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Stimulation of mitochondrial aspartate aminotransferase (mAAT) activity by testosterone was determined in organ cultures of rat ventral prostate. The effect of testosterone on citrate accumulation in the culture medium was also determined. Testosterone stimulation of citrate accumulation and mAAT occurred in a dose dependent manner. Stimulation of mAAT activity occurred after a 1-3 h lag period and appeared to involve the synthesis of specific RNA since the response was inhibited by actinomycin D. Studies utilizing [3H]L-leucine indicated that unlike the total tissue, testosterone stimulated the incorporation of [3H]leucine into proteins of the mitochondrial fraction. The results suggested that mitochondrial proteins may be more sensitive to testosterone stimulation than cytosol proteins. The response was specific for mAAT since testosterone had no effect on mitochondrial malic dehydrogenase activity. The data suggested that testosterone may regulate prostate citrate content by the induction of mAAT in prostate mitochondria, which results in a source oxalacetic acid for citrate synthesis through transamination of aspartate by alpha ketoglutarate.

The net production of citrate from exogenous substrates by rat ventral prostate was investigated. The preparation of isolated prostate epithelial cells was described. These cells were capable of oxidizing pyruvate (5 mmol/l) as a source of acetyl coenzyme A. The addition of aspartate + alpha-ketoglutarate (5 mmol/l) in the presence of pyruvate resulted in significant net production of citrate and excess oxalacetate. In the presence of aspartate and glutamate, the cells were capable of producing citrate without excessive oxalacetate production. Neither glucose alone nor glucose plus pyruvate resulted in net citrate production. The results demonstrated that aspartate could serve as a 4-carbon source of oxalacetate for citrate synthesis. Furthermore, the results indicate the intramitochondrial operation of a glutamate-aspartate-citrate pathway involving mitochondrial aspartate aminotransferase and glutamic dehydrogenase activities in prostate epithelial cells.

The effects of FireMaster BP-6 and several pure isomers on hepatic microsomal mixed-function oxidase (MFO) activity in rainbow trout have been investigated. After one parenteral injection of these agents at 150 mg/kg and sacrifice 5 d later, there was no elevation of hepatic cytochrome P-450. The noncoplanar isomers 2,4,5,2',4',5'-hexa and 2,3,4,5,2',4',5'-heptabromobiphenyl did not increase any MFO activity generally associated with an increase in cytochrome P-450 levels, including the dealkylations of ethoxycoumarin and benzphetamine. The coplanar isomer 3,4,5,3',4',5'-hexabromobiphenyl produced an increase in O-deethylation of both ethoxycoumarin and ethoxyresorufin, suggesting enhancement of cytochrome P-448 associated activity, but produced no increase in benzphetamine N-dealkylation. The "mixed inducer" 2,4,5,3',4',5'-hexabromobiphenyl, produced a small but insignificant elevation of cytochrome P-448 associated MFO activities and no increase in cytochrome P-450 associated activities. At both 150 and 500 mg/kg, the commercial mixture of polybrominated biphenyls FireMaster BP-6 produced marked elevations of both ethoxycoumarin and ethoxy-resorufin dealkylations but had no effect on demethylation of benzphetamine. When microsomes from rainbow trout treated with FireMaster BP-6 were examined electrophoretically, A coomassie Blue-staining band at 57,000 daltons was intensified, as seen after treatment of fish with the cytochrome P-448 inducer beta-naphthoflavone. It is concluded that coplanar polybrominated isomers produce an induction of hepatic cytochrome P-448 associated activity, while noncoplanar isomers are ineffective as inducers of cytochrome P-450 related MFO activities in rainbow trout.

We compared a new, rapid, qualitative test for rotavirus (TestPack Rotavirus; Abbott Laboratories, North Chicago, Ill.) with another enzyme immunoassay (Pathfinder Rotavirus; Kallestad Laboratories, Inc., Austin, Tex.) and electron microscopy to determine its clinical utility in a population of symptomatic hospitalized children. In the first part of the study, 100 frozen stool samples were tested. The results after resolution with a blocking reagent showed a sensitivity of only 50% and a specificity of 88% for TestPack Rotavirus. In the second part of the study, we tested TestPack Rotavirus on 100 fresh, unfrozen samples. The results (sensitivity/specificity) were as follows: TestPack Rotavirus, 95/90%; Pathfinder Rotavirus, 84/98%; direct electron microscopy, 63/100%. Although it was not as sensitive or specific as immune electron microscopy, TestPack Rotavirus was more sensitive than direct electron microscopy or Kallestad Pathfinder Rotavirus. TestPack Rotavirus represents a rapid, qualitative method for the detection of rotavirus in stools of symptomatic children.

The prostate gland is unusual in that it has a high content of citric acid compared with other soft tissues. The mechanism of citrate accumulation in prostate is unknown. One possibility is that aspartate aminotransferase (AAT) could supply an extra Krebs cycle source of oxaloacetate for citrate formation. Since testosterone regulates citrate production and secretion and strong transaminase activity exists in prostate, a relationship between AAT and citrate production could exist if testosterone also regulates AAT activity. To investigate this possibility, animals were castrated and given testosterone. After a total castration period of 72 hours, animals were sacrificed and mitochondrial AAT activity determined. Results revealed that castration decreased AAT activity and testosterone replacement stimulated activity. Neither castration nor testosterone affected malate dehydrogenase (MDH) activity. Both actinomycin D and cycloheximide blocked the testosterone stimulation of AAT activity. These studies indicate that testosterone may regulate AAT activity in rat ventral prostate. Furthermore, regulation may occur through testosterone stimulation of specific RNA and protein synthesis.

The prostate gland (human, rat ventral prostate) has the major function of accumulating and secreting extremely high levels of citrate. This function requires unique and specialized metabolic pathways associated with prostate secretory epithelial cells by which exogenous substrates must be utilized as the six-carbon sources of citrate. Recent studies demonstrated that aspartate can serve as the four-carbon source of oxalacetate for citrate synthesis. Identification of the two-carbon source of acetyl CoA (AcCoA) had not been established. The present study investigated the probability that exogenous glucose, via pyruvate oxidation, is a physiological source of AcCoA for net citrate production by isolated epithelial cells from rat ventral prostate. Under adequate oxygenation, 5 mM glucose in the presence of aspartate plus glutamate markedly stimulated citrate production. Exogenous and endogenous pyruvate also stimulated net citrate production. We propose that glucose via aerobic glycolysis and pyruvate oxidation provides AcCoA, which condenses with oxalacetate obtained from aspartate transamination for citrate synthesis. Prostate epithelial cells do not readily oxidize citrate, which permits accumulation and secretion of the synthesized citrate.

The effects of testosterone on mitochondrial aspartate aminotransferase (mAAT) synthesis in rat ventral prostate was investigated. Procedures for the isolation, purification and characterization of AAT isozymes were developed and described. Purified mAAT preparations contained no demonstrable contaminating proteins. Prostatic mAAT was characterized as a cationic protein with an estimated mol. wt of 120,000. Cytoplasmic AAT (cAAT) isozyme was identified as an anionic protein with an estimated mol. wt of 132,000. A cytosolic cationic isozyme, similar to mAAT, was also identified as pre-mAAT. Testosterone administration to castrated rats resulted in significant increases in leucine incorporation into mAAT, in the level of mAAT, and in mAAT activity. These effects of testosterone were observed within 2 h of administration. Conversely, testosterone administration had none of these effects on cAAT or on non-AAT protein pool. Testosterone treatment did appear to increase leucine incorporation into pre-mAAT. Testosterone treatment in organ cultures and in prostate epithelial cell cultures resulted in the same stimulatory effects on mAAT as observed in the in vivo studies. The hormone was effective at the physiological concentration of 2 X 10(-9) M. These results indicated that testosterone has a rapid and specific effect on the biosynthesis of mAAT. This continues to support our proposal that testosterone regulates prostate citrate production via a stimulatory effect on mAAT which results in increased mitochondrial synthesis of citrate from aspartate.

The effects of liposome-encapsulated diethylstilbestrol (DES) on the size of the ventral prostate gland and total testosterone levels in rats were studied. DES was encapsulated in phosphatidycholine-cholesterol-stearylamine-alpha-tocopherol containing multilamellar liposomes in a molar ratio of 4:5:1:0.01. The liposome suspension or free DES plus blank liposomes were administered sc on days 1, 3, and 5 at a dose of 400, 87, or 12 micrograms/kg. Plasma testosterone values showed a decrease with dose for animals treated with either entrapped DES or free DES. Prostate gland weight per g animal and prostatic protein mg per g animal on day 7 were significantly decreased in the treated animals compared to those in controls dosed with DES plus buffer containing liposomes. In addition, there was a body weight loss for the high dose DES-encapsulated animals and for high and medium dose DES plus blank liposome-treated and blank liposome-treated animals over the treatment period, although only the DES-encapsulated group value was statistically significant (P less than 0.05). Animals at the other doses did not lose weight, but exhibited weight gains which were less than those observed for sham-injected animals. Rats that received 7 micrograms/kg liposome-entrapped DES for 4 weeks had significantly lower prostate gland weights, prostatic protein levels, and testosterone levels than control animals receiving the same regimen of free DES plus blank liposomes. Results from the studies indicate that liposome-entrapped DES was more effective in decreasing the size of the prostate gland and that the desired therapeutic end point (prostate ablation) may be attained with a lower dose of DES, which would decrease the risk of weight loss in the experimental animals.

Naphthalene and 2-methylnaphthalene cause a highly organo- and species-selective lesion of the pulmonary bronchiolar epithelium in mice. Naphthalene- but not 2-methylnaphthalene-induced pulmonary bronchiolar injury is blocked by prior administration of the cytochrome P-450 monooxygenase inhibitor piperonyl butoxide, thus suggesting that metabolism by enzymes other than the P-450 monooxygenase inhibitor piperonyl butoxide, thus suggesting that metabolism by enzymes other than the P-450 monooxygenases may be important in 2-methylnaphthalene-induced lung injury. Since many of the polycyclic aromatic hydrocarbons are metabolized by the prostaglandin endoperoxide synthetase system and because detectable xenobiotic metabolizing activity has been associated with the prostaglandin synthetases in the Clara cell, the studies reported here were done to compare NADPH-versus arachidonate-dependent metabolism of naphthalene and 2-methylnaphthalene in vitro and to determine whether indomethacin, a potent inhibitor of prostaglandin biosynthesis, was capable of blocking the in vivo toxicity of these two aromatic hydrocarbons. The NADPH-dependent metabolism of naphthalene and 2-methylnaphthalene to covalently bound metabolites in lung or liver microsomal incubations occurred at easily measurable rates. Renal microsomal NADPH-dependent metabolism of either substrate was not detected. The formation of covalently bound naphthalene or 2-methylnaphthalene metabolites was dependent upon NADPH and was inhibited by the addition of reduced glutathione, piperonyl butoxide, and SKF 525A. Covalent binding of radioactivity from [14C]2-methylnaphthalene also was strongly inhibited by incubation in a nitrogen atmosphere or at 2 degree. The arachidonic acid-dependent formation of reactive metabolites from naphthalene or 2-methylnaphthalene was undetectable in microsomal incubations from lung, liver or kidney. Indomethacin, 1 hr before and 6 hr after the administration of 300 mg/kg naphthalene or 2-methylnaphthalene, failed to block the pulmonary bronchiolar injury induced by these aromatic hydrocarbons. These studies suggest that the major enzymes involved in the metabolic activation of naphthalene or 2-methylnaphthalene in vitro are the cytochrome P-450 monooxygenases and that cooxidative metabolism by the prostaglandin synthetases appears to play little role in the formation of reactive metabolites in vitro.

Constant intravenous infusion (65 to 260 ng/min) of prostaglandin E2 (PGE2) produced an elevation of the plasma calcium concentration in unanesthetized rats in a period of 4 h. The increase in plasma calcium was related to the amount of PGE2 infused, and the hypercalcemia persisted for at least 10 h during continuous infusion. There was also a small increase in plasma inorganic phosphorus concentration. The increase in plasma calcium was not due to hemoconcentration; in fact, there was a small decrease in hematocrit during infusion of PGE2, and no change in total plasma protein concentration was detected. Infusion of PGF2, did not raise plasma calcium concentration. We conclude that under appropriate conditions, the exogenous administration of PGE2 can cause an elevation of plasma calcium in the intact rat.

The use of animal models to study drug dependence and tolerance requires that the species used metabolizes the drugs like man, a condition frequently not fulfilled by non-primate species. The metabolic fate of several amphetamine drugs, namely amphetamine, norephedrine, chlorphentermine and phenmetrazine, in the rhesus monkey and the tamarin and two non-primate species has been investigated and compared to that found for man. The findings show that the two primate species metabolize these drugs in a manner similar to that in man.

Earlier studies demonstrating marked differences in the profile of polar metabolites formed during incubations of glutathione, naphthalene and microsomes from target (lung) and nontarget (liver and kidney) tissues of the mouse suggested that the formation of a particular reactive metabolite may be the underlying basis for the highly organ selective toxicity of this hydrocarbon. The studies reported here were done to characterize more fully the microsomal metabolism of naphthalene to 1,2-dihydro-1,2-dihydroxynaphthalene and to three glutathione-derived conjugates that were separated by high-pressure liquid chromatography. The microsomal formation of polar naphthalene metabolites was linear with time and microsomal protein; the relative proportions of each of the metabolites remained relatively stable over the range of time and protein concentrations studied. The rate of formation of naphthalene glutathione adducts, but not the dihydrodiol, was dependent upon the amount of 100,000 X g supernatant protein added. Addition of lung cytosol to liver microsomal incubations or liver cytosol to lung microsomal incubations altered the overall rate of conjugate formation but not the relative proportions of each of the three conjugate peaks. Epoxide hydrolase induction by dietary butylated hydroxyanisole or inhibition by cyclohexene oxide altered the rate of hepatic microsomal formation of naphthalene dihydrodiol in the expected manner and increased the production of conjugate peak 2. Butylated hydroxyanisole or cyclohexene oxide failed to alter the rate of formation of conjugate peak 1 or 3. Addition of piperonyl butoxide or SKF 525-A to hepatic microsomal incubations markedly decreased covalent binding of naphthalene metabolites but only slightly decreased glutathione adduct formation. Dihydrodiol formation was increased by both inhibitors. Phenobarbital or 3-methylcholanthrene pretreatment produced a marked increase in the pulmonary microsome-catalyzed formation of all four polar naphthalene metabolites. In comparison, phenobarbital increased the rates of formation of the dihydrodiol, conjugate peaks 1 and 2 but not 3 in hepatic microsomes. 3-Methylcholanthrene increased the rate of formation of the dihydrodiol and conjugate peak 2 but not 1 or 3. These studies indicate that the predominant formation of conjugate peak 2 in lung microsomal incubations in comparison to liver microsomal incubations is due to the regio- or stereoselectivity of naphthalene metabolism by cytochrome P-450 monooxygenases or epoxide hydrolases but not by the glutathione transferases.

The oxidative metabolites of 2-methylnaphthalene (2-MN) were extracted from rat liver microsome suspensions. One monohydroxylated and three isomeric dihydrodiol metabolites of 2-MN were isolated and purified by HPLC. The metabolites were characterized by GC-MS together with 1H Fourier transform (1HFT) NMR. The identification of a 2-MN-monohydroxylated product as 2-hydroxymethylnaphthalene was confirmed by comparison of its HPLC retention time and NMR spectrum with that of a synthetic standard. The three isomeric dihydrodiol metabolites had different HPLC retention times, fragmentation patterns, ultraviolet, and 1H NMR spectra. GC-MS of the silylated dihydrodiols revealed the consistent presence of an ion peak at m/z 320, indicative of a disilylated dihydrodiol. Analysis of the 1H FT NMR spectra revealed the metabolites to be the 3,4-dihydrodiol, 5,6-dihydrodiol, and 7,8-dihydrodiol of 2-methylnaphthalene.

Acute, subchronic, and chronic toxicity studies were conducted with isomazole, a new (investigational) inotropic agent with significant vasodilator properties. When given acutely to either young adult rats or mice, the oral median lethal dose was approximately 135 or 525 mg/kg, respectively. Clinical signs of toxicity were leg weakness, hypoactivity, tremors, clonic convulsions, and ataxia. Fischer 344 rats (15/sex/group) were fed diets containing isomazole in concentrations of 0, 0.03, 0.1, or 0.3% for 3 months with no resulting mortality or clinical signs of toxicity. The average daily intake of the compound was approximately 0, 20, 65, or 198 mg/kg in both sexes. Body weight gain, food consumption, and efficiency of food utilization were significantly reduced only in males in the 198 mg/kg dose group. There were no changes of toxicological significance in any of the hematology, clinical chemistry, or urine parameters. Isomazole produced significant increases in hepatic p-nitroanisole O-demethylase activity and relative liver weight primarily at the 65 and 198 mg/kg treatment levels. These effects were consistent with induction of the hepatic drug-metabolizing enzyme system. Histopathologic findings consisted of centrilobular fat deposition in the livers of 9 of 15 males in the 198 mg/kg dose group, and periarteritis in the adventitia of small and medium-sized arteries in the mesentery in 3 of 30 and 12 of 30 animals from the 65 and 198 mg/kg dose groups, respectively. The plasma levels of isomazole had a tendency to drop after 90 days compared to Day 2 in all dose groups and was more apparent in male rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The phenomenon of unidirectional non-cross tolerance (UNCT) to etorphine was observed in adult male rats tolerant to morphine. Using a morphine pellet (four 75 mg morphine pellets) implantation protocol, a 10-fold increase in the analgesic ED50 value, obtained by tail-flick assay, was observed for morphine sulfate, with no alteration in analgesic ED50 value for etorphine. After etorphine pellet (two 60 micrograms pellets) implantation, no significant change in the ED50 value for etorphine was observed, but the ED50 value for morphine increased 15-fold. Both morphine and etorphine pellet-implanted animals, challenged with naloxone, showed similar qualitative signs of precipitated withdrawal. This seeming dissociation obtained by demonstration of physical dependence in the absence of tolerance to etorphine has its basis in the UNCT phenomenon. Thus, appropriate experimental paradigms must be included when evaluating tolerance to avoid specious conclusions.