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A sensitive HPLC assay for measurement of the antipsychotic drug, olanzapine, in plasma has been developed. The assay has a limit of quantitation of 1 ng ml-1 in plasma and utilizes solid-phase extraction and electrochemical detection. The method provides a linear response for olanzapine over a concentration range of 1-100 ng ml-1 with coefficients of determination greater than 0.9912. The inter-assay precision was 15.9% at the limit of detection and ranged from 7.33% to 8.47% over the range of 5-100 ng ml-1. The intra-assay precision was in the range 0.97%-26.0%. The inter-assay accuracy ranged from 98.9 to 118% and the intra-assay accuracy ranged from 92.5% to 125% of the theoretical value. In addition, the assay was extended to measure the plasma levels of two metabolites of olanzapine, namely the N-desmethyl- and the 2-hydroxymethyl analogs. The utility of the assay was demonstrated following the administration of a single oral dose of 14C-olanzapine to rats where, at several time-points after dosing, the plasma was assayed for total radioactivity, levels of olanzapine, and the two metabolites. Olanzapine and two of its metabolites accounted for less than 50% of the total plasma radiocarbon; olanzapine accounting for approximately 39% at the Cmax, N-desmethyl for 5% and 2-hydroxymethyl for 8% respectively. The plasma elimination half-times for olanzapine and the two metabolites were approximately the same, ranging from 3.3 to 4.4 h.

The induction of DNA repair (the "SOS Response") by X rays has been studied in E. coli GE94, irradiated under euoxic versus anoxic conditions. GE94 contains a recA-lacZ gene fusion that allows the radiation-induced transcription of recA to be followed by measuring the changes in beta-galactosidase levels. At doses up to about 450 Gray, more recA transcription (indicating more DNA damage capable of inducing SOS repair activity) occurs in cells irradiated in air instead of 100% nitrogen. Although the presence of dissolved oxygen cannot change the initial number of DNA radicals (initial "energy-deposition events"), apparently more of these radicals become stable lesions when oxygen is present.

Citrate production and accumulation are characteristic physiological functions of the prostate gland that are regulated by testosterone and prolactin. Results reported here show that treatment of LNCaP cells with dihydrotestosterone (DHT) resulted in increased intracellular citrate and increased citrate accumulation in the medium. Moreover, DHT also caused an increase in both mitochondrial aspartate aminotransferase (mAAT) activity and the steady state level of pmAAT (precursor) mRNA. Androgen treatment increased the rate of citrate oxidation by LNCaP cells as it does in rat ventral prostate which suggests that DHT increased aconitase activity in LNCaP cells. The results reported here are consistent with the operation of the glutamate-aspartate-citrate pathway that we described for rat ventral prostate. In addition, these results provide the first evidence that androgen responsive functions associated with citrate metabolism are retained in LNCaP cells. In addition, and more important, these results suggest that the more aggressive PC-3 carcinoma cell line has a higher rate of citrate oxidation than the less aggressive LNCaP cell line. This could have significant implications for our understanding of the relationship between alterations in prostate citrate metabolism and expression of the malignant phenotype.

Mitochondria (m-)aconitase is a rate-limiting regulatory enzyme in prostate epithelial cells which minimizes citrate oxidation by these cells. This unique metabolite characteristic is responsible for the ability of the prostate to accumulate and secrete extraordinarily high levels of citrate. Testosterone is a major regulator of prostate growth and function, and stimulates citrate oxidation. Therefore, an important action of testosterone might be its stimulation of m-aconitase in prostate epithelial cells. Studies were conducted with rat ventral prostate (VP) epithelial cells to establish the effect of testosterone on the level of m-aconitase and corresponding citrate oxidation. Physiological concentrations (10(-7)-10(-10) M) of testosterone in vitro markedly increased the level of m-aconitase in freshly prepared isolated prostate epithelial cells. This increase was apparent within 3 h of exposure to the hormone. The stimulatory effect of testosterone on m-aconitase was abolished by actinomycin D and by cycloheximide. Both the level of m-aconitase enzyme and the level of m-aconitase activity were similarly increased by testosterone treatment. Correspondingly, testosterone increased the rate of mitochondrial citrate oxidation while having no effect on the rate of isocitrate oxidation, thereby demonstrating that the action of testosterone is specifically targeted at the m-aconitase reaction. In vivo studies revealed that castration markedly decreased and testosterone administration increased the m-aconitase level of prostate epithelial cells. In contrast, neither liver nor kidney m-aconitase level was altered by castration. These studies demonstrate that testosterone regulates the biosynthesis of m-aconitase in prostate epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondrial aspartate aminotransferase (mAAT) is one of two key enzymes in the pathway of citrate production in prostate. Expression of mAAT is modulated by testosterone and prolactin in prostate. We cloned the promoter and 5'-flanking region of the rat mAAT gene and sequenced 2.0 kilobases of the DNA. This fragment contains the 5'-regulatory promoter region that lacks a TATA and a CCAAT box but is G+C rich. The 5'-upstream flanking region contains sequences that have high homology with the consensus glucocorticoid response element/androgen response element (ARE) and a reported ARE sequence that is different from the consensus sequence. Functional transcription studies showed that a 481-base region containing the two ARE sequences was sufficient for androgen-regulated gene expression. There are multiple transcription start sites that are regulated by testosterone in prostate. In liver, on the other hand, castration did not affect transcription from any of the start sites. Therefore, these data provide evidence that transcriptional regulation of the rat pmAAT gene occurs through an ARE located in the 5'-region. In addition, not only is gene expression modulated by testosterone, but the effect of testosterone on transcription is cell specific.

Prolactin is an important regulator of prostate citrate production. In rats this regulatory effect of prolactin is specific for lateral prostate, and has no effect on either ventral or dorsal prostate. The mechanisms by which prolactin regulates prostate citrate production have not been elucidated. Two key regulatory enzymes involved in citrate synthesis by prostate epithelial cells are mitochondrial aspartate aminotransferase (mAAT) which provides oxalacetate, and PDH E1 alpha (pyruvate dehydrogenase) which provides acetyl CoA for citrate synthesis. Our previous studies demonstrated that prolactin regulates mAAT. However, an increase in citrate synthesis would require an increase in both oxalacetate and acetyl CoA. Therefore, we investigated the possibility that prolactin might also regulate PDH E1 alpha in LP epithelial cells. The present studies demonstrate that prolactin administration (1 mg/rat) to rats resulted in an increased level of E1 alpha in LP epithelial cells within 6 hr, but had no effect on the E1 alpha level of VP epithelial cells. In vitro studies demonstrated that exposure of freshly prepared LP epithelial cells to prolactin (0.1-1.0 microgram/ml) resulted in increased levels of E1 alpha. Prolactin had no effect on either VP or DP epithelial cells. The stimulatory effect of prolactin on E1 alpha was inhibited by actinomycin and cycloheximide, thereby indicating that prolactin stimulated the biosynthesis of E1 alpha. The studies reveal that prolactin specifically stimulates E1 alpha levels of LP epithelial cells, whereas testosterone specifically stimulates E1 alpha levels of VP epithelial cells. At this time, we propose that the effects of prolactin and testosterone involve increased expression of the E1 alpha gene of LP and VP epithelial cells, respectively.

Normal and benign prostate hyperplasia (BPH) prostate is characterized by the presence of extraordinarily high levels of citrate. Presumably, this results from the inability of the prostate epithelial cells to oxidize citrate due to a limiting mitochondrial (m-) aconitase. In contrast, prostate carcinoma (CA) is not characterized by high citrate levels. Malignant prostate epithelial cells apparently undergo a metabolic transformation from citrate-producing to citrate-oxidizing cells. A consequence of citrate production in normal and BPH cells is an inefficient and low level of ATP production. It is proposed that the process of malignancy necessitates an energy production that cannot be provided by citrate-producing cells. Consequently, the transformation of prostate epithelial cells to citrate-oxidizing cells which increases the energy production capability is essential to the process of malignancy and metastasis. The metabolic transformation likely occurs as a premalignant or early malignant stage. This bioenergetic theory of prostate malignancy, if correct, will provide new approaches to the diagnosis and treatment of CA.

Tissue distribution studies, utilizing whole-body autoradiography and organ dissection techniques, were conducted in male Fischer 344 rats following the oral administration of 14C-dapoxetine HCl, a potent serotonin reuptake inhibitor. The preliminary study using whole-body autoradiography proved invaluable in locating radioactivity in an organ not usually harvested in a tissue distribution study, namely the preputial gland. Selected organs, based on whole-body autoradiography findings, were dissected from rats and analyzed for radiocarbon content by liquid scintillation counting and for parent drug and N-dealkylated metabolites by extraction and HPLC analysis. Highest concentrations of radiocarbon were observed in the organs of absorption and elimination (ileum, cecum, stomach, duodenum, liver, colon, and kidney) but notable quantities were observed in the lung and preputial and Harderian glands. Most tissues had returned to background radioactive levels 72 h after dosing but persistent concentrations of radiocarbon were present in the preputial gland and liver one week after the single dose of 14C-dapoxetine. Analysis by HPLC demonstrated the presence of parent drug and N-desmethyl metabolite (nor-dapoxetine) in those organs examined; however, the majority of the radioactivity remained unidentified.

We propose that: 1. Prolactin is a trophic hormone required for normal development and growth of prostate as well as other tissues. 2. Citrate production, the major function of prostate, is directly regulated by prolactin. 3. The mechanisms of prolactin regulation of citrate production by prostate epithelial cells include a. Regulation of the pmAAT gene, which results in an increase in the biosynthesis of mAAT and, ultimately, an increase in citrate synthesis (the prolactin regulation of gene transcription is mediated via PKC). b. Inhibition of citrate oxidation, possibly via zinc inhibition of m-aconitase. c. Stimulation of aspartate transport, possibly via regulation of the biosynthesis of the high-affinity aspartate transporter. 4. These relationships apply to normal human prostate, and might have important implications in the pathogenesis of prostate neoplasms. 5. The regulation of prostate citrate production is the reproductive function of prolactin in males.

A major and unique function of the prostate is the production of extremely high levels of citrate. Testosterone is an important regulator of this function. This regulation is achieved by hormonal stimulation of citrate synthesis by prostate secretory epithelial cells. Previous studies have established that testosterone stimulates the mitochondrial transamination of aspartate to OAA for citrate synthesis. However, increased citrate synthesis would also require an increased availability of AcCoA which is derived from pyruvate. The present studies were concerned with the in vivo effect of testosterone on pyruvate dehydrogenase (PDH) activity of prostate mitochondria isolated from rat ventral prostate (VP). The results demonstrated that testosterone administration to castrated rats stimulates PDH activity and citrate production from pyruvate in the presence of OAA. Therefore testosterone increases both OAA and acetyl CoA production which results in increased citrate synthesis. The mechanism of testosterone regulation of PDH can now be investigated.

The prostate gland produces and secretes extraordinarily high levels of citrate. Studies with rat ventral prostate (VP) have demonstrated that aspartate can serve as a four-carbon source of oxalacetate in the synthesis of citrate. To achieve this, prostate secretory epithelial cells must contain a transport system for the active uptake of aspartate from circulation. The present studies with VP epithelial cells confirm the existence of a Na(+)-dependent high-affinity L-aspartate transporter. The transporter has an optimal pH approximately 7.5 and is temperature dependent. It appears to be an anionic amino acid transporter capable of transporting L-glutamate but not basic or neutral amino acids. The transporter is inhibited by ATPase inhibitors, thereby indicating its dependency on a Na+ gradient. The characteristics of the high-affinity L-aspartate transporter are consistent with its operation at the basilar membrane for the transport of circulating aspartate into the cell. Castration (24 hr) resulted in a significant decrease in the ability of VP epithelial cells to transport L-aspartate. The administration of testosterone to castrated rats completely restored L-aspartate transport. In addition, in vitro testosterone addition (10(-8) M for 30 min) to isolated prostate epithelial cells markedly increased L-aspartate transport. Both cycloheximide and actinomycin inhibited the testosterone effect. The studies reveal that testosterone is a regulator of this Na(+)-dependent high-affinity L-aspartate transporter. The mechanism of this testosterone effect appears to involve both RNA and protein synthesis. We now have a model system to elucidate this novel effect of testosterone.

The effect of testosterone on the precursor mitochondrial aspartate aminotransferase (pmAAT) gene and on pmAAT-mRNA was studied in rat ventral prostate (VP) and pig prostate epithelial cells. Castration significantly decreased the level of nuclear pmAAT transcripts in VP; whereas testosterone treatment of castrated animals restored the level of pmAAT transcripts. Correspondingly, castration resulted in a marked decrease in the transcription rate of the pmAAT gene; whereas testosterone treatment markedly increased the transcription rate. In vitro studies with isolated pig prostate epithelial cells demonstrated that testosterone directly and rapidly induced a transient increase in the transcription rate of the pmAAT gene. The increase in transcription was associated with an increase in the steady-state level of pmAAT-mRNA. Similar in vitro effects were observed with isolated VP epithelial cells. In addition to its stimulatory effect on transcription of the pmAAT gene, testosterone also increased the half-life of pmAAT-mRNA from 2 h in the absence of hormone to 16 h in its presence. Consequently, testosterone appears to stabilize the pmAAT-mRNA. The combination of its immediate effect on stimulating the transcription of the pmAAT gene and its stabilizing effect on pmAAT-mRNA would account for the increase in the steady-state level of pmAAT-mRNA by testosterone. These studies support our proposal that, through these effects, testosterone increases the biosynthesis of mAAT thereby increasing the transamination of aspartate to oxaloacetate and ultimately increasing the synthesis of citrate. This appears to provide at least one of the mechanisms by which testosterone regulates prostate citrate production.

Prolactin (PRL) has been reported to stimulate citrate production and the activity of mitochondrial aspartate aminotransferase (mAAT) and its precursor form pmAAT in prostate epithelial cells. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused the same result as PRL, which suggests that the PRL effect on mAAT activity might be mediated by protein kinase C (PKC) stimulation of pmAAT gene transcription. Both PRL and TPA increased the level of pmAAT mRNA by 2.5- to 3-fold in pig prostate cells. The PKC inhibitor gossypol completely inhibited the PRL and TPA induced increases. In addition, the effects of both PRL and TPA were inhibited by down-regulation of prostate PKC. Nuclear run-off assays indicated that PRL and TPA induction of pmAAT occurred primarily at the transcriptional level. The stimulation of pmAAT transcription by TPA suggests that the pmAAT gene contains a TPA response element. Thus, these results are consistent with our previous observation that PRL directly induces pmAAT and that the mechanism of this PRL effect might involve stimulation of PKC.

Accumulation and secretion of extraordinarily high levels of citrate are principal functions of the prostate gland of humans and other animals. To achieve this, prostate secretory cells must possess unique metabolic relationships which distinguish them from virtually all other cells. Furthermore, citrate metabolism is markedly altered in benign prostatic hyperplasia (BPH) and in prostatic carcinoma (CA). This review assimilates existing information and presents current concepts related to 1) the pathway of metabolism associated with net citrate production, 2) the involvement of transporting mechanisms associated with citrate secretion, 3) energy implications of citrate production, 4) altered metabolic relationships in BPH and CA, and 5) the importance of citrate relationships as biochemical markers for characterizing prostate secretory epithelial cells. It is hoped that this review will bring attention to the importance and urgency of elucidating and understanding the metabolic relationships associated with citrate production by normal and neoplastic prostate epithelial cells. Research in these areas has been severely neglected despite the fact that the combined incidence of BPH and CA constitutes the most prevalent neoplastic disease among men.

Following the oral administration of [14C]quinelorane, a potent and highly specific dopamine (D2) agonist, to rats, mice, and monkeys, the compound was well absorbed, with 50% or more of the radioactivity appearing in the urine within 24 hr. Dogs were pretreated with 22 consecutive daily doses of quinelorane by the oral route (in order to induce tachyphylaxis to the emetic effect) before receiving an iv dose of [14C]quinelorane; just over 80% of the radioactivity was excreted into the urine. A tissue-distribution study in rats receiving a single oral dose of 0.1 mg/kg [14C]quinelorane indicated a widespread distribution of radioactivity, with levels being notably low in the blood and plasma and high in the salivary gland, adrenals, pancreas, and spleen; levels were highest in the stomach and kidneys. The Tmax of radiocarbon in the 22 tissues varied between 0.5 and 6 hr, with some tissues showing a plateau of radioactivity between these time-points. After 8 hr, levels of radioactivity were clearly decreasing, and by 48 hr, background levels were attained. Following the oral and iv administration of quinelorane to rats, the systemic bioavailability was calculated to be 16% and the volume of distribution was found to approximate that of total extracellular water, i.e. approximately 300 ml/kg. Since absorption was satisfactory and the tissue distribution study indicated widespread radioactivity, the low bioavailability may be due to first-pass metabolism. Rats excreted marginally more of the N-despropyl metabolite than unchanged drug into the urine, and dogs excreted principally unchanged quinelorane into their urine, followed by the N-despropyl metabolite.(ABSTRACT TRUNCATED AT 250 WORDS)

A unique and major function of prostate secretory epithelial cells is to synthesize, accumulate, and secrete extraordinarily high levels of citrate. This function is regulated by testosterone and by prolactin. Concepts of the mechanisms of hormonal regulation are presented. The relationship of testosterone and prolactin to the origin and homologies of different prostate cell lines is described. The metabolic differentiation of citrate and non-citrate producing prostate secretory epithelial cells is discussed. Concepts of the pathogenesis of prostatic neoplasms are presented based on hormonal, metabolic, and homologous relationships associated with citrate production. Characterization of normal and neoplastic secretory epithelial cells by their citrate function is emphasized. The urgency and necessity for research relating to all aspects of prostate citrate production in normal and pathological prostate are emphasized.

The effect of testosterone on precursor mitochondrial aspartate aminotransferase (pmAAT) mRNA was studied in rat ventral prostate and primary cell cultures of mini-pig prostate. Testosterone induced a 2-3-fold increase in pmAAT mRNA level in both rat ventral prostate and mini-pig prostate cultures. The pmAAT mRNA induction occurred 30 min after testosterone treatment and was maximal by 1.5 h. Prostatic mAAT activity was also induced by testosterone with a 1-2 h lag period. The time-course of induction of pmAAT mRNA, pmAAT activity and mAAT activity was consistent with stimulation of mRNA synthesis followed by increased synthesis and import of pmAAT into mitochondria. The effect of testosterone on pmAAT mRNA was specific because the increase in pmAAT mRNA was at least 2-fold greater than the increase in poly (A+) RNA. These results suggest that testosterone stimulated mAAT activity by induction of pmAAT mRNA. This continues to support our proposal that a major physiological effect of testosterone is increased pmAAT mRNA steady-state levels which result in increased pmAAT synthesis and increased mAAT activity. These changes ultimately result in increased citrate production by prostate epithelial cells.

Prolactin, in vitro, significantly increased citrate production, mAAT (mitochondrial aspartate aminotransferase) and pmAAT (precursor form of mAAT) activity of prostate epithelial cells derived from rat lateral prostate (LP) and pig prostate cultures. In contrast, prolactin had no effect on the cytosolic isozyme, cAAT. This prolactin effect appeared to be independent of testosterone. The phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate) induced the same effects as prolactin thereby indicating the involvement of protein kinase C. This report demonstrates that prolactin directly regulates citrate production of prostate epithelial cells and the availability of an in vitro model to elucidate the mechanism of action of prolactin.

The transport of aspartate was measured in isolated prostate cells. Two kinetically distinct uptake systems of high (Km = 10.0 microM) and low (Km = 0.8 mM) affinity were observed. Transport by both systems was Na+ dependent and saturable. The tissue distribution of aspartate was also determined by measuring the washout kinetics of endogenous and radiolabeled aspartate from prostate fragments. The distribution of aspartate resulted in 47% of aspartate in the intracellular compartment and 52% in the extracellular (presumably) lumenal compartment. The distribution of aspartate resulted in a calculated intracellular to plasma concentration gradient of approximately 43. We concluded that prostate cells maintain a considerable intracellular to plasma gradient for aspartate and that these cells contain a transport mechanism capable of uptake of aspartate against this considerable gradient.

Intraperitoneal doses of 2-methylnaphthalene (2-MN) have been shown to cause pulmonary toxicity in DBA/2J mice. Pretreatment with the monooxygenase inducers sodium phenobarbital and 3-methylcholanthrene (3-MC) failed to protect the DBA/2J mice from the toxic effect of 2-methylnaphthalene. Pretreatment of DBA/2J mice with the monooxygenase inhibitors, SKF 525-A and piperonyl butoxide also failed to enhance or attentuate the pulmonary lesions. Pulmonary and hepatic microsomes from DBA/2J mice metabolized 2-methylnaphthalene to three dihydrodiols, 2-naphthyl alcohol and other unidentified metabolites. Kidney microsomes produced 2-naphthyl alcohol but no detectable dihydrodiols. In comparison to control animals, hepatic microsomes from animals pretreated with sodium phenobarbital produced more of the least polar dihydrodiol, while amounts of the other two dihydrodiols were unaffected. 3-Methylcholanthrene, piperonyl butoxide and diethylmaleate failed to affect dihydrodiol formation in both pulmonary and hepatic microsomes. After the administration of a lung toxic dose (400 mg/kg, i.p.) of 2-MN, irreversible binding was highest in the liver, followed by the kidney, the lung and lastly skeletal muscle. Of the pretreatments given to the mice, only phenobarbital demonstrated a significant effect, and this elevation was apparent only in the liver. A pulmonary toxic dose of 2-MN (400 mg/kg, i.p.) administered to DBA/2J mice significantly depleted reduced GSH in the liver and lung and to a lesser extent, in the kidney. There appeared no good correlation between the pulmonary toxicity of 2-MN-dihydrodiol and/or alcohol formation or the in vivo irreversible binding to macromolecules. These results are compared with those reported previously in C57BL/6J mice.

We report here the first development of a continuous cell line in tissue culture of an animal pancreatic duct adenocarcinoma that is histologically similar to human pancreatic duct adenocarcinoma. A primary pancreatic duct adenocarcinoma, induced in a male Syrian golden hamster after 23 weeks of weekly subcutaneous injection of N-nitrobis (2-hydroxypropyl)amine, was minced and injected subcutaneously into three hamsters. After 8 weeks, a single tumor was apparent. Subsequent passages of fragments into the cheek pouches were performed at 3- to 4-week intervals. After five passages, minced fragments of a tumor were placed in tissue culture. Colonies appeared by 7 days; an epitheloid cell line, without fibroblasts, was established by 60 days. Single-cell suspensions, injected into hamster cheek pouches or subcutaneously, produced tumors in a dose-dependent fashion. Spent culture medium of tissue culture cells and saline extracts of freshly excised tumors contained pancreatic oncofetal antigen-like activity.

Mini-pig prostate epithelial cells exhibited the unique metabolic characteristics associated with the specialized function of production and secretion of high levels of citric acid. Epithelial cell suspensions from mini-pig prostate were successfully grown in primary and secondary cultures. The cultured epithelial cells exhibited rapid proliferation reaching confluency in approximately 6 days. Growth and proliferation of fibroblasts were markedly restricted by the dominance of epithelial cell growth. Confluent cultures could be maintained for approximately 6 weeks. The epithelial cells retained their polymorphic appearance in primary and secondary cultures and exhibited the characteristic formalin-resistant acid phosphatase reaction. Testosterone stimulated mitochondrial aspartate aminotransferase (mAAT) activity and citrate production by confluent epithelial cell cultures. These initial results indicate that cultured epithelial cells derived from mini-pig prostate might be an excellent model related to human for studies of prostate biology and hormonal regulation.

The induction of hepatic cytochrome P-450-linked monooxygenases has been studied after the twice daily, oral administration of two imidazo[4,5-c]pyridine-containing compounds and one imidazo[4,5-b]pyridine-containing drug. The compounds were administered by the oral route, at different doses, for 6 days after which time hepatic microsomes were prepared. In vitro biochemical assays revealed that all three compounds increased the O-deethylation of 7-ethoxyresorufin in a dose-dependent manner while not significantly affecting either the O-dealkylation of 7-ethoxycoumarin or the levels of NADPH-cytochrome c reductase. Ethylmorphine N-demethylation was decreased after dosing with the imidazo[4,5-b]pyridine-containing drug. Levels of cytochrome(s) P-450 and liver-to-body weight ratios were not significantly altered. The imidazo[4,5-b]pyridine-containing compound was more potent in terms of the induction of 7-ethoxyresorufin than either of the imidazo[4,5-c]pyridine-containing compounds but was approximately fourfold less active in this regard than 3-methylcholanthrene. No induction of cytochrome-P-450-linked monooxygenase activities was evident at a twice daily dose of 5 mg/kg for 6 days for all three compounds tested, constituting a no-effect level. The imidazo[4,5-c]pyridine-containing compounds exhibited modified Type II difference spectra when added to a suspension of rat hepatic microsomes. The imidazo[4,5-b]pyridine-containing compound has previously been reported to be a rapid and potent inducer of monooxygenase activity and have a Type II difference spectrum.

Aconitase activity and citrate oxidation were determined in mitochondrial preparations from rat ventral prostate. Aconitase activity in prostate mitochondria was approximately 10% of the activity observed in kidney preparations. The prostate aconitase activity was completely inhibited by the addition of 1.0 mmol/l zinc and kidney aconitase was inhibited by approximately 50%. The presence of 1.0 mmol/l zinc also markedly inhibited the formation of isocitrate from cis-aconitate. The reduction of NADP by citrate was markedly inhibited (approx. 95%) by 1.0 mmol/l zinc as compared with a 70 and 40% inhibition of aconitate--and isocitrate--stimulated reduction, respectively. These results indicate that zinc might be a strong inhibitor of aconitase activity with an inhibitory effect on isocitrate dehydrogenase also. The effect of 1.0 mmol/l zinc on citrate oxidation was studied by determining 14CO2 production from 6-14C-citrate. 14CO2 was significantly inhibited by zinc. These results further corroborate the conclusion that citrate accumulation in prostate might be the result of limited citrate oxidation which results from a combination of low aconitase activity and possibly zinc inhibition.

Recent studies of a number of volatile aromatic hydrocarbons have suggested that the formation of covalently bound metabolites arises solely through the intermediate formation of phenols. This study further examines the involvement of 1-naphthol in the in vivo and in vitro formation of covalently bound metabolites and pulmonary bronchiolar necrosis by naphthalene. Marked differences were observed in the rate of 1-naphthol formation in lung and liver microsomal incubations without correspondingly large differences between the rates of formation of covalently bound metabolites from naphthalene and 1-naphthol. Glutathione decreased covalent binding in hepatic microsomal incubations containing 14[C]1-naphthol but did not result in the formation of any of the glutathione adducts isolated from identical incubations containing 14[C]naphthalene. Tissue levels of covalently bound radioactivity in mice treated with 14[C]1-naphthol or 14[C]naphthalene were similar; however, in contrast to studies with naphthalene, 1-naphthol administration did not deplete tissue glutathione nor result in detectable tissue injury. These studies indicate that 1-naphthol is not an obligate intermediate in the formation of covalently bound metabolites from naphthalene nor does it appear to be a more proximate lung toxic metabolite.

The metabolism of 2-methylnaphthalene in rats (in vivo and in vitro) and rainbow trout (in vitro) has been investigated. Three isomeric dihydrodiols were formed by microsomal preparations and these were isolated and identified by high-pressure liquid chromatography and gas chromatography/mass spectroscopy. The temperature, microsomal protein content, and incubation time were varied to obtain the optimum conditions for their formation. The conversion of 2-methylnaphthalene to both monohydroxylated compounds and dihydrodiols was reduced by incubation with carbon monoxide, the omission of NADPH, or use of heat-denatured microsomes, implying the involvement of cytochrome(s) P-450-linked mixed-function oxidase activity. The three dihydrodiols obtained in vitro in microsomes from both rat and fish were identical with those compounds isolated from rat urine. Pretreatment with phenobarbital and beta-naphthoflavone selectively altered the rate of formation of specific dihydrodiols by rat liver microsomes. Although phenobarbital had no significant effect on the rate of dihydrodiol formation in trout liver microsomes, beta-naphthoflavone was a strong inducer and its selectivity in increasing the rate of dihydrodiol formation was similar to that seen with rat liver microsomes.

Male and female Sprague-Dawley rats were treated once with either crude or purified hexachlorobenzene (HCB) or crude or purified hexabromobenzene (HBB) at 150 mg/kg, intraperitoneally. Examination of hepatic microsomes 4 d later revealed an increase in cytochrome P-450 levels in both HCB- and HBB-pretreated animals. HBB produced a slight but statistically significant hypsochromic shift. Both HCB and HBB produced an increase in benzphetamine N-dealkylation: HCB produced a greater effect than HBB, and male rat microsomes produced more HCHO than female rat microsomes from the N-demethylation of benzphetamine. HCB and HBB both enhanced ethoxycoumarin and ethoxyresorufin O-dealkylation. Liver-to-body weight ratios were not significantly affected by pretreatment with either halogenated compound. There appeared to be no difference between the crude and purified halogenated compounds. Electrophoresis of microsomes from male rats pretreated with purified HBB indicated the presence of a band at 53,000 daltons, which was also seen in microsomes from rats pretreated with 3-methylcholanthrene. This band was absent in microsomes from rats pretreated with phenobarbital. Evidence from other laboratories has demonstrated the mixed type of P-450(s) induction after HCB administration, as does this report using enzymic and electrophoretic data.

Mitochondrial aspartate transamination was investigated as a major source of oxalacetate for citrate synthesis in rat ventral prostate. Citrate accumulation was measured in isolated mitochondria incubated with acetyl coenzyme A and various combinations of amino acids. Aspartate plus alpha ketoglutarate in the presence of acetyl coenzyme A resulted in significant citrate accumulation. Neither aspartate nor alpha ketoglutarate alone resulted in any significant citrate accumulation. Aspartate and alpha ketoglutarate use was comparable to glutamate and citrate production. The results indicated the presence of a mitochondrial aspartate aminotransferase. Castration (3 days) caused a significant decrease in citrate production from aspartate plus alpha ketoglutarate as well as a decrease in mitochondrial AAT activity in prostate although no effect on kidney activity occurred. A single injection of 1 mg. testosterone propionate to castrate rats significantly increased prostate mitochondrial AAT activity within 24 hours while MDH activity was unaltered. A double reciprocal plot indicated that testosterone might regulate the level of mitochondrial AAT in prostate. Ventral prostate also contain a uniquely high level of endogenous aspartate. These studies indicate that aspartate might be the major 4-carbon source of oxalacetate for citrate synthesis. Also testosterone possibly regulated prostate citrate production by its effect on the level of mitochondrial AAT activity.

Acute, subchronic, and chronic toxicity studies were conducted in dogs with the new vasodilator/cardiotonic drug isomazole (IMZ) to support, in part, clinical investigations of this agent in humans. Single oral doses of IMZ of 25, 50, or 100 mg/kg given to English pointer dogs (2/dose) caused a marked drop in systemic blood pressure and reflex-induced increases in heart rate to values well over 200 beats per minute. These responses were maintained for 12 to 22 hr depending on the dose given. One of the dogs receiving 100 mg/kg died at 4.5 hr postdose. Results of subchronic (3 months) and chronic (1 year) studies in beagle dogs (4/sex/dose group), in which measurable plasma levels of the drug and its metabolites were found, indicated that IMZ did not produce any discernible adverse findings when given in doses up to 16 mg/kg, other than expected cardiotoxic effects. The plasma t1/2 of IMZ at 16 mg/kg increased to between 4 and 8 hr from 2 hr noted at lower doses. In the 1-year study, at all doses and in both sexes, plasma levels of IMZ declined over the first month, stabilizing (at the 2 and 6 mg/kg doses) thereafter for the duration of the study. At the high dose of 16 mg/kg, after 1 year plasma levels of IMZ exceeded (females) or equaled (males) the 1-month values. At peak plasma levels of IMZ (2 hr postdose), plasma levels of parent drug increased linearly with the dose. The cardiotoxic effects consisted of substantial postdose increases in heart rate throughout the course of treatment (5 mg/kg and above), significant increases in heart weight (6 mg/kg and above), and multifocal myocardial fibrosis (6 mg/kg and above). There was a decline in basal heart rate at doses of 12.5 mg/kg and higher. The results of these studies demonstrated that repeated IMZ administration, as expected, was cardiotoxic to the dog, a species relatively sensitive to the pharmacological activity and hemodynamic changes induced by vasodilator/cardiotonic drugs. The no-effect dose level for cardiotoxicity in the repeated dose studies was considered to be 2 mg/kg, the lowest dose tested.

Citrate oxidation by rat ventral prostate was reduced by castration and increased by testosterone administration. Similarly, the mitochondrial aconitase activity was decreased by castration; whereas cytosol aconitase was unaffected. The rate of citrate oxidation is extremely low in prostate. Castration also decreased mitochondrial aspartate aminotransferase activity while having no effect on the cytosol isoenzyme. Testosterone markedly stimulated the net production of citrate from aspartate plus glutamate by prostate mitochondria. These studies support the proposal that aspartate is a major source of oxalacetate for citrate production, and that a "glutamate-aspartate-citrate" pathway may be functional in prostate mitochondria. In addition, testosterone can regulate citrate production by a specific effect on mitochondrial aspartate aminotransferase activity. Testosterone also regulates the flux of citrate through the Krebs cycle, but this represents only a small proportion of the citrate accumulated. These conditions would be consistent with the function of prostate epithelium in accumulating and secreting citrate.

When [14C] (+/-)-amphetamine sulphate (0.3 mg/kg) was injected into Tamarin monkeys, some 70-80% of the 14C was excreted in the urine in 24 hours. Some 75% of the 24-hour excretion was unchanged drug, 7% was free and conjugated benzoic acid and 7% was hydroxylated in the aromatic ring to give mainly 4-hydroxyamphetamine (Paredrine). With similar doses of [14C] norephedrine hydrochloride nearly 90% of the 14C was excreted in 24 hours, most of this being unchanged drug. Only 3-4% was metabolized at the side chain and there was no aromatic hydroxylation. The results were compared with those obtained earlier in man, rat, rabbit and guinea pig. Quantitatively the metabolites of amphetamine, norephedrine and preludin in the tamarin were more like those in man than in the other three species.

1. The metabolism and disposition of 14C-indolidan, a potent, orally-active positive inotrope with vasodilator properties, has been studied after single dose oral administration to rats, mice, dogs and monkeys. 2. Excretion of 14C in all 4 species was mostly via the urine, largely as parent drug together with two other major metabolites. 3. The two metabolites have been isolated and identified, by mass spectroscopy and 1H-n.m.r., as a dehydro-compound, with a double bond in the pyridazanone ring, and a hydroxylated derivative of the parent drug. 4. Plasma t 1/2 values, based on 14C, were 14 h in dog, 5 h in mouse and 8 h in monkey. Plasma t 1/2 of parent drug, by h.p.l.c. was 10 h in dog, approx. 5 h in rodents, and 8 h in monkeys. 5. Tissue distribution in rats showed no accumulation in any tissue; 14C concn. in all tissues were indistinguishable from background 48 h after dosage. 14C peaked at 6-8 h for most tissues but in blood and plasma, 14C was maximal 1 h after dosing.

An inhibitor of adrenal steroid biosynthesis, aminoglutethimide, was administered to seven patients with low renin essential hypertension, and the antihypertensive action of the drug was compared with its effects on adrenal steroid production. In all patients aldosterone concentrations in plasma and urine were within normal limits before the study. Mean arterial pressure was reduced from a pretreatment value of 117+/-2 (mean+/-SE) mm Hg to 108+/-3 mm Hg after 4 days of aminoglutethimide therapy and further to 99+/-3 mm Hg when drug administration was stopped (usually 21 days). Body weight was also reduced from 81.6+/-7.2 kg in the control period to 80.6+/-7.0 kg after 4 days of drug treatment and to 80.1+/-6.7 kg at the termination of therapy. Plasma renin activity was not significantly increased after 4 days of treatment but had risen to the normal range by the termination of aminoglutethimide therapy. Mean plasma concentrations of deoxycorticosterone and cortisol were unchanged during aminoglutethimide treatment whereas those of 18-hydroxydeoxycorticosterone, progesterone, 17alpha-hydroxyprogesterone, and 11-deoxycortisol were increased as compared to pretreatment values. In contrast, aminoglutethimide treatment reduced mean plasma aldosterone concentrations to about 30% of control values. Excretion rates of 16beta-hydroxydehydroepiandrosterone, 16-oxo-androstenediol, 17-hydroxycorticosteroids and 17-ketosteroids, and the secretion rate of 16beta-hydroxydehydroepiandrosterone were not significantly altered by aminoglutethimide treatment whereas the excretion rate of aldosterone was reduced from 3.62+/-0.5 (mean+/-SE) in the control period to 0.9+/-0.2 mug/24 h after 4 days and to 1.1+/-0.3 mug/24 h at the termination of aminoglutethimide treatment. The gradual lowering of blood pressure and body weight during aminoglutethimide therapy is consistent with the view that the antihypertensive effect of the drug is mediated through a reduction in the patients' extracellular fluid volume, probably secondary to the persistent decrease in aldosterone production. The observation that chronic administration of aminoglutethimide lowered blood pressure in these patients and elevated their plasma renin activity to the normal range without decreasing production of the adrenal steroids, deoxycorticosterone, 18-hydroxydeoxycorticosterone, and 16beta-hydroxydehydroepiandrosterone, makes it unlikely that these steroids are responsible either for the decreased renin or the elevated blood pressure in patients with low renin essential hypertension.

Prostate rudiments from Wistar rats were cultured in MEM supplemented with 15% FBS and insulin (2 microgram/ml). In situ prostate acinar epithelium consists of tall columnar and low basal cells. Ultrastructure of the columnar cells is that of typical secretory cells; however, the basal cells are poor in cell organelle. During the first week of culture many secretory cells degenerated and were replaced by cuboidal or low columnar cells. In the presence of testosterone (2 microgram/ml) the secretory cells remained viable for 8-10 days before undergoing necrosis. At the termination of the experiment the acinar epithelium consisted of low cuboidal cells. The cultures showed a pattern of citrate production which reached a maximum at 5 days and remained-relatively constant thereafter. Ultrastructure of 1-week cultures exhibited Golgi complexes with dilated cisternae. Although a paucity of cell organelle was found in 7-day cultures, older cultures (19 days) exhibited morphologic characteristics of normal secretory cells.

Prostatic aconitase was investigated utilizing fragments of rat ventral prostate. The rate of citrate formation from isocitrate was used as an index of aconitase activity. Inhibition of citrate formation from isocitrate by fluorocitrate indicated that aconitase activity was being measured. The results indicated that whereas both kidney and prostate formed citrate from isocitrate, the aconitase activity of prostate was independent of both medium isocitrate and citrate concentration. In addition the aconitase activity of prostate seemed to favor citrate formation to a much greater degree than kidney. The results showed that the aconitase catalyzed conversion of citrate to isocitrate is limited in prostate and suggested that the deficient citrate oxidation observed in prostate is the result of a unique aconitase activity.

The number of studies in aquatic toxicology has rapidly increased due to the importance of fish as a food source and also because of the increasing awareness of long-term health hazards of aquatic pollution of humans as well as to aquatic flora and fauna. The bioaccumulation and persistence of certain chemicals in fish have been studied for some time but with the characterization of an active hepatic mixed function oxidase system, comparable in many respects to that found in mammals, the role of metabolism in the disposition, toxicity, and persistence of certain chemicals can be studied. The recent observation that rainbow trout and their eggs are very sensitive to aflatoxin B1, a carcinogen that apparently must be activated to a chemically reactive intermediate to exert its effect, has led to the development of fish being considered appropriate animal models for toxicity testing. Since most predictive toxicology in the past has been restricted to nonaquatic species, this review has attempted to describe some of the similarities and dissimilarities between mammals and fish in terms of absorption, distribution, metabolism, and excretion of xenobiotics.

Glutamate dehydrogenase activity was determined in mitochondrial preparations from rat ventral prostate and rat kidney. Kinetic parameters of the ventral prostate enzyme were comparable to those for the kidney enzyme. Glutamate dehydrogenase activity in the direction of glutamate oxidative deamination was inhibited by alpha-ketoglutarate. However, the characteristics of alpha-ketoglutarate inhibition indicated that glutamate oxidation via glutamate dehydrogenase can occur at in vivo prostatic alpha-ketoglutarate levels. These results suggest that glutamate dehydrogenase activity in prostate may provide a continuous source of alpha-ketoglutarate for aspartate transamination to oxalacetate and ultimate citrate synthesis. In addition prostate mitochondria are able to couple the glutamic dehydrogenase reaction to aspartate aminotransferase. Under these conditions aspartate in the presence of glutamate and acetyl coenzyme A will result in a net synthesis of citrate. Consequently we propose an aspartate-glutamate pathway for citrate synthesis in prostate.