alter the isomerization kinetics of isocyclosporin A to cyclosporin A.     

RASSF1A基因启动子甲基化与贲门癌的相关性

目的 探讨抑癌基因RAS相关区域家族1基因(RASSF1A)在贲门癌组织中的表达与RASSF1A基因肩动子甲基化的关系.方法 运用RT-PCR方法检测35例贲门癌组织(A组)和20例贲门正常组织(B组)中RASSF1A的表达,并运用甲基化特异PCR(MSP)方法检测这些组织中RASSF1A基因启动子的甲基化情况.结果 RASSF1A基因在A组中有25例(71%)存在表达缺失,明显多于B组的3例(15%)(P<0.05).A组的RASSF1A基因启动子甲基化频率62%,明显高于B组的20%(P<0.05).结论 贲门癌组织中RASSF1A基因启动子的高甲基化是引起RASSF1A基因表达下调的重要原因,在肿瘤的发生和发展中起重要的调控作用.     

Biological Evaluation of Androstene Derivatives

The effect of several new dihydroepiandrosterone ester derivatives A2 – A6 was demonstrated using female cycling mice, which were synchronized for estrus with luteinizing hormone‐releasing hormone (LHRH) and injected with the steroids. The binding to the progesterone receptor (PR), was obtained from the cytosol of uteri from adult estrogen‐primed rabbits. A1 binds to the PR and inhibited the ovulation in cycling mice stimulated with LHRH. The activity of the endometrium and mammary glands in these mice was markedly reduced as compared to the control. A2 , A4 , and A5 were not active; nevertheless, A3 binds to the PR with high affinity. However, this steroid did not produce any effect as compared to that observed for the control in the endometrial and mammary glands. A6 binds to the PR with the highest affinity and induces a synergistic activity with progesterone in these tissues. Furthermore, A6 inhibited the ovulation in the same manner as A1 . These results suggested that A1 and A6 are blocking the gonadotropin secretion. A1 inhibited the conversion of progesterone to 5α‐progesterone. As a result of this, a blockage of the ductal and alveolar epithelial cell proliferation in the mammary and endometrial glands, which depends on 5α‐progesterone, was also observed.     

Effect of concanavalin A on serotonin transport into blood platelets: possible involvement of protein kinase C

Possible involvement of protein kinases in the serotonin (5-HT) transport system in platelets and the inhibitory effect of concanavalin A (Con A) on platelet 5-HT uptake were investigated. Staurosporine and K-252a, highly active inhibitors of protein kinases, did not inhibit 5-HT transport, but they antagonized the inhibitory effect of Con A on 5-HT uptake. KT5720, a protein kinase A inhibitor that has no effect on protein kinase C, neither affected 5-HT transport nor antagonized the inhibitory effect of Con A on 5-HT uptake. The Con A effect on 5-HT uptake was also antagonized by LaCl3, a Ca++ entry blocker. When the activity of Ca++ transport into platelets was estimated, Con A was shown to have a stimulative effect, which was antagonized by alpha-methyl-D-mannoside, a specific antagonist of Con A binding to cell membrane glycoproteins. Furthermore, Con A was shown to stimulate the protein kinase C activity of platelets, which phosphorylates a 40-kDa platelet protein; the Con A effects were antagonized by alpha-methyl-D-mannoside, staurosporine and K-252a, but not by KT5720. We suggest that the activation of protein kinase C and phosphorylation of 40-kDa protein might be involved in the inhibitory effect of Con A on platelet 5-HT transport.     

五味子甲素对大鼠肝微粒体CYP3A活性的影响

目的：通过体外药物代谢实验探讨五味子甲素对CYP3A活性的影响。方法：以大鼠肝微粒体为载体,选取咪达唑仑（MDZ）作为药物“探针”,建立高效液相色谱（HPLC）检测方法,体外给药测定五味子甲素对MDZ的IC50值以及相关酶动力学参数。结果：孵育体系内源性物质不干扰测定,方法快捷、稳定、灵敏度高。在肝微粒中,五味子甲素对MDZ的IC50浓度为6．26μmol／mL,相关酶动力学参数：Km=15．77μmol／L,K1=5．50μmol／mL。结论：五味子甲素对大鼠肝微粒体CYP3A活性具有抑制作用,其抑制作用为混合型,即：非竞争与反竞争抑制。     

Possible role of orexin A in nonadrenergic, noncholinergic inhibitory response of muscle of the mouse small intestine.

The effect of a novel peptide, orexin A, on longitudinal muscle of ICR mouse small intestine was examined in vitro. Exogenous orexin A induced a transient contraction in duodenal, jejunal and ileal segments. Atropine and tetrodotoxin completely inhibited the contractions. Contraction of longitudinal muscle of jejunal segments induced by electrical field stimulation was still observed after the jejunal segment had been desensitized to orexin A, suggesting that orexin A is not a final neurotransmitter to induce the contraction. On the other hand, in the presence of atropine and guanethidine, orexin A induced a transient gradual relaxation in duodenal, jejunal and ileal segments. Electrical field stimulation also induced significant relaxation of the muscle in jejunal segments. The electrical field stimulation-induced relaxation was inhibited by 55% after the desensitization of the segments to orexin A. Although the electrical field stimulation-induced relaxation was inhibited by 47% by a nitric oxide synthesis inhibitor, NG-nitro-L-arginine (L-NOARG), orexin desensitization did not affect the relaxation which persisted after L-NOARG treatment. The exogenous orexin A-induced relaxation was completely inhibited by L-NOARG. The results suggest that orexin A partially mediates nonadrenergic, noncholinergic (NANC) relaxation via activation of nitrergic neurones in longitudinal muscle of ICR mouse small intestine.     

Conformation of cyclosporin A in polar solvents

A major conformation of cyclosporin A in methanol and in aqueous methanol was revealed by some simple NMR experiments. Thus, a stepwise transition of cyclosporin A conformation from 100% CDCl₃ to 100% CD₃OD was followed by ¹H NMR, which showed that the chloroform conformation of cyclosporin A was still the major one in methanol. Employing the same technique, it was also shown that the chloroform conformation of cyclosporin A was one of the major conformations in 50% aqueous methanol. This may be the first experimental determination of a major conformation of cyclosporin A in polar solvents.     

Stimulation of acetylcholine output from brain slices caused by the ionophores BrX-537A and A23187.

1 The effect of two ionophores, BrX-537A (Bromolasolacid) and A 23187, on acetylcholine (ACh) output from brain slices was studied. 2 The slices were prepared from rat cerebral cortex, incubated in Krebs solution containing physostigmine and ACh output determined by bioassay. 3 Both ionophores enhanced ACh output. BrX-537A exerted its maximal effect, a six fold increase, at a concentration of 1.8 micron, while A 23187 caused a three fold increase at a concentration of 58 micron. 4 When the slices were incubated in a Ca-free medium, the effect of A 23187 on ACh output was only reduced, BrX-537A was abolished while that of BrX-537A was also active when disodium edetate (EDTA) was added to the the Ca-free medium. 5 The activity of BrX-537A was not affected by the presence of tetrodotoxin in the incubation medium. 6 The stimulation of ACh output elicited by KCl (25 mM) was increased further by hyoscine, but not by BrX-537A. Hyoscine however had no effect when ACh output was stimulated by BrX-537A. 7 The effect of BrX-537A on ACh output was potentiated by the addition of Mg2+ (9.3 mM) to the incubation medium and was reduced in a Mg-free medium. 8 It is concluded that A 23187 stimulates ACh output by transporting extracellular Ca2+ into cholinergic nerve endings. The effect of BrX-537A does not depend only on Ca2+ but also on other mechanisms.     

Comparative responses of the central adrenaline- and noradrenaline-containing neurons after reserpine injections

The responses of the noradrenaline (NA)- and adrenaline (A)-containing neurons to a reserpine treatment have been studied in the rat brain by using biochemical indices of the neuronal activity. Three days after multiple reserpine injections, tyrosine hydroxylase activity was significantly increased in the locus coeruleus (LC), A1-C1 and C2 regions. No change in this activity was observed in the A2 region. Furthermore, the NA and A endogenous levels were markedly reduced both in NA and A cell bodies and/or terminals, suggesting a reserpine action on NA and A neurons. The NA turnover was unchanged in all the regions analyzed. Conversely, the A turnover was reduced in the LC, A2 and C2 regions and in the nucleus periventricularis of the hypothalamus. This result suggests a different degree of sensitivity and/or response of the NA and A neurons following reserpine administration.     

Non-invasive method to detect induction of CYP3A4 in chimeric mice with a humanized liver

Chimeric mice with a humanized liver have been previously established by the transplantation of human hepatocytes to urokinase-type plasminogen activator/severe combined immunodeficiency mice. A non-invasive method to detect the induction of cytochrome P450 (CYP) 3A4 was evaluated in chimeric mice with a humanized liver. Dexamethasone (DEX) was used as a probe drug to detect induction; and rifampicin was used as a model drug to induce CYP3A4. Before and after rifampicin treatment (50 mg kg(-1), intraperitoneal injection once a day for 4 days) in the chimeric mice, DEX was subcutaneously injected and the urinary excretion of 6beta-hydroxydexamethason (6betaOHD) and DEX was determined. The metabolic ratio (6betaOHD/DEX) significantly increased after rifampicin treatment. Livers from the control and rifampicin-treated chimeric mice were stained immunohistolochemically with antibodies against CYP3A4 and CYP3A5. CYP3A4 and CYP3A5 were detected in the area of humanized liver, but staining was intense for CYP3A4 and very weak for CYP3A5. Only the staining of CYP3A4 was increased after rifampicin treatment. Formation of 6betaOHD by human liver microsomes was higher than that formed by mouse liver microsomes. Metabolite formation was catalysed by both CYP3A4 and CYP3A5 and the intrinsic clearance (V(max)/K(m)) by CYP3A4 was found to be 50-fold higher than that of CYP3A5. The results of the present study indicate that estimation of the changes of the urinary metabolic ratio (6betaOHD/DEX) in the chimeric mice with a humanized liver is a very useful tool for detecting the induction of CYP3A4 by a non-invasive method.