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We have demonstrated that zinc exposure induces apoptosis in human prostate cancer cells (PC-3) and benign hyperplasia cells (BPH), but not in normal prostate cells (HPR-1). However, the mechanisms underlying the effects of zinc on prostate cancer cell growth and zinc homeostasis remain unclear. To explore the zinc effect on gene expression profiles in normal (HPR-1) and malignant prostate cells (PC-3), we conducted a time course study of Zn treatment with microarray analysis. Microarray data were evaluated and profiled using computational approach for the primary and secondary data analyses. Final analyses were focused on the genes (1) highly sensitive to zinc; (2) associated with zinc homeostasis, i.e., metallothioneins (MTs), solute zinc carriers (ZIPs) and zinc exporters (ZnTs); (3) relevant to several oncogenic pathways. Zinc-mediated mRNA levels of MT isotypes were further validated by semi-quantitative RT-PCR. Results showed that zinc effect on genome-wide expression patterns was cell-type specific, and zinc appeared to have mainly down-regulatory effects on thousands of genes (1953 in HPR-1; 3534 in PC-3) with a threshold of +/-2.5-fold, while fewer genes were up-regulated (872 in HPR-1; 571 in PC-3). The patterns of zinc effect on functional MT genes' expression provided evidence for the cell type-dependent zinc accumulation and zinc-induced apoptosis in prostate cells. In PC-3 cells, zinc significantly up-regulated the expression of MT-1 isotypes MT-1J and MT-1M, denoted previously as "nonfunctional" MT genes, and now a depictive molecular structure of MT-1J was proposed. Examination of genes involved in oncogenic pathways indicated that certain genes, e.g., Fos, Akt1, Jak3 and PI3K, were highly regulated by zinc with cell-type specificity. This work provided an extensive database on zinc-related prostate cancer research. The strategy of data analysis was devoted to finding genes highly sensitive to Zn, and the genes associated with zinc accumulation and zinc-induced apoptosis. The results indicate that zinc regulation of gene expression is cell-type specific, and MT genes play important roles in prostate malignancy.

Zinc is an essential trace element that is involved in diverse metabolic and signaling pathways. Zinc deficiency is associated with retardation of bone growth. Previous in vitro studies have suggested a direct effect of zinc on both the proliferation and differentiation of osteoblast-like cells. However, the mechanisms for uptake of zinc into osteoblasts have not been examined in detail. Several families of zinc transporters have previously been characterized in mammalian cells; such transporters function in the uptake, intracellular sequestration or efflux of zinc. In the current study, we examined zinc transport in osteoprogenitor cells and have attempted to define a functional role for a zinc transport mechanism in osteogenic differentiation. We identified at least two zinc transporters in both human mesenchymal stem cells (MSCs) and in osteoblastic cells--the ubiquitous zinc transporter, ZIP1, and LIV-1, which was previously characterized as a protein that is expressed in breast cancer cells. The subcellular localization of both these zinc transporters suggested distribution in both the plasma membrane and also diffusely in the cytoplasm. During the differentiation process of pluripotent MSCs into osteoblast-like cells, both zinc uptake and expression of the ZIP1 protein were increased. An adenoviral-mediated overexpression of ZIP1 in MSCs resulted in Alizarin-red-positive mineralization and also increased expression of specific osteoblast-associated markers, such as alkaline phosphatase, and of several osteoblast differentiation genes, including osteopontin, Cbfa1/Runx2, promyelocytic leukemia zinc finger and bone sialoprotein. An siRNA-mediated reduction of ZIP1 protein expression in MSCs caused decreased zinc uptake and inhibition of osteoblastic differentiation under osteogenic culture conditions. Finally, following overexpression of ZIP1 in MSCs, cDNA microarray analysis revealed differential regulation of several genes associated with the proliferation of osteoprogenitor cells and osteoblast differentiation. In conclusion, these studies provide important insights into the role of a plasma membrane zinc transporter in the initiation of an osteogenic lineage from MSCs.

Human prostate secretory epithelial cells have the uniquely specialized function of accumulating and secreting extremely high levels of citrate. This is achieved by their ability to accumulate high cellular levels of zinc that inhibit citrate oxidation. This process of net citrate production requires unique metabolic/bioenergetic mitochondrial relationships. In prostate cancer, the malignant cells undergo a metabolic transformation from zinc-accumulating citrate-producing sane cells to citrate-oxidizing malignant cells that lost the ability to accumulate zinc. This review describes the metabolic/bioenergetic, zinc and mitochondrial relationships involved in normal and malignant prostate. Hopefully, this report will generate much needed interest and research in this neglected, but critically important, area of investigation.

The compound, 5-{4-[3-(4-cyclohexyl-2-propylphenoxy)propoxy]phenyl}-1,3-oxazolidine-2,4-dione (compound A) is a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist. PPARgamma agonists have proven useful in the treatment of type 2 diabetes, which is characterized by hyperglycemia, insulin resistance and/or abnormal insulin secretion. The metabolism of this oxazolidinedione (OZD) was investigated in male rat, dog, monkey and human liver microsomes, and recombinant human cytochrome P450 enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in the presence of NADPH. Routes of metabolism included monohydroxylation of the cyclohexane ring at multiple positions, monohydroxylation of the n-propyl side chain or the tether linkage, and OZD ring opening, giving rise to the keto amide and alcohol amide entities. Liver microsomes showed subtle qualitative and quantitative metabolic differences among rat, dog, monkey and human preparations. Further, CYP2C8 and CYP2C19 did not display different regioselectivity for hydroxylation on the cyclohexane ring with both of them giving rise to C-3 and C-4 hydroxy metabolites, but they did display different stereoselectivity with CYP2C8 preferring cyclohexane hydroxylation in equatorial positions and CYP2C19 in axial positions.

Although the total zinc content of cells generally approximates 0.2 mM, the cytosolic free zinc ion concentration is negligible (subnanomolar concentrations). However, all reported studies of effects of zinc on cellular respiration and terminal oxidation involved microM-mM levels of free zinc ions. Prostate cells and their mitochondria accumulate 3-10 fold more zinc than other mammalian cells. We considered that a cytosolic pool of mobile reactive low molecular weight zinc ligands could inhibit respiration and terminal oxidation. The effects of ZnLigands, especially ZnCitrate, versus free Zn++ ions on respiration and terminal oxidation were studied with prostate and liver mitochondria. ZnLigands were equally as effective as free Zn++ ions in the inhibition of respiration and terminal oxidation of both prostate and liver mitochondria, which supports our concept that zinc can be transferred from cytosolic donor ZnLigands directly to zinc-binding sites of terminal oxidation components. Also, the respiration and specific activities of terminal oxidation components of prostate mitochondria are 20-50% of liver mitochondria. Zinc inhibition and inherently low levels of electron transport components are likely major factors responsible for the low respiration that characterizes prostate cells.

Objectives: The function of the prostate gland in many animals, including humans, is to accumulate and secrete large quantities of citrate. This function derives from the metabolic characteristics of the prostate secretory epithelial cells. These cells possess a uniquely limiting mitochondrial aconitase (m-aconitase) that minimizes citrate oxidation and thus permits citrate to accumulate. Unfortunately, the characteristics of prostate m-aconitase and its manner of regulation have not been established. The hormones testosterone and prolactin, however, are significantly involved in regulating prostate citrate production. Thus it is reasonable to hypothesize that these hormones may be involved in the regulation of both m-aconitase and citrate oxidation., Methods: Using freshly prepared pig prostate epithelial cells, we attempted to determine the effects of testosterone and prolactin treatment on the level of m-aconitase enzyme, on the level of m-aconitase activity, and on citrate utilization. The epithelial cells were incubated for 3 hours with either testosterone (10(-9) M), prolactin (1 microgram/mL), or vehicle (control)., Results: Both hormone applications caused a marked increase in the level of m-aconitase. In contrast, neither hormone had any effect on the m-aconitase level of pig seminal vesicle cells, which are also citrate-producing cells. Moreover, neither hormone had any effect on pyruvate dehydrogenase E1a. These findings suggest that testosterone and prolactin regulation of prostate m-aconitase is a highly specific effect. Along with the increase in the level of m-aconitase enzyme, both hormones also increased m-aconitase activity and prostate-cell utilization of citrate., Conclusions: These studies demonstrate that testosterone and prolactin can regulate m-aconitase and sub-sequent citrate oxidation of specific prostate epithelial cells. This unique aconitase relationship is not observed in other mammalian cells.

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.

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.