Effect of trimethylamine-carbomethoxyborane on growth traits and lipid metabolism in lines of mice selected for high and low fat content.

The purpose of this study was to determine the effects of the amine carboxyborane, trimethylamine-carbomethoxyborane (B), on growth, body composition and lipid metabolism in lines of mice differing in fat content as a result of directional selection: high fat content (HF); low fat content (LF); and random control (RC). Mice were injected i.p. daily with either 20 mg/kg of B dissolved in 1% (w/v) carbomethoxycellulose as the vehicle or just vehicle (C) from 26 to 42 days of age. Growth rate, feed intake, feed efficiency, epididymal fat pad weight/BW and percentage body water were not affected by B treatment, but liver weight/BW was increased (P < 0.001). HF mice had larger epididymal fat pad weight/BW and less percentage body water when compared to LF mice (P < 0.001). Treatment with B lowered (P < 0.01) serum triglyceride and cholesterol levels, and selection for divergence in fat content led to positive divergence (P < 0.01, (HF-LF) > 0) in serum triglyceride and serum cholesterol levels; divergence x treatment interactions (P < 0.01) were due to administration of B eliminating the line differences present when C was administered. Treatment with B elevated (P < 0.001) HDL cholesterol level and lowered (P < 0.001) chylomicron, LDL and VLDL cholesterol levels, while positive divergence was found for all four fractions. Fecal triglyceride and cholesterol levels were increased (P < 0.01) by administering B, but were not affected by selection. Hepatic enzyme activity of cholesterol-7 alpha-hydroxylase was elevated (P < 0.001) by treatment with B while activities of other hepatic enzymes were depressed, e.g., acyl-CoA cholesterol acyltransferase. Line differences in activity of several hepatic enzymes also were found. These results indicate that B acts as a hypolipidemic agent in both high-fat and low-fat mouse genotypes, lowering serum cholesterol and triglyceride levels. However, at the administered dose, B had no affect on growth rate, feed intake, feed efficiency or body fat content.

Anti-inflammatory activity of amine-carboxyboranes in rodents.

Amine-carboxyboranes are potent anti-inflammatory agents reducing induced edema and pleural effusion at 8 mg/Kg, i.p. They protect against LPS (Salmonella) induced septic shock from 2-8 mg/Kg/day and are effective in blocking pain mediated both locally and centrally. The mode of action of these agents is by blocking release of cytokines from macrophages, thus reducing lysosomal hydrolytic and proteolytic enzyme activities of affected cells. The agents also reduce prostaglandin and leukotriene synthesis by blocking the activities of regulatory enzymes of the respective pathways.

The anti-osteoporotic activity of amine-carboxyboranes in rodents.

Amine-carboxyboranes have been shown to prevent osteoporosis and loss of bone mass in rodents. In vitro studies using CF1 mouse pup calvaria and rat UMR-106 osteosarcoma cells showed that amine-carboxyborane derivatives reduced significantly the loss of intracellular calcium into the growth medium from 10(-4) to 10(-8) M over 48 hours. Amine-carboxyborane derivatives were more effective than calcitonin or simple boron salts. Calcium incorporation into these cells and proline incorporation into collagen was accelerated in the presence of amine carboxyboranes. The amine-carboxyborane derivatives effectively inhibited lysosomal and proteolytic enzymes as well as activities of serine elastase, prostaglandin cyclooxygenase, and 5'-lipoxygenase in mouse macrophages, human PMNs, leukocytes and Be Sal cells. IC50 values were in the range of 10(-6) M. In lactating ovariectomized female rats after administered amine-carboxyboranes for 14 days at 8 mg/kg/day orally, the femur and humerus showed increased volume, weight, density and ash weight. Serum calcium levels were elevated significantly with minimum reductions on serum inorganic phosphate levels. Femur calcium levels were elevated after treatment with amine-carboxyborane derivatives, but not with etidronate. Humerus total lipids after 14 days were slightly elevated probably due to increased levels of triglycerides and phospholipids.

The cytotoxic activity of 1-acyl- and 1,2-diacyl-4,4-diethyl-3,5-pyrazolidinediones.

The 1-acyl- and 1,2-diacyl-4,4-diethyl-3,5-pyrazolinediones proved to be cytotoxic against the growth of a number of cell lines, including murine and human leukemias. HeLa suspended carcinoma, colon adencarcinoma SW480, KB nasopharynx and glioma tumors. Selected compounds were also active in the human lung bronchogenic MB-9812, and osteosarcoma TE418 screens. These derivatives were active in vivo in the Ehrlich ascites carcinoma screen in CF-1 mice at 8 mg/kg/day I.P. The mode of action in Tmol3 leukemia cells showed that the compounds reduced de novo synthesis of purines and pyrimidines and inhibited dihydrofolate reductase and ribonucleoside reductase activities. The DNA molecule was not a target although limited DNA strand scission may be possible.

Hypolipidemic, anti-obesity, anti-inflammatory, anti-osteoporotic, and anti-neoplastic properties of amine carboxyboranes.

The amine-carboxyborane derivatives were shown to be effective antineoplastic/cytotoxic agents with selective activity against single-cell and solid tumors derived from murine and human leukemias, lymphomas, sarcomas, and carcinomas. The agents inhibited DNA and RNA synthesis in preference to protein synthesis in L1210 lymphoid leukemia cells. Inosine-monophosphate dehydrogenase apparently is a target site of the compounds; similar effects on phosphoribosyl-pyrophosphate amido transferase, orotidine-monophosphate decarboxylase, and both nucleoside and nucleotide kinases were observed. Deoxyribonucleotide pool levels were reduced in the cells; DNA strand scission was observed with the agents. In rodents, the amine carboxyboranes were potent hypolipidemic agents, lowering both serum cholesterol and triglyceride concentrations, in addition to lowering cholesterol content of very low-density lipoprotein and low-density lipoprotein (LDL) and elevating high-density lipoprotein (HDL) cholesterol concentrations. De novo regulatory enzymes involved in lipid synthesis were also inhibited (e.g., hypocholesterolemic 3-hydroxy-3-methyl-Coenzyme A reductase, acyl-Coenzyme A cholesterol acyltransferase, and sn-glycerol-3-phosphate acyltransferase). Concurrently, the agents modulated LDL and HDL receptor binding, internalization, and degradation, so that less cholesterol was delivered to the plaques and more broken down from esters and conducted to the liver for biliary excretion. Tissue lipids in the aorta wall of the rat were reduced and fewer atherosclerotic morphologic lesions were present in quail aortas after treatment with the agents. Cholesterol resorption from the rat intestine was reduced in the presence of drug. Genetic hyperlipidemic mice demonstrated the same types of reduction after treatment with the agents. The agents would effectively lower lipids in tissue based on the inhibition of regulatory enzymes in pigs. These findings should help improve domestic meat supplies from fowl and pigs. The amine-carboxyboranes were effective anti-inflammatory agents against septic shock, induced edema, pleurisy, and chronic arthritis at 2.5 to 8 mg/kg. Lysosomal and proteolytic enzyme activities were also inhibited. More significantly, the agents were dual inhibitors of prostaglandin cyclooxygenase and 5'-lipoxygenase activities. These compounds also affected cytokine release and white cell migration. Subsequent studies showed that the amine-carboxyboranes were potent anti-osteoporotic agents reducing calcium resorption as well as increasing calcium and proline incorporation into mouse pup calvaria and rat UMR-106 collagen.

Anti-inflammatory and anti-osteoporotic activities of base-boronated nucleosides and phosphate-boronated nucleotides in rodents.

The 2'-deoxyribonucleoside cyanoboranes were effective anti-inflammatory agents in rodents at 2-8 mg/kg; they blocked induced edema, septic shock, and pleurisy. Overall compounds 3',5'-O-(bis- (triisopropylsilyl)-2'-deoxyinosine (1), 3',5'-O-bis(triisopropylsilyl)-2'-deoxycytidine (10), N3-(cyanoboryl)-2'-deoxycytidine (11), N7-(cyanoboryl)-N2-isobutyryl- 3',5'-O-bis(triisopropylsilyl)-2'-deoxyguanosine (20), and N7-(cyanoboryl)-N2- isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-(triisopropylsilyl)-2' -deoxyguanosine (22) were the most active when all the anti-inflammatory screens are considered. The agents also blocked both local and central pain caused by inflammation. These nucleosides blocked calcium resorption but were less effective compared to other amine carboxyboranes. The inflammation process appeared blocked by these compounds because of their effectiveness in reducing both hydrolytic lysosomal enzyme and proteolytic enzyme activities. The agents were also dual inhibitors of prostaglandin cyclooxygenase and 5'-lipoxygenase activities in leukocytes and macrophages. These agents at 10(-4) M demonstrated no specific organ toxicity to ileum mucosa cells grown in tissue culture.

An investigation of 2'-deoxyribonucleoside cyanoboranes in mice for therapeutic safety.

A standard acute toxicity study was undertaken to assess 2'-deoxyribonucleoside cyanoboranes for therapeutic safety. 2'-Deoxyribonucleoside cyanoboranes and related derivatives were nontoxic at doses required for anti-neoplastic and hypolipidemic activities. At higher doses (50 and 100 mg/kg/day IP for 7 days), all treated animals survived with slight reductions in total body weight and small decrements in daily food consumption. No clinical chemistry value was elevated to a magnitude suggesting onset of organ specific toxicity. However, agents appeared to modulate subpopulations of white blood cells, i.e., more lymphocytes than PMNs were present in blood from treated animals as determined by differential cell counts. This modulation is correlated with increases in granulomatous foci in the spleen and mesentery of treated animals after 7 days. The kidney was damaged only by Compound 5 at 50 and 100 mg/kg/day; Compound 5 had the most potent anti-neoplastic activity. The compounds demonstrated no in vitro toxicity against human HCT-8 ileum cells. LD(50) values were greater than 1000 mg/kg, IP, for all compounds.

The cytotoxicity of copper(II) complexes of heterocyclic thiosemicarbazones and 2-substituted pyridine N-oxides.

Thiosemicarbazone complexes of copper(II) were shown to be potent cytotoxic/antineoplastic agents against the growth of murine and human tumor cells. Selectivity of some agents was demonstrated against specific solid tumor growth. In L1210 lymphoid leukemia cells the copper complexes preferentially inhibited DNA synthesis with their major effects on the purine de novo pathway at PRPP amido transferase, IMP dehydrogenase and dihydrofolate reductase. The reductions of purines correlated positively with inhibition of DNA synthesis and cytotoxicity of the agents tested. DNA itself was fragmented after incubation with the drug; however, no binding of the agent to nucleotide bases or intercalation between base pairs was evident.

The cytotoxicity of heterocyclic thiosemicarbazones and their metal complexes on human and murine tissue culture cells.

Heterocyclic thiosemicarbazones, thioureas and 2-substituted pyridine N-oxides as well as representative nickel, cobalt and copper complexes were shown to be potent antineoplastic/cytotoxic agents. The cytotoxicity was demonstrated against single cell leukemia as well as cell lines derived from solid tissue (colon adenocarcinoma, HeLa, KB, skin, bronchogenic lung, bone osteosarcoma and glioma). In L1210 cells, DNA synthesis and subsequently RNA synthesis were particularly inhibited by the agents. IMP dehydrogenase activity and thus purine de novo synthesis was reduced significantly by the agents. Dihydrofolate reductase, ribonucleoside reductase, nucleoside kinase and DNA polymerase alpha activities were inhibited by the agents. d(NTP) pool levels were reduced by most of the agents. DNA strand scission was present with all of the derivatives; however, there was no evidence of intercalation, cross linking or alkylation/binding to bases of DNA. This new group of compounds may offer novel exploratory derivatives for future investigations in the treatment of cancer.

Hypolipidemic activity of boronated nucleosides and nucleotides in rodents.

Base-boronated nucleoside and phosphate-boronated nucleotides were potent hypolipidemic agents in rodents, lowering both serum cholesterol and triglyceride levels. Rat VLDL and LDL cholesterol levels were generally reduced and HDL cholesterol levels were significantly elevated after 14 days dosing at 8 mg/kg/day. Tissue cholesterol, triglyceride and phospholipid levels were reduced by selected derivatives. Increased fecal excretion of lipids did not appear to be a mechanism by which these derivatives lowered serum lipids in rodents. Rather, the agents suppressed appetite and reduced the activities of rate-limiting enzymes for de novo lipid synthesis, specifically cytoplasmic acetyl CoA synthetase, squalene synthetase, and phosphatidylate phosphohydrolase with IC50 values of approximately 10(-5) m.

Estrogenic effect of phenol red in MCF-7 cells is achieved through activation of estrogen receptor by interacting with a site distinct from the steroid binding site.

MCF-7 cells serially subcultured in media containing phenol red show poor stimulation of progesterone receptor (PR) synthesis in response to estradiol compared to cells grown in phenol red-free media. Phenol red, when added to cytosol, did not compete with [3H]estradiol for estrogen binding sites in concentrations ranging from 2 microM-1 mM. However 25 microM of the dye was sufficient to increase nuclear translocation of estrogen receptor (ER) in the intact cell. Phenol red activates cytoplasmic ER as indicated by DNA-cellulose binding studies. When cells grown in phenol red-free medium were exposed to phenol red for 48 h, PR levels increased in a dose dependent manner. From these data, it may be concluded that phenol red causes estrogenic effect in MCF-7 cells through activation of cytoplasmic receptor by interacting at a site distinct from the steroid binding site.

Are estrogen receptors cytoplasmic or nuclear? Some immunocytochemical and biochemical studies.

The subcellular localization of estradiol receptor (ER) has been examined using various experimental approaches. Immunocytochemical studies using the monoclonal antibody JS 34/32, raised against calf uterine cytosolic ER, yielded only equivocal results. In general, cells and tissues pretreated with estradiol showed positive immunostaining in the nuclei whereas those not exposed to the steroid did not show any staining. Nuclear translocation of ER was examined in intact MCF-7 cells using compounds which are known to influence receptor activation. When MCF-7 cells were exposed to molybdate (20 mM), nuclear translocation was completely inhibited while dithiothreitol (20 mM), dibutyryl cAMP (1 microM) and dibutyryl cGMP (1 microM) increased the translocation 2-3-fold. Phenol red, at the range of concentrations generally used in tissue culture media, also increased translocation. The physiological validity of such translocation was examined using cellular progesterone receptor (PR) synthesis as a specific parameter. When MCF-7 cells were grown in media containing phenol red for 48 h, the PR synthesis increased significantly. We further examined whether cytoskeletal proteins are involved in the translocation of ER. Colchicine, an inhibitor of microtubule assembly, inhibited translocation of ER in MCF-7 cells at 1-10 microM. PR synthesis was also inhibited by colchicine in a dose-dependent manner. It may be concluded from these and other published data that ER may not be located at all times in a single subcellular compartment but may rather exist in a dynamic equilibrium between the plasma membrane, cytoplasm and nucleus.

The role of plasma lipoproteins in steroidogenic response of rat luteal cells during gonadotropin-induced refractory states.

Administration of human chorionic gonadotropin (hCG) to pregnant mare's serum gonadotropin--hCG primed rats results in the loss of in vitro responsiveness of the ovaries to exogenous gonadotropins for progesterone production. This state is associated with a loss of membrane receptors for hCG and a concomitant increase in lipoprotein receptors. Although lipoproteins potentiated gonadotropin response in ovaries from saline-injected rats, no stimulation was observed in hCG-desensitized ovarian cells. Examination of the time course for the loss of lipoprotein response after hCG injection revealed that injection with 50 IU of hCG results in a loss of gonadotropin response as early as 1 h after injection, but exogenous cholesterol-carrying lipoprotein fractions, LDL and HDL, were capable of stimulating progesterone production up to 4 h after hormone injection. Measurement of endogenous cholesteryl ester content showed that there was a 72% decline during this period with a concomitant increase in the basal progesterone production. One hour after hCG injection there was no stimulation of steroidogenesis by hCG in the presence or absence of exogenous lipoproteins. The refractoriness to exogenous hCG appeared only 4 h later when the hCG dose was reduced to 10 IU, whereas with 25 IU of hCG, the effect was similar to that observed using 50 IU of hCG. Such diverse steroidogenic stimuli as hCG, LH, LDL, cAMP, and cholera enterotoxin failed to stimulate progesterone synthesis in vitro in luteal cells of rats injected with 50 IU of hCG 48 h prior to sacrifice.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, degradation, and utilization of plasma high density and low density lipoproteins for progesterone production in cultured rat luteal cells.

These studies were intended to examine the binding and degradation of plasma lipoprotein fractions and the utilization of lipoprotein-bound cholesterol for progesterone production in cultured rat luteal cells. These cells bound [125I]human low density lipoprotein ([125I]iodo-hLDL),[125I]human high density lipoprotein ([125I]iodo-hHDL), and [125I]iodorat HDL ([125I] iodo-rHDL) with high affinity. The equilibrium dissociation constants of the binding of labeled rHDL, hHDL, and hLDL were 90.5, 78.3, and 36.8 micrograms/ml, respectively. All three lipoproteins were also degraded in a concentration-dependent manner, with apparent Km values of 18.3, 17.5, and 22.4 micrograms/ml for rHDL, hHDL, and hLDL, respectively. The degradation of the lipoproteins was inhibited by lysosomotropic agents, transglutaminase inhibitors, and metabolic inhibitors, suggesting that these lipoproteins undergo internalization and lysosomal degradation. In addition, all three lipoproteins also augmented the hCG-stimulated steroidogenesis. When cells were incubated with reconstituted LDL (the cholesterol ester in the LDL was replaced with [3H]cholesteryl linoleate) and the steroids produced identified, incorporation of tritium label in the progesterone fraction was observed in a time- and concentration-dependent manner. The incorporation of tritium into progesterone was increased by hCG and inhibited by an excess of unlabeled LDL in the incubation medium. These results show that the ovarian cells use lipoproteins as a source of cholesterol for steroidogenesis through receptor-mediated uptake and internalization, and the evidence suggests that the lipoproteins are intracellularly degraded.

Evidence for the existence of gonadotropin receptors in the nuclei isolated from rat ovary.

Specific binding of radiolabeled human chorionic gonadotropin (hCG) to nuclei isolated from pseudopregnant rat ovaries was studied. Incubation of cultured luteal cells or isolated nuclei with fluorescein isothiocyanate conjugated hCG showed concentration of fluorescence in the nuclear region. Isolated nuclei exhibited saturable high affinity binding of radiolabeled hCG with an apparent Kd of 3.42 X 10(-10) M. The binding was inhibited by increasing concentrations of unlabeled hCG. Under dissociating conditions, the bound hCG was dissociated from the nuclei. However, unlike the plasma membranes, the hCG bound to nuclei was not degraded before dissociation. Radiolabeled hCG bound to the nuclei could also be dissociated by brief exposure to MgCl2 or acidic incubation medium. The bound hCG was not extractable with 4M KCl or 2% Triton X-100. The available evidence suggest that nuclear receptors are distinct from plasma membrane receptors for hCG.

Studies on the mode of action of gonadotrophin-inhibiting material (anti-LH) isolated from human urine.

Gonadotrophin-inhibiting material (GIM) was able to inhibit the binding of 125I-hCG to rat Leydig cells, suggesting that its inhibiting properties are due to its ability to complete for the hCG binding sites on Leydig cells. Binding of fluorescein isothiocyanate-tagged GIM to Leydig cells was also seen. The effect of GIM on hCG-induced adenylate cyclase activation and testosterone production was also studied. It was found that there was a dose-dependent inhibition of both these effects.

Mammary gland differentiation in adult male rat--effect of prenatal exposure to cyproterone acetate.

Breast tissue of adult male Holtzman rats exposed to cyproterone acetate during embryonic differentiation showed presence of specific estradiol receptor proteins and C-19 steroid aromatase. We reported similar findings in gynecomastia in man. It is therefore proposed that gynecomastia probably results from failure of adequate testosterone action on the breast primordia during embryonic differentiation.

Oestradiol receptors in non-neoplastic gynaecomastic tissue of phenotypic males.

Cytoplasmic oestradiol receptors have been reported in breast tumor tissue but not in the uninvolved tissue from the same patients. Since gynaecomastic tissue is highly predisposed to neoplastic transformation, such receptors were looked for in this tissue from 13 phenotypic males of whom 6 had Klinefelter's syndrome. High affinity saturable binding (Kd approximately 10(-10) M) of 3H-oestradiol-17 beta was found in the breast tissue from 12 of these patients by gel elution and dextran-coated charcoal techniques. The concentration of binding sites were found to range from 16 to 359 fmol/mg cytosol protein. Previous studies by the present authors showing steroid aromatising capacity and the present finding of specific oestradiol receptors in a 'high-risk tissue' in the absence of any histologically detectable neoplasms might be relevant in elucidating the natural history of breast cancer in such individuals.