Hepatic effects of <Emphasis Type="Italic">Cimicifuga racemosa</Emphasis> extract <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

Extracts of Cimicifuga racemosa are used frequently for menopausal complaints. Cimicifuga is well tolerated but can occasionally cause liver injury. To assess hepatotoxicity of cimicifuga in more detail, ethanolic C. racemosa extract was administered orally to rats, and liver sections were analyzed by electron microscopy. Tests for cytotoxicity, mitochondrial toxicity and apoptosis/necrosis were performed using HepG2 cells. Mitochondrial toxicity was studied using isolated rat liver mitochondria. Microvesicular steatosis was found in rats treated with > 1,000 mg/kg [DOSAGE ERROR CORRECTED] body weight cimicifuga extract. In vitro, cytotoxicity was apparent at concentrations > or =75 microg/mL, and mitochondrial beta-oxidation was impaired at concentrations > or =10 microg/mL. The mitochondrial membrane potential was decreased at concentrations > or =100 microg/mL, and oxidative phosphorylation was impaired at concenq trations > or =300 microg/mL. The mechanism of cell death was predominantly apoptosis. C. racemosa exerts toxicity in vivo and in vitro, eventually resulting in apoptotic cell death. The results are compatible with idiosyncratic hepatotoxicity as observed in patients treated with cimicifuga extracts.     

Absorption of double labeled (glycerol 3H-linoleic acid 14C) native bile by phosphatidylcholines]

Double-labeled bile ([U. 3H glycerol] [1. 14C linoleic acid])--in which about 70% of labeling 14C and 80% of labeling 3H of total lipids were borne by phosphatidylcholines (PC), (isotopic ratio of these PC was equal to 1)--was introduced into the duodenum of test Rats, some of them with a bile fistula. As low amounts of the hydrolysis products of biliary PC were found in the intestinal lumen, a higher hydrolysis must occur further (brush border, enterocyte ?) because, in the mucosa, the highest labeling 14C was present as triglycerides and PC have an isotopic ratio 3H/14C higher than 1. As in the lumen the isotopic ratio 3H/14C of PC was higher than 1 and increased with the time elapsed, this finding suggests that mucosal PC were added to biliary PC (secretion or desquamation ?) unless these modifications were due to luminal micro-organisms. As test Rat bile was poorly labeled a very weak enterohepatic circulation of biliary diunsaturated PC may exist.     

Metabolism of 1,3,7-trimethyldihydrouric acid in the rat: New metabolic pathway of caffeine

Summary [1-CH₃-¹⁴C] 1,3,7-trimethyldihydrouric acid which, in quantity, is the most important caffeine metabolite, was isolated and purified from the urine of rats fed with [1-CH₃-¹⁴C] caffeine. The oral administration of this metabolite to rats showed that 1,3,7-trimethyldihydrouric acid was excreted unchanged in urine and was therefore an end product of caffeine metabolism. This result implies a new metabolic pathway of caffeine.     

Insect tissues, not microorganisms, produce linoleic acid in the house cricket and the American cockroach

Biosynthesis of linoleic acid, 18:2 (n-6), was unambiguously demonstrated to occur in the cockroach, Periplaneta americana, and the cricket, Acheta domesticus. Axenic tissue from both of these insect species was demonstrated by radio-gas-liquid chromatography (radio-GLC) and radio-high-performance liquid chromatography (radio-HPLC) to incorporate [1-14C]acetate and [1-14C]oleate into this essential fatty acid.     

Adenosine and its derivatives released by neocortex synaptosomes of the guinea pig

Nerve endings (synaptosomes) prepared from guinea-pig neocortex were loaded with [14C] adenosine at 37 degrees C during a 30 min incubation. After the removal of the extracellular adenosine, the preparations were superfused and adenosine derivatives were analyzed in the superfusion fluid. The measurement of the amounts of [14C] nucleotides released in the presence or absence of metabolic inhibitor allowed us to conclude that adenosine and to a smaller extent inosine, are the molecules released from the synaptosomes during superfusion.     

Interaction of BW755C, a potent inhibitor of lipoxygenase and cyclo-oxygenase, with mitochondrial cytochrome oxidase

The interaction between BW755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline), a potent inhibitor of both lipoxygenase and cyclo-oxygenase, and respiratory chain in mitochondria and electron transport particles (ETP) from rat livers was examined. BW755C accelerated the oxygen uptake by mitochondria without the addition of substrate for the respiratory chain. Spectrophotometric study revealed that BW755C was quickly oxidized by cytochrome oxidase in mitochondria to a compound possessing an absorption maximum at 524 nm. p-Phenylenediamine (p-diaminobenzene, PPDA), which, like BW755C, serves as an electron donor to cytochrome oxidase, was shown to inhibit the generation of active oxygen in macrophages; the inhibition was stronger than that of BW755C. These results strongly suggest that the oxidative conversion of BW755C by mitochondrial cytochrome oxidase is associated with its potentially inhibitory action on the active oxygen-generating system in phagocytes.     

Interaction of BW755C,a potent inhibitor of lipoxygenase and cyclo-oxygenase,with mitochondrial cytochrome oxidase

Summary The interaction between BW755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline), a potent inhibitor of both lipoxygenase and cyclo-oxygenase, and respiratory chain in mitochondria and electron transport particles (ETP) from rat livers was examined. BW755C accelerated the oxygen uptake by mitochondria without the addition of substrate for the respiratory chain. Spectrophotometric study revealed that BW755C was quickly oxidized by cytochrome oxidase in mitochondria to a compound possessing an absorption maximum at 524 nm. p-Phenylenediamine (p-diaminobenzene, PPDA), which, like BW755C, serves as an electron donor to cytoschrome oxidase, was shown to inhibit the generation of active oxygen in macrophages; the inhibition was stronger than that of BW755C. These results strongly suggest that the oxidative conversion of BW755C by mitochondrial cytochrome oxidase is associated with its potentially inhibitory action on the active oxygen-generating system in phagocytes.The authors are indebted to Dr M. Hori, Gifu College of Pharmacy and to Dr Y. Orii, Kyoto University for their kind supplies of BW755C and pure cytochrome oxidase, respectively.     

Relationship between regeneration of cell surface glycoproteins in trypsin-treated chick embryo fibroblasts and cell adhesion to the substratum

The ability of cells to adhere to a substratum was altered by treatment with trypsin but was restored after a 1.5-h culture. A concomitant incorporation of [3H] leucine and [14C] glucosamine in the trypsin-sensitive cell surface glycoproteins was observed and almost reached a plateau within 1.50 h following the treatment with trypsin.     

Oxidative photochemical decarboxylation of zaragozic acid A

A unique decomposition reaction of the novel squalene synthase inhibitors called zaragozic acids has been studied. Under very mild conditions, e.g. by merely exposing their solutions to air and visible light at ambient temperature, these compounds, characterized by the 2,8-dioxabicyclo[3.2.1]octane-4,6,7-trihydroxy-3,4,5-tricarboxylic acid core, rapidly decompose. As relatively stable intermediates in the cascade of decomposition, the biologically active 2,8-dioxabicyclo[3.2.1]octane-6,7-dihydroxy-4-keto-5-caroxylic acid (or 3,4-decarboxy-4-dehydro) derivatives of these compounds have been isolated in ca. 20% yield. Derivatization on the highly reactive 4-carbonyl group yields stable derivatives, several of which are potent inhibitors of squalene synthase. Further decomposition results in the elimination of C3 and C4 atoms and the carboxylic acid on C5, the oxidation of C5 to carboxylic acid and the liberation of the oxo group on C1. Specific results obtained with zaragozic acid A, a key representative of the family of these potent cholesterol-lowering agents, are presented in this study.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptor-negative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [3H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10(-6) M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10(-5) M linoleic acid or 10(-5) M arachidonic acid but not by 10(-6) M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (Kd 0.54 nM +/- 0.11 nM; n = 6) than growth in medium supplemented with untreated serum (complete medium) (Kd = 1.68 nM +/- 0.48 nM; n = 6) (p less than 0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10(-5) M linoleic acid or 10(-5) M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10(-5) M stearic acid or 10(-5) M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10(-5) M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [3H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Aberrant, canavanyl protein formation and the ability to tolerate or utilize L-canavanine

L-Canavanine, 2-amino-4-(guanidinooxy)butyric acid, and L-arginine incorporation into de novo synthesized proteins was compared in six organisms. Utilizing L-[guanidinooxy14C]canavanine and L-[guanidino14C]arginine at substrate saturation, the canavanine to arginine incorporation ratio was determined in de novo synthesized proteins. Caryedes brasiliensis and Sternechus tuberculatus, canavanine utilizing insects; Canavalia ensiformis, a canavanine storing plant; and to a lesser extent Heliothis virescens, a canavanine resistant insect, failed to accumulate significant canavanyl proteins. By contrast, Manduca sexta, a canavanine-sensitive insect, and Glycine max, a canavanine free plant, readily incorporated canavanine into newly synthesized proteins. This study supports the contention that the incorporation of canavanine into proteins in place of arginine contributes significantly to canavanine's antimetabolic properties.     

Insect tissues,not microorganisms,produce linoleic acid in the house cricket and the American cockroach

Summary Biosynthesis of linoleic acid, 182(n–6), was unambiguously demonstrated to occur in the cockroach,Periplaneta americana, and the cricket,Acheta domesticus. Axenic tissue from both of these insect species was demonstrated by radio-gas-liquid chromatography (radio-GLC) and radio-high-performance liquid chromatography (radio-HPLC) to incorporate [1-¹⁴C]acetate and [1-¹⁴C]oleate into this essential fatty acid.This work was supported by the National Science Foundation under grant DCB-8914417. We would like to thank Coby Schal for his generous gift of American cockroaches and Tania Kellermeyer for her excellent technical assistance.     

Synthesis and some characteristics of [13C]-specially enriched tetragastrin and the related compound

[13C]-enriched tetragastrin and the related compound were synthesized in solution. Conversion of S-[13C]methylated tetragastrin to the enriched tetragastrin gave 10.5 ppm upfield chemical shift of Cepsilon resonance. The potency of the synthetic tetragastrin to stimulate gastric acid secretion was virtually identical with that of pentagastrin (ICI).     

Transfer of oligosaccharide from oligosaccharide pyrophosphoryl dolichol to endogenous acceptor proteins in human breast malignant and normal tissues

We have prepared dolichylpyrophosphoryl-[14C]-oligosaccharide (Dol-PP-oligosaccharide) from calf thyroid. Microsomal fractions from human breast tissues catalyzed the transfer of labeled oligosaccharide to endogenous acceptor proteins. Malignant tumors showed higher activity of the oligosaccharide transferring enzyme than normal tissue. With kojibiose (Kj), an inhibitor of (Glc3)-glucosidase, an increase in the radioactivity associated with glycoprotein was observed.     

Oxidation of malate, NADH and glycine in C3 and C4 plant mitochondria]

Spinach leaf mitochondria (C3 plant) were capable of oxidizing Glycine. This oxidation was linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport inhibitors. In marked contrast however, neither mitochondria from dark grown plants nor mitochondria from C4 leaf plants were capable of oxidizing this amino acid.     

Protein anabolism in endometrium and myometrium during the growth of induced deciduoma in rats

Summary Protein anabolism in the endometrium and myometrium was studied during the growth of induced deciduoma in terms of incorporation of [¹⁴C]-leucine into proteins. The data show that the specific activity of the proteins was highest 2 days after decidualisation. Protein synthesis in the endometrium studied on that day as a function of time after injection of the labelled amino acid showed a steady increase during the first 2 h. Cycloheximide (2 mg/kg b.wt) administration produced nearly 95% inhibition of protein synthesis in endometrium as well as in myometrium.     

Serum and liver radioactivity levels in mice after intraperitoneal and subcutaneous injection of [14C]orotic acid

Summary [¹⁴C]Orotic acid was rapidly distributed in blood after, both i.p. and s.c. injection but was not completely absorbed from the peritoneal cavity until 20 min after injection. S.c. injection should be an acceptable alternative to i.p. injection although the incorporation into the liver acid soluble- and RNA-fractions was somewhat delayed after the s.c. injection.This work was supported by grants from the Carl Trygger Foundation and the Foundation of Director Albert Påhlsson.     

Aberrant,canavanyl protein formation and the ability to tolerate or utilize L-canavanine

Summary L-Canavanine, 2-amino-4-(guanidinooxy)butyric acid, and L-arginine incorporation into de novo synthesized proteins was compared in six organisms. Utilizing L-[guanidinooxy¹⁴C]canavanine and L-[guanidino¹⁴C]arginine at substrate saturation, the canavanine to arginine incorporation ratio was determined in de, novo synthesized proteins.Caryedes brasiliensis andSternechus tuberculatus, canavanine utilizing insects;Canavalia ensiformis, a canavanine storing plant; and to a lesser extentHeliothis virescens, a canavanine resistant insect, failed to accumulate significant canavanyl proteins. By contrast,Manduca sexta, a canavanine-sensitive insect, andGlycine max, a canavanine free plant, readily incorporated canavanine into newly synthesized proteins. This study supports the contention that the incorporation of canavanine into proteins in place of arginine contributes significantly to canavanine's antimetabolic properties.     

Transfer of oligosaccharide from oligosaccharide pyrophosphoryl dolichol to endogenous acceptor proteins in human breast malignant and normal tissues

Summary We have prepared dolichylpyrophosphoryl-[¹⁴C]-oligosaccharide (Dol-PP-oligosaccharide) from calf thyroid. Microsomal fractions from human breast tissues catalyzed the transfer of labeled oligosaccharide to endogenous acceptor proteins. Malignant tumors showed higher activity of the oligosaccharide transferring enzyme than normal tissue. With kojibiose (Kj), and inhibitor of (Glc₃)-glucosidase, an increase in the radioactivity associated with glycoprotein was observed.     

Cholesterol esterification activities in the intestines and pancreas of the albino rat

Summary Cholesterol esterification activities in intestines and pancreas are much greater with unsaturated fatty acids than with the saturated ones; the maximum activity is with arachidonic acid in intestines and with oleic acid in pancreas. The pancreatic cholesterol esterification activity is higher than the intestinal one.