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A major issue relating to many cancers is the absence of effective chemotherapeutic agents; so that most often untreatable morbidity and death are prevalent once the cancer has been detected and has advanced. The search for efficacious anticancer agents is imperative. One potential agent is zinc, which is decreased in the development of some cancers in order to avoid its cytotoxic/tumor suppressor effects on the malignant cells. This provides the basis and opportunity to employ a treatment regimen that restores elevated zinc levels in the malignant cells and elicits the cytotoxic/tumor suppressor effects of zinc. The enigma is that this approach and expectation has not reached fruition. The question is "why?". This article provides a discussion of relevant zinc issues that need to be considered and resolved. Important areas of research are identified as being essential for the successful application of zinc cytotoxicity/tumor suppression actions for the treatment of specific cancers.

It has been known for about seventy years that bone, in all vertebrates, contains uniquely high citrate levels. However, the role of citrate, its source, its regulation, and its implication in normal bone formation and in bone disorders have remained largely unknown. For the past thirty-five years, the relationship of citrate in bone has been a neglected area of attention and research. It has recently been discovered that citrate is critical for the structure of the apatite nanocrystal, and is required to impart the important properties of bone such as its stability, strength, and resistance to fracture. This brings to focus the need for a renewed interest and research into the relationships of citrate in bone formation. A most fundamental question that must be resolved is "What is the source of citrate in bone?". This presentation provides a historical review of the early research to the present status of citrate implications in bone. This leads to a new concept of the role of osteoblasts as specialized citrate producing cells that provide the source of citrate in bone formation; i.e. the "osteoblast citration" process. This also brings into focus a new insight into the role of zinc in bone in relation to osteoblast citrate production. The genetic/hormonal/metabolic relationships of "net citrate production" are described. The intent of this presentation is to provide the background for a new perspective of the important implications of osteoblasts and citrate in bone formation; which, hopefully, will stimulate a renewed interest and essential research.

Prostate cancer is the second leading cause of cancer deaths among men. The availability of animal models that represent the events and factors that exist in the natural history and biology of human prostate cancer is essential in dealing with prostate cancer. In recent decades and presently, emphasis has been directed at the development and employment of prostate cancer induced in transgenic mice. However, the important consistent hallmark characteristic and event of decrease in zinc and citrate and downregulation of ZIP1 zinc transporter in prostate malignancy has not been studied or identified in any animal model. We investigated the status of these parameters in TRAMP tumors as compared with human prostate cancer. The results show that citrate levels are markedly decreased in the developing and advancing stages of malignancy in TRAMP. Zinc levels are also decreased and ZIP1 transporter is lost in TRAMP tumors. In vitro studies show that zinc treatment of TRAMP C2 cells exhibits cytotoxic effects. Collectively, these results mimic the ZIP1, zinc, and citrate status and relationship that exist in human prostate cancer. This is the first report that establishes the existence of the human prostate zinc/citrate hallmark characteristic and relationship in an animal model. It now appears that the TRAMP model will be suitable for studies relating to the implications and role of zinc- and citrate-related metabolism in the development and progression of human prostate cancer.

Quantifying bacterial abundance and biomass is fundamental to many microbiological studies. Directly counting via epifluorescence microscopy has become the method of choice, especially for environmental samples, and conventional techniques require filtration of cells onto black polycarbonate membrane filters. We investigated the utility of instead capturing stained bacterial suspensions on bioadhesive slides, performing tests using pure cultures of bacteria, mixtures of cultured bacteria, and environmental samples from five habitat types. When compared to the standard filtration and flow cytometric approaches, bioadhesive slides were found to be an accurate and precise platform for rapid enumeration of bacteria. Total bacterial counts made using the three methods were positively correlated for acridine orange and Live/Dead® (L/D) staining (0.81≤r≤0.95, all p≤0.002). All platforms had similar precision, though counts obtained using bioadhesive slides were significantly higher than those made with polycarbonate filters and flow cytometry. The specific bioadhesive slides we used resulted in substantial cell mortality for certain pure cultures and river water samples, limiting their use for L/D determination. Cell enumeration using bioadhesive slides is particularly effective because it is highly precise at a wide range of cell concentrations, allows observation of cells that are not readily discernible on filters, reduces the number of steps and processing materials associated with sample analysis, and increases throughput., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: A marked decrease in the level of zinc is a consistent characteristic of prostate cancer; which results from down-regulation of ZIP1 zinc transporter. The aim of this study was to determine if RREB-1 transcription is involved in the down-regulation of ZIP1 gene expression; and to determine the expression of RREB-1 in benign and cancerous prostate in situ., Methods: Overexpression and siRNA knock down of RREB-1 were used to determine the effect of RREB-1 on hZIP1 abundance in PC-3 cells. Immunohistochemistry with tissue microarrays (TMAs) and tissue sections was used to determine the levels of RREB-1 expression in prostate in situ., Results: Overexpression of RREB-1 resulted in a decrease in the abundance of hZIP1 in the plasma membrane of PC-3 cells; whereas siRNA knock down significantly increased hZIP1 expression. Prostate TMAs and tissue sections showed an inverse relationship between RREB-1 and hZIP1 staining., Conclusions: RREB-1 overexpression results in down-regulation of hZIP1 and contributes to the loss of hZIP1 expression and zinc in prostate cancer. This is an early event in prostate carcinogenesis., (Copyright © 2011 Wiley-Liss, Inc.)

Pancreatic adenocarcinoma is an untreatable deadly cancer. The factors involved in its early development remain unknown; which contributes to the absence of biomarkers for early detection of malignancy or at-risk subjects, and the absence of efficacious therapeutic agents. Because zinc changes are implicated in some cancers, we determined if it might be involved in the development of pancreatic adenocarcinoma. With in situ Dithizone and Zinquin staining of normal pancreas and adenocarcinoma tissue sections, we show for the first time, a consistent major loss of zinc in ductal and acinar epithelium in adenocarcinoma compared to the normal epithelium. This decrease in zinc is evident in well-differentiated through poorly-differentiated stages of malignancy. Immunohistochemistry identified ZIP3 as the basilar membrane zinc uptake transporter in normal ductal/acinar epithelium; and that the transporter is absent in adenocarcinoma. In situ Rt-PCR revealed that ZIP3 gene expression is silenced in adenocarcinoma. The ZIP3 down regulation accompanied the loss of zinc in early and progressing malignancy. RREB1 transcription factor was down regulated along with ZIP3; and might be involved in the silencing of ZIP3 expression. Zinc treatment was cytotoxic to malignant Panc1 cells. The combination of concurrent zinc, ZIP3, and RREB-1 changes represent early events in the development of adenocarcinoma; and suggest that zinc might be a tumor suppressor of pancreatic cancer. This report provides the clinical foundation for further mechanistic studies that will provide important insight into pancreatic carcinogenesis, and can lead to the development of effective early biomarkers and effective therapeutic agents for pancreatic cancer.

In addition to its critical role in normal cell function, growth, and metabolism, zinc is implicated as a major factor in the development and progression of many pathological conditions and diseases. Despite this importance of zinc, many important factors, processes, and mechanisms of the physiology, biochemistry, and molecular biology of zinc remain unknown. Especially important is the unresolved issue regarding the mechanism and process of the trafficking, transport, and reactivity of zinc in cells; especially in mammalian cells. This presentation focuses on the concept that, due to the existence of a negligible pool of free Zn(2+) ions in the mammalian cell environment, the trafficking, transport and reactivity of zinc occurs via a direct exchange of zinc from donor Zn-ligands to acceptor ligands. This Zn exchange process occurs without the requirement for production of free Zn(2+) ions. The direct evidence from mammalian cell studies is presented in support of the operation of the direct Zn-ligand exchange mechanism. The paper also provides important information and conditions that should be considered and employed in the conduct of studies regarding the role and effects of zinc in biological/biomedical research; and in its clinical interpretation and application., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Introduction: The human nuclear receptors pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) are known to regulate gene expression of the cytochrome P450 (CYP) enzymes, 3A4, 2B6, and 1A2, respectively. In conventional CYP induction studies, the activity of each CYP enzyme is assessed in a separate incubation with the appropriate marker substrate. The objective of this study was to assess, simultaneously, the induction of CYP3A4, CYP2B6, and CYP1A2 activity in cultured human hepatocytes treated with various prototypical ligands of PXR, CAR, and AhR by utilizing an optimized substrate cocktail, as well as a rapid, sensitive liquid chromatography-mass spectrometry method., Methods: To evaluate the xenobiotic-mediated induction of hepatocellular gene expression, the prototypical inducers rifampicin (10 μM) and phenobarbital (1 mM) were used for CYP3A4, CITCO (1 μM) and artemisinin (50 μM) were used for CYP2B6, and 3-methylcholanthrene (1 μM) and omeprazole (50 μM) were utilized for induction of CYP1A2. Primary human hepatocytes were treated with each compound for 48h, followed by a 30-min incubation of the hepatocyte culture along with the addition of three marker substrates for specific CYP activity: midazolam (CYP3A4; 5 μM), bupropion (CYP2B6; 50 μM), and phenacetin (CYP1A2; 100μM). The assessment of CYP activity was performed with a rapid, sensitive liquid chromatography-tandem mass spectrometry method which simultaneously assessed activity of CYP3A4, CYP2B6, and CYP1A2 in a single 3-min method by examining the formation of the probe substrate metabolites, 1'-hydroxymidazolam, hydroxybupropion, and acetaminophen, respectively., Results: The average fold-induction of CYP3A4, CYP2B6, and CYP1A2 activity was comparable between the cocktail and the conventional assay., Discussion: The combination of three marker substrates in a single 30-min incubation, in addition to a rapid, sensitive LC-MS/MS method, resulted in an efficient and robust method for assessing cytochrome P450 induction as compared to the conventional methodology., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Lack of success in restoring the native Eastern oyster, Crassostrea virginica, to Chesapeake Bay has been linked to the low occurrence of oyster larval setting in tributaries to the Bay. Among the many potential factors that could affect efforts to produce oysters through aquaculture or supplementation of shell beds is substratum condition. The present study examined larval setting on field-produced biofilms from Little Wicomico River (Virginia, USA) to assess whether bacterial community structure (examined by terminal restriction fragment length polymorphism, T-RFLP) or other characteristics of contemporary biofilms in this tributary (biofilm age and mass, algal abundance, and percentage organic matter) inhibited setting of larval oysters. The structure of the natural and heterogenous bacterial community in the biofilms and the success of oyster set were correlated, suggesting that specific microbial species may play a role in oyster setting. Larval set increased with biofilm age and mass, suggesting that established field-produced biofilms have no inhibitory effect. In contrast, the percentage of organic matter was negatively correlated with oyster set, whereas chlorophyll a concentration had no observed effect. The study expands prior knowledge by providing a more realistic timeframe for biofilm development (weeks as opposed to days), recounting effects of biofilms that are more representative of the natural dynamic and disturbance processes that would be expected to occur on submerged structures, and by incorporating seasonal and spatial variation. An important negative effect observed during the study period was heavy predation by Stylochus ellipticus on newly set oysters. Overall, the results of this study, which is the first assessment of the effects of biofilms produced naturally within a Chesapeake Bay tributary, suggest that the absence of large numbers of oysters in Little Wicomico River is not related to microbes or other specific characteristics of biofilms that develop on suitable setting substrata, but rather to heavy predation of newly set larvae.

A validated method for the simultaneous characterization of xenobiotic compound-mediated inhibition of seven major cytochrome P450 (CYP) enzymes in pooled human liver microsomes through the use of specific CYP probe substrates (cocktail assay) with low protein content, and a rapid, three minute LC-MS/MS analytical method is described. The specific CYP substrates used in this cocktail assay included phenacetin (CYP1A2), bupropion (CYP2B6), amodiaquine (CYP2C8), tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A4/5). The LC-MS method incorporated the aforementioned seven CYP substrates along with their respective major metabolites, and one internal standard, labetalol. In a cross-validation analysis, the concentrations of each CYP probe substrate in the assay had minimal effect (i.e., inhibition or activation) on the other CYP activities. Furthermore, the assay conditions for the multiple probe substrate, ie., cocktail assay, were validated against the single probe substrate assay using 18 compounds with known CYP inhibition liabilities and 10 proprietary compounds. The inhibitory constant (Ki) determined with this cocktail assay was highly correlated (R(2) ≥ 0.77 for each individual probe substrate) with that of the single probe substrate assay for all 27 CYP inhibitors. This seven CYP inhibition cocktail assay has increased the efficiency to assess compounds for inhibition of the major CYP isoforms in a high throughput, drug discovery setting.

This minireview is prompted by the recent report of Banas et al. (J Biol Inorg Chem 15:1147-1155, 2010), which purports to show and concludes that zinc levels are increased in prostate cancer. Such a conclusion conflicts with the overwhelming corroborating clinical and experimental evidence that has amassed from numerous reports over the past approximately 60 years; these consistently show that prostate zinc levels are decreased in the development and progression of prostate cancer. We submit that this is an established relationship in prostate cancer that must be considered and described in any studies that purport to identify results that are inconsistent with this established relationship. In support of this relationship, we provide a minireview of the information that has led to the establishment of this relationship. As with most established clinical relationships, exceptions and anomalies often exist. However, these must be described and explained in the context of the established relationship, and not in the context of refutation of the established relationship, at least not until sufficient corroborating evidence overwhelms the existing evidence. This provides a background to address and to critique the report of Banas et al. Of broader and more serious implications are the widespread recalcitrance and/or lack of knowledge within the clinical and biomedical research community for recognition that zinc decrease in prostate cancer is an established relationship. This leads to misinformation and misinterpretations regarding clinical, experimental, and epidemiological issues that do not serve the best interests of the scientific, medical, and public communities.

Background: Normal prostate accumulates extremely high levels of zinc compared to other soft tissues. In contrast, the level of zinc in the prostate decreases significantly in prostate cancer. We have shown that down-regulation of the expression of the zinc transporter hZIP1 in prostate cancer is an important event that is responsible for the decrease of zinc levels. However, the mechanism of hZIP1 down-regulation is not known. We have hypothesized that hZIP1 is down-regulated through transcriptional regulation., Methods: The hZIP1 promoter was studied using luciferase reporter assays, site-directed mutagenesis, gel shift, and ChIP assay., Results: We have characterized a promoter region, downstream of the transcription start site, responsible for repression of hZIP1 transcription. We demonstrate that this region contains a binding site for the Ras-Responsive Element Binding protein 1 (RREB-1) and that the binding of RREB-1 to the hZIP1 promoter is involved in the decrease of hZIP1 transcription in PC-3 cells., Conclusion: The Ras pathway and activation of RREB-1 are involved in hZIP1 down-regulation and may play a role in the decrease of the transporter expression in prostate cancer., (Copyright 2009 Wiley-Liss, Inc.)

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 a trace element that is essential for the normal function of cells. It is a cofactor for the structure and function of a wide range of cellular proteins including enzymes, transcription factors, and structural proteins. Recent studies have shown that zinc plays a role in the development of various cancers. Unfortunately no established common relationships of zinc with cancer development and progression have been identified. Zinc is known to have systemic effects such as regulation of the immune system as well as direct cellular effects resulting in regulation of gene expression, bioenergetics, metabolic pathways, signal transduction and cell invasion. Zinc is also reported to regulate cell proliferation and growth. In this review presentation we focus on the effects of zinc that are involved in the regulation of apoptosis in malignant cells. We selected the apoptotic effects of zinc because zinc is reported to both induce apoptosis in some cancers and to protect other cancer cells against apoptosis induced by other factors. The effects of zinc in the regulation of apoptosis appear to be cell type specific. More importantly the reported effects of zinc on cancer cells must be viewed from the perspective of the physiological regulation of zinc homeostasis. Thus one must be mindful of the experimental conditions under which zinc effects are investigated relative to the physiological and pathological conditions of cellular zinc distribution and concentrations that can exist in situ.

Background: The development and progression of prostate cancer requires the transformation of normal zinc-accumulating epithelial cells to malignant cells that have lost the ability to accumulate zinc. This metabolic transformation is essential so that the tumor suppressive effects of zinc can be eliminated and the malignant process can proceed. One of the major effects of zinc is its prevention of prostate cell growth by its induction of apoptosis. The accumulation of cellular zinc has a direct effect on the mitochondria that results in the release of cytochrome c, which initiates the caspase cascade that leads to apoptosis. This effect is associated with the mitochondrial pore-forming process, but the mechanism by which zinc induces the release of cytochrome c and induces mitochondrial apoptogenesis has not been resolved. The present report provides for the first time information that implicates Bax in the zinc induction of mitochondrial apoptogenesis., Results: The effects of zinc treatment on the Bax levels of PC-3 cells and on the mitochondria were determined. The exposure of isolated mitochondria to zinc results in an increase in membrane bound Bax, which is due to the mitochondrial insertion of endogenous resident Bax. The mitochondrial Bax/Bcl-2 ratio is increased by zinc treatment. Zinc treatment of PC-3 cells also increases the mitochondrial level of Bax. In addition, zinc treatment increases the cellular level of Bax and the cellular Bax/Bcl2 ratio. Down regulation of Bax in PC-3 cells eliminates the zinc induction of apoptosis. The increase in cellular Bax level appears to involve zinc induction of Bax gene expression., Conclusion: This report extends and confirms that physiological levels of zinc induce apoptosis in prostate cells. The study provides evidence that zinc is directly involved in facilitating a Bax-associated pore formation process that initiates mitochondrial apoptogenesis. This is enhanced by an additional effect of zinc on increasing the cellular level of Bax. To avoid the anti-tumor apoptogenic effects of zinc, the malignant cells in prostate cancer posses genetic/metabolic adaptations that prevent the cellular accumulation of zinc.

Midazolam is a potent benzodiazepine derivative with sedative, hypnotic, anticonvulsant, muscle-relaxant, and anxiolytic activities. It undergoes oxidative metabolism catalyzed almost exclusively by the CYP3A subfamily to a major metabolite, 1'-hydroxymidazolam, which is equipotent to midazolam. 1'-Hydroxymidazolam is subject to glucuronidation followed by renal excretion. To date, the glucuronidation of 1'-hydroxymidazolam has not been evaluated in detail. In the current study, we identified an unreported quaternary N-glucuronide, as well as the known O-glucuronide, from incubations of 1'-hydroxymidazolam in human liver microsomes enriched with uridine 5'-diphosphoglucuronic acid (UDPGA). The structure of the N-glucuronide was confirmed by nuclear magnetic resonance analysis, which showed that glucuronidation had occurred at N-2 (the imidazole nitrogen that is not a part of the benzodiazepine ring). In a separate study, in which midazolam was used as the substrate, an analogous N-glucuronide also was detected from incubations with human liver microsomes in the presence of UDPGA. Investigation of the kinetics of 1'-hydroxymidazolam glucuronidation in human liver microsomes indicated autoactivation kinetics (Hill coefficient, n = 1.2-1.5). The apparent S(50) values for the formation of O- and N-glucuronides were 43 and 18 microM, respectively, and the corresponding apparent V(max) values were 363 and 21 pmol/mg of microsomal protein/min. Incubations with recombinant human uridine diphosphate glucuronosyltransferases (UGTs) indicated that the O-glucuronidation was catalyzed by UGT2B4 and UGT2B7, whereas the N-glucuronidation was catalyzed by UGT1A4. Consistent with these observations, hecogenin, a selective inhibitor of UGT1A4, selectively inhibited the N-glucuronidation, whereas diclofenac, a potent inhibitor of UGT2B7, had a greater inhibitory effect on the O-glucuronidation than on the N-glucuronidation. In summary, our study provides the first demonstration of N-glucuronidation of 1'-hydroxymidazolam in human liver microsomes.

Background: The disturbance of zinc homeostasis featured with a significant decrease of cellular zinc level was well documented to associate with the development and progression of human prostate malignancy. We have previously reported that zinc treatment induces prostate malignant cell apoptosis through mitochondrial pathway. Metallothionein (MT) is a major receptor/donor of zinc in the cells. However, the studies on the expression of MT in association with the prostate pathological and malignant status are very limited, and the zinc regulation of MT isoform expression in prostate cells remains elusive. The goals of this study were to define the expression of endogenous MTs, the isoforms of MT 1, 2, 3 at both messenger ribonucleic acid (mRNA) and protein levels; and to investigate the zinc effect on MT expression in normal prostate, benign prostatic hyperplasia (BPH) and malignant PC-3 cells, and in relevant human tissues. Cellular MT proteins were detected by immunohistochemistry, fluorescence staining and Western blot analysis; reverse transcription polymerase chain reaction (RT-PCR) was used to determine the MT isoform-specific mRNAs., Results: Our results demonstrated a significant suppression of endogenous levels of MT1/2 in malignant PC-3 cells (95% reduction compared to the normal prostate cells) and in human adenocarcinoma tissues (73% MT1/2 negative). A moderate reduction of MT1/2 expression was observed in BPH. Zinc treatment remarkably induced MT1/2 expression in PC-3 and BPH cells, which was accordant with the restored cellular zinc level. MT 3, as a growth inhibitory factor, was detected and up-regulated by zinc mainly in BPH cells., Conclusion: This study provided evidence of the association of attenuated MT1/2 with prostate tumor progression, and the zinc induction of MT1/2 expression resulting in cellular zinc restoration. The results suggest the potential of MT1/2 as a candidate biomarker for prostate cancer and the utilization of zinc in prostate cancer prevention and treatment.

Despite the prevalence of prostate cancer, the etiology and factors associated with its development and progression are largely unknown. An important relationship in prostate cancer is the role of zinc. Clinical evidence and experimental evidence have established that prostate cancer is associated with a decrease in the zinc uptake and accumulation in the malignant cells; and that the accumulation of zinc in the prostate cells prevents malignancy. In contrast to this established consistent clinical relationship, numerous epidemiology studies and reports of the effect of dietary and supplemental zinc on the incidence of prostate cancer have provided divergent, inconsistent, and inconclusive results; which range from adverse effects of zinc, protective effects of zinc, and no effect of zinc on the risk of prostate cancer. Despite these divergent and inconclusive results, a prevailing view and public warning has evolved from unsubstantiated and uncorroborated epidemiology studies that zinc consumption increases the risk of developing advanced stage prostate cancer. Such a conclusion is not well-founded and has serious, confusing and erroneous implications for the medical/scientific community and for the public-at-large. The admonition of Dimitrios Trichopoulos over a decade ago [1] that, " … (epidemiology) studies will inevitably generate false positive and false negative results with disturbing frequency. …, when (people) do take us seriously, we may unintentionally do more harm than good " can be applied to the situation that is the subject of this report. Therefore it is extremely important to review the epidemiology studies that have lead to the conclusion of an adverse effect of zinc, and also that have produced such inconsistent and divergent results. This critical review defines issues, problems, and shortcomings that exist in the conduct, conclusions, and dissemination of the epidemiology studies. We caution that one should be knowledgeable and understanding of these issues in assessing the validity and the conclusiveness of the outcomes from the epidemiology studies of purported associations of dietary and supplemental zinc on the risk of prostate cancer; particularly when the unsubstantiated conclusions are at odds with clinical and experimental evidence. It is in the interest of the medical, scientific and public communities that this critical review is undertaken. We hope that this review will generate an open, objective, scientific and medical discussion and assessment of this important issue.

Background: In normal prostate epithelial cells low m-aconitase activity decreases citrate oxidation leading to citrate accumulation. In prostate cancer cells m-aconitase activity is increased and citrate content is lower. The effect of inhibition of m-aconitase on ATP production by prostate cancer cells (PC3) is not known nor is the contribution of glycolysis versus respiration., Methods: ATP content of PC3 cells as affected by inhibition of m-aconitase (fluoroacetate (FA), zinc), inhibition of glycolysis (2DxG), or respiration (DNP, oligomycin) was determined. The ability to maintain ATP using glucose or glutamine as sole substrate was also determined. Intermediates including ATP, lactate, glucose, and glutamine were assayed in neutralized perchloric acid (PCA) cell extracts, virgin, and conditioned medium by enzymatic fluorometry., Results: Data show that inhibition of m-aconitase, glycolysis, or respiration alone did not decrease ATP content. Inhibition of both glycolysis and respiration were required to decrease ATP content. PC3 cells were able to produce ATP with either glucose or glutamine as sole substrate. Though FA clearly inhibited m-aconitase there was no evidence that zinc had a similar effect., Conclusion: PC3 cells can support ATP production when m-aconitase is inhibited by using glycolysis or oxidation of substrate (e.g., glutamine) entering the TCA cycle distal to citrate., ((c) 2007 Wiley-Liss, Inc.)

Human prostate glandular epithelial cells have the unique capability of accumulating high levels of zinc. This is essential to inhibit m-aconitase activity so that citrate can accumulate for secretion into prostatic fluid, which is a major function of the prostate gland. As a result, the Krebs cycle is truncated with the consequence of the lost ATP production that would result from citrate oxidation. The cellular accumulation of zinc also inhibits mitochondrial terminal oxidation and respiration. In addition to these metabolic effects, zinc accumulation exhibits anti-proliferative effects via its induction of mitochondrial apoptogenesis. Zinc accumulation also inhibits the invasive/migration activities in malignant prostate cells. The anti-proliferative effects and the effects on invasion and migration occur through zinc activation of specific intracellular signaling pathways. Consequently, these effects impose anti-tumor actions by zinc. The ability of prostate cells to accumulate zinc is due to the expression and activity of the zinc uptake transporter, ZIP1. To avoid the anti-tumor effects of zinc, in prostate cancer the malignant prostate cells exhibit a silencing of ZIP1 gene expression accompanied by a depletion of cellular zinc. Therefore we regard ZIP1 as a tumor suppressor gene in prostate cancer. In addition to prostate cells, similar tumor suppressor effects of zinc have been identified in several other types of tumors.

Background: The normal human prostate glandular epithelium has the unique function of accumulating high levels of zinc. In prostate cancer this capability is lost as an early event in the development of the malignant cells. The mechanism and factors responsible for the ability of the normal epithelial cells to accumulate zinc and the loss of this capability in the malignant cells need to be identified. We previously reported that Zip1 is an important zinc uptake transporter in prostate cells and is down regulated in the malignant cells in situ along with the depletion of zinc levels. In this report we investigated the expression of two other Zip family zinc transporters, Zip2 and Zip3 in malignant versus nonmalignant (normal and BPH) glands. Zip2 and Zip3 relative protein levels were determined by immunohistochemistry analysis of human prostate tissue sections., Results: Normal and BPH glandular epithelium consistently exhibited the strong presence of both Zip 2 and Zip3; whereas both transporters consistently were essentially non-detectable in the malignant glands. This represents the first report of the expression of Zip3 in human prostate tissue; and more importantly, reveals that ZiP2 and Zip3 are down regulated in malignant cells in situ as we also had demonstrated for Zip1. Zip2 and Zip3 transporter proteins were localized predominantly at the apical cell membrane, which is in contrast to the Zip1 localization at the basolateral membrane. Zip2 and Zip3 seemingly are associated with the re-uptake of zinc from prostatic fluid., Conclusion: These results coupled with previous reports implicate Zip2 and Zip3 along with Zip1 as important zinc uptake transporters involved in the unique ability of prostate cells to accumulate high cellular zinc levels. Zip1 is important for the extraction of zinc from circulation as the primary source of cellular zinc. Zip 2 and Zip3 appear to be important for retention of the zinc in the cellular compartment. The down regulation of all three transporters in the malignant cells is consistent with the loss of zinc accumulation in these cells. Since zinc imposes tumor suppressor effects, the silencing of the gene expression for these transporters is a required event for the manifestation of the malignant activities of the neoplastic cells. This now provides new insights into the genetic/molecular events associated with the development of prostate cancer; and supports our concept of Zip1, and now Zip2 and Zip3, as tumor suppressor genes and zinc as a tumor suppressor agent.

MK-0524 is a potent, selective and orally active Prosglandin D(2) Receptor 1 (DP(1)) antagonist currently under clinical development for the treatment of niacin-induced flushing. Experiments to study the pharmacokinetics, metabolism and excretion of MK-0524 were conducted in rats, dogs and monkeys. MK-0524 displayed linear kinetics and rapid absorption following an oral dose. Following intravenous (i.v.) administration of MK-0524 to rats and dogs (1 and 5 mg/kg), the mean Cl(p) was approximately 2 and approximately 6 ml/min/kg, the T(1/2) was approximately 7 and approximately 13 h and the Vd(ss) was approximately 1 and approximately 5 L/kg, respectively. In monkeys dosed i.v. at 3 mg/kg, the corresponding values were 8 ml/min/kg, 3 h and 1 L/kg, respectively. Following oral dosing of MK-0524 to rats (5, 25 and 100 mg/kg), dogs (5 mg/kg) and monkeys (3 mg/kg), the absorption was rapid with the mean C(max) occurring between 1 and 4 h. Absolute oral bioavailability values in rats, dogs and monkeys were 50, 70 and 8%, respectively. The major circulating metabolite was the acyl glucuronide of MK-0524 (M2), with ratios of glucuronide to the parent aglycone being highest in the monkey followed by dog and rat. In bile duct-cannulated rats and dogs, MK-0524 was eliminated primarily via acyl glucuronidation followed by biliary excretion of the acyl glucuronide, M2, the major drug-related entity in bile.

(3R)-4-(4-Chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl acetic acid (MK-0524) is a potent orally active human prostaglandin D(2) receptor 1 antagonist that is currently under development for the prevention of niacin-induced flushing. The major in vitro and in vivo metabolite of MK-0524 is the acyl glucuronic acid conjugate of the parent compound, M2. To compare metabolism of MK-0524 across preclinical species and humans, studies were undertaken to determine the in vitro kinetic parameters (K(m) and V(max)) for the glucuronidation of MK-0524 in Sprague-Dawley rat, beagle dog, cynomolgus monkey, and human liver microsomes, human intestinal microsomes, and in recombinant human UDP glucuronosyltransferases (UGT). A comparison of K(m) values indicated that UGT1A9 has the potential to catalyze the glucuronidation of MK-0524 in the liver, whereas UGT1A3 and UGT2B7 have the potential to catalyze the glucuronidation in the intestine. MK-0524 also was subject to phase I oxidative metabolism; however, the rate was significantly lower than that of glucuronidation. The rate of phase I metabolism was ranked as follows: rat approximately monkey > human intestine > dog > human liver with qualitatively similar metabolite profiles across species. In all the cases, the major metabolites were the monohydroxylated epimers (M1 and M4) and the keto-metabolite, M3. Use of inhibitory monoclonal antibodies and recombinant human cytochromes P450 suggested that CYP3A4 was the major isozyme involved in the oxidative metabolism of MK-0524, with a minor contribution from CYP2C9. The major metabolite in hepatocyte preparations was the acyl glucuronide, M2, with minor amounts of M1, M3, M4, and their corresponding glucuronides. Overall, the in vivo metabolism of MK-0524 is expected to proceed via glucuronidation, with minor contributions from oxidative pathways.

The proteins from the ZIP and the CDF families of zinc transporters contain a histidine-rich sequence in a loop domain located between transmembrane domains III and IV for the ZIP family and transmembrane domains IV and V for the CDF family. Topological predictions suggest that these loops are located in the cytoplasm. The loops contain a histidine-rich sequence with a variable number of histidine residues depending on the transporter. The histidine-rich sequence was postulated to serve as an extra-membrane metal binding site in these proteins. hZip1 is a human zinc transporter ubiquitously expressed. The histidine-rich motif located in the large loop of this transporter is composed of the following sequence, H(158)WHD(161). To determine if this motif is involved in the zinc transport activity of the protein, we performed site directed-mutagenesis to replace the loop histidines with alanines. Results suggest that both histidines are necessary for the zinc transport function and are not involved in the plasma membrane localization of the transporter as has been reported for the Zrt1 transporter in yeast. In addition, two histidine residues in transmembrane domains IV and V are also important in the zinc transport function. The results support an intermolecular exchange mechanism of zinc transport.

MK-0767 (KRP-297; 2-methoxy-5-(2,4-dioxo-5-thiazolidinyl)-N-[[4-(trifluoromethyl)phenyl] methyl]benzamide) is a thiazolidinedione (TZD)-containing dual agonist of the peroxisome proliferator-activated receptors alpha and gamma that has been studied as a potential treatment for patients with type 2 diabetes. The metabolism and excretion of [14C]MK-0767 were evaluated in six human volunteers after a 5-mg (200 microCi) oral dose. Excretion of 14C radioactivity was found to be nearly equal into the urine (approximately 50%) and feces (approximately 40%). Elimination of [14C]MK-0767 was primarily by metabolism, with minimal excretion of parent compound into the urine (<0.5% of dose) and feces (approximately 14% of the dose). [14C]MK-0767 was the major circulating compound-related entity (>96% of radioactivity) through 48 h postdose. It was also found that approximately 91% of the total radioactivity area under the curve was due to intact MK-0767. Several minor metabolites were detected in plasma (<1% of radioactivity, each), formed by cleavage of the TZD ring and subsequent S-methylation and oxidation. All the metabolites excreted into urine were formed by TZD cleavage, whereas the major metabolite in feces was the O-demethylated derivative of MK-0767.

The relationship between functional redundancy and microbial community structure-diversity was examined using laboratory incubations to ensure constant environmental conditions. Serial dilutions of a sewage microbial community were prepared, used to inoculate sterile sewage, and maintained in batch culture. Probability suggests that dilution of the initial community should remove rare organism types, creating mixtures of cells differing in diversity. Regrowth of the diluted mixtures generated communities similar in abundance but differing in community structure and relative diversity (as determined using two DNA fingerprinting techniques and dilution-to-extinction analysis of community-level physiological profiles). The in situ function of each regrown community was examined by monitoring the short-term uptake of five different (14)C-labeled compounds (glucose, acetate, citrate, palmitic acid, and an amino acid mixture). No significant differences were detected between treatments in either the rate of uptake of a substrate or the efficiency with which each community assimilated each compound. The fact that the activity of the original community was the same as that of a community regrown from an inoculum containing fewer that 100 cells (10(-6) dilution) indicates that functional redundancy was quite high in this system. For each organism type eliminated during the dilution process, at least one of the remaining types was able to provide the same function at the same level as the lost one. Further research is necessary to determine what impact this functional redundancy may have on overall ecosystem function and stability.

Background: Mitochondrial (m) aconitase plays an important role in the unique pathway of citrate accumulation in prostate epithelial cells through its limited activity. In the current study, we characterized the human m-aconitase gene promoter., Methods: A 1,411-bp 5'-flanking fragment of the human m-aconitase gene was cloned, followed by 5' serial deletion analysis of promoter activity. Transcriptional start sties and transcription factors bound to the promoter were identified by 5' RACE, DNA pull-down assay and transcription factor array analysis., Results: Two transcriptional start sites were identified. The promoter fragment pulled down 15 transcription factors, some without consensus sequences in the promoter. Deletion of one Sp1 site eliminated all promoter activity., Conclusions: The m-aconitase promoter is contained in a 153-bp 5' fragment lacking a TATA or CAAT sequence. Sp1 binding to a specific Sp1 site is required for promoter activity while other transcription factors are recruited through protein-protein interactions., (Copyright 2005 Wiley-Liss, Inc.)

Background: The genetic and molecular mechanisms responsible for and associated specifically with the development and progression of malignant prostate cells are largely unidentified. In addition, despite its implication in virtually all malignant cells, the role of altered cellular metabolism as an essential factor in prostate malignancy has been largely ignored. Moreover, the intermediary metabolism of normal prostate as well as malignant prostate cells is among the least studied and most poorly understood of all mammalian cells. Some important factors, especially the role of zinc, have been identified and implicated in the development and progression of prostate malignancy. In this review, we provide a current and updated integrated assessment of the relationships of intermediary metabolism in normal prostate and in prostate cancer. The experimental and clinical evidence that leads to the formulation of concepts of normal and malignant prostate metabolism is presented. The evidence for a concept of zinc as a tumor suppressor agent and Zip1 zinc transporter as a tumor-suppressor gene is described., Results: The specialized function of the normal prostate glandular epithelium to produce and secrete enormously high levels of citrate involves and requires unique intermediary metabolism activities that are not generally associated with other normal mammalian cells. The accumulation of zinc by these cells is an essential factor in this unique metabolic relationship. In malignancy, the normal zinc-accumulating citrate-producing epithelial cells are metabolically transformed to citrate-oxidizing cells that lose the ability to accumulate zinc. A genetic alteration in the expression of ZIP1 zinc transporter is associated with this metabolic transformation. These genetic/metabolic relationships have important consequences on citrate-related metabolism, bioenergetics, cell proliferation and invasive capabilities of the malignant cells, which result in tumor-suppression characteristics., Conclusion: The genetic/metabolic relationships in normal prostate glandular epithelium are driven by the unique function to accumulate and secrete citrate. The genetic/metabolic transformation of the prostate malignant cells is driven by the metabolic/bioenergetic, growth/proliferative, and invasive/migration requirements of the malignant process. Zinc is critical to these relationships. An understanding of these genetic/metabolic relationships provides new directions and opportunities for development of regimens for the prevention and treatment of prostate cancer. Important insight into the genetic/metabolic requirements of the prostate malignant process is now evolving. Most importantly at this time, an appreciation and recognition of the genetic/metabolic significance and implications in the development of prostate malignancy is imperative; and much needed research in this area is essential. Hopefully, this review will help to achieve these goals.

Background: In prostate cancer, normal citrate-producing glandular secretory epithelial cells undergo a metabolic transformation to malignant citrate-oxidizing cells. m-Aconitase is the critical step involved in this altered citrate metabolism that is essential to prostate malignancy. The limiting m-aconitase activity in prostate epithelial cells could be the result of a decreased level of m-aconitase enzyme and/or the inhibition of existing m-aconitase. Earlier studies identified zinc as an inhibitor of m-aconitase activity in prostate cells; and that the depletion of zinc in malignant cells is an important factor in this metabolic transformation. However, a possibility remains that an altered expression and level of m-aconitase enzyme might also be involved in this metabolic transformation. To address this issue, the in situ level of m-aconitase enzyme was determined by immunohistochemical analysis of prostate cancer tissue sections and malignant prostate cell lines., Results: The immunocytochemical procedure successfully identified the presence of m-aconitase localized in the mitochondrial compartment in PC-3, LNCaP, and DU-145 malignant prostate cell lines. The examination of prostate tissue sections from prostate cancer subjects demonstrated that m-aconitase enzyme is present in the glandular epithelium of normal glands, hyperplastic glands, adenocrcinomatous glands, and prostatic intraepithelial neoplastic foci. Quantitative analysis of the relative level of m-aconitase in the glandular epithelium of citrate-producing adenomatous glands versus the citrate-oxidizing adenocarcinomatous glands revealed no significant difference in m-aconitase enzyme levels. This is in contrast to the down-regulation of ZIP1 zinc transporter in the malignant glands versus hyperplastic glands that exists in the same tissue samples., Conclusion: The results demonstrate the existence of m-aconitase enzyme in the citrate-producing glandular epithelial cells; so that deficient m-aconitase enzyme is not associated with the limiting m-aconitase activity that prevents citrate oxidation in these cells. The level of m-aconitase is maintained in the malignant cells; so that an altered enzyme level is not associated with the increased m-aconitase activity. Consequently, the elevated zinc level that inhibits m-aconitase enzyme is responsible for the impaired citrate oxidation in normal and hyperplastic prostate glandular epithelial cells. Moreover, the down-regulation of ZIP1 zinc transporter and corresponding depletion of zinc results in the increase in the activity of the existing m-aconitase activity in the malignant prostate cells. The studies now define the mechanism for the metabolic transformation that characterizes the essential transition of normal citrate-producing epithelial cells to malignant citrate-oxidizing cells.

Background: Prostate epithelial cells accumulate a high level of aspartate that is utilized as a substrate for their unique function of production and secretion of enormously high levels of citrate. In most mammalian cells aspartate is synthesized; and, therefore is a non-essential amino acid. In contrast, in citrate-producing prostate cells, aspartate is an essential amino acid that must be derived from circulation. The prostate intracellular/extracellular conditions present a 40:1 concentration gradient. Therefore, these cells must possess a plasma membrane-associated aspartate uptake transport process to achieve their functional activity. In earlier kinetic studies we identified the existence of a unique Na+-dependent high-affinity L-aspartate transport process in rat prostate secretory epithelial cells. The present report is concerned with the identification of this putative L-aspartate transporter in rat and human prostate cells., Results: The studies show for the first time that EAAC1 is expressed in normal rat prostate epithelial cells, in normal and hyperplastic human prostate glands, and in human malignant prostate cell lines. EAAC1 expression and high-affinity L-aspartate transport are correspondingly down-regulated by EAAC1 siRNA knock down. Exposure of prostate cells to physiological levels of prolactin or testosterone results in an up-regulation of EAAC1 expression and a corresponding increase in the high-affinity transport of L-aspartate into the cells., Conclusion: This study shows that EAAC1 functions as the high-affinity L-aspartate transporter that is responsible for the uptake and accumulation of aspartate in prostate cells. In other cells (predominantly excitable tissue cells), EAAC1 has been reported to function as a glutamate transporter rather than as an aspartate transporter. The regulation of EAAC1 expression and L-aspartate transport by testosterone and prolactin is consistent with their regulation of citrate production in prostate cells. The identification of EAAC1 as the high-affinity L-aspartate transporter now permits studies to elucidate the mechanism of hormonal regulation of EAAC1 gene expression, and to investigate the mechanism by which the cellular environment effects the functioning of EAAC1 as an aspartate transporter or as a glutamate transporter.

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.

Mutations in mitochondrial DNA are frequent in cancer and the accompanying mitochondrial dysfunction and altered intermediary metabolism might contribute to, or signal, tumour pathogenesis. The metabolism of human prostate peripheral zone glandular epithelial cells is unique. Compared with many other soft tissues, these glandular epithelial cells accumulate high concentrations of zinc, which inhibits the activity of m-aconitase, an enzyme involved in citrate metabolism through Krebs cycle. This causes Krebs cycle truncation and accumulation of high concentrations of citrate to be secreted in prostatic fluid. The accumulation of zinc also inhibits terminal oxidation. Therefore, these cells exhibit inefficient energy production. In contrast, malignant transformation of the prostate is associated with an early metabolic switch, leading to decreased zinc accumulation and increased citrate oxidation. The efficient energy production in these transformed cells implies increased electron transport chain activity, increased oxygen consumption, and perhaps, excess reactive oxygen species (ROS) production compared with normal prostate epithelial cells. Because ROS have deleterious effects on DNA, proteins, and lipids, the altered intermediary metabolism may be linked with ROS production and accelerated mitochondrial DNA mutations in prostate cancer.

A species difference was observed in the excretion pathway of 2-[[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy]-2-methylpropanoic acid (MRL-C), an alpha-weighted dual peroxisome proliferator-activated receptor alpha/gamma agonist. After intravenous or oral administration of [14C]MRL-C to rats and dogs, radioactivity was excreted mainly into the bile as the acyl glucuronide metabolite of the parent compound. In contrast, when [14C]MRL-C was administered to monkeys, radioactivity was excreted into both the bile and the urine as the acyl glucuronide metabolite, together with several oxidative metabolites and their ether or acyl glucuronides. Incubations in hepatocytes from rats, dogs, monkeys, and humans showed the formation of the acyl glucuronide of the parent compound as the major metabolite in all species. The acyl glucuronide and several hydroxylated products, some which were glucuronidated at the carboxylic acid moiety, were observed in incubations of MRL-C with NADPH- and uridine 5'-diphosphoglucuronic acid-fortified liver microsomes. However, metabolism was more extensive in the monkey microsomes than in those from the other species. When the acyl glucuronide metabolite of MRL-C was incubated with NADPH-fortified liver microsomes, in the presence of saccharo-1,4-lactone, it underwent extensive oxidative metabolism in the monkey but considerably less in the rat, dog, and human liver microsomes. Collectively, these data suggested that the oxidative metabolism of the acyl glucuronide might have contributed to the observed in vivo species differences in the metabolism and excretion of MRL-C.

The re-emergence of interest in intermediary metabolism and the development of metabolomics in relation to cancer and other diseases provide a timely reason to revisit issues of tumour cell metabolism. In this review, we address the issue of the role of high aerobic glycolysis, which is commonly associated with the metabolism of many tumour cells. The concept presented emphasises the importance of the glycolysis-citrate-lipogenesis pathway in providing the synthetic and bioenergetic requirements that are essential for the growth and proliferation of tumour cells. We hope that our discussion will be informative and instructive, and will stimulate interest and research regarding the intermediary metabolism and its regulation in tumour cells. We express our appreciation to the many pioneering and contemporary researchers whose studies provide much of the basis for this presentation.

The role of zinc in the development and progression of prostate malignancy and its potential application in the prevention and treatment of prostate cancer (PCa) are contemporary critical issues for the medical/scientific community and the public-at-large. The overwhelming clinical and experimental evidence provides a compelling rational basis for the expectation and concept that prostate zinc accumulation is an important factor in the development and progression of prostate malignancy; and that zinc could be efficacious in the prevention and treatment of PCa. In contrast, various epidemiologic studies have produced divergent and conflicting results regarding the efficacy of dietary and supplemental zinc against PCa. Before reaching any definitive conclusions regarding this complex issue, one should have a complete understanding of the clinical and experimental evidence associated with the involvement of zinc in the normal and malignant prostate. Also, an understanding of interacting effects of confounding factors on the absorption, assimilation, and bioavailability of supplemental dietary zinc is important. The purpose of this review is to present the current state of the clinical and experimental information regarding zinc relationships in the normal prostate and in the pathogenesis PCa. The evidence in support of a potential beneficial effect of zinc supplement versus potential harmful effects on PCa is assessed. A discussion of the divergent results of the epidemiologic studies is presented along with a description of important factors and conditions that impact or mask the effects of dietary zinc on PCa development and progression. We also hope to bring more attention to the medical and research community of the critical need for concerted clinical and basic research regarding zinc and PCa, for the development of appropriate human prostate models to investigate these relationships, for further appropriately designed epidemiologic studies, and for future well-controlled clinical trials.

Background: The genetic and molecular mechanisms responsible for and associated with the development and progression of prostate malignancy are largely unidentified. The peripheral zone is the major region of the human prostate gland where malignancy develops. The normal peripheral zone glandular epithelium has the unique function of accumulating high levels of zinc. In contrast, the ability to accumulate zinc is lost in the malignant cells. The lost ability of the neoplastic epithelial cells to accumulate zinc is a consistent factor in their development of malignancy. Recent studies identified ZIP1 (SLC39A1) as an important zinc transporter involved in zinc accumulation in prostate cells. Therefore, we investigated the possibility that down-regulation of hZIP1 gene expression might be involved in the inability of malignant prostate cells to accumulate zinc. To address this issue, the expression of hZIP1 and the depletion of zinc in malignant versus non-malignant prostate glands of prostate cancer tissue sections were analyzed. hZIP1 expression was also determined in malignant prostate cell lines., Results: hZIP1 gene expression, ZIP1 transporter protein, and cellular zinc were prominent in normal peripheral zone glandular epithelium and in benign hyperplastic glands (also zinc accumulating glands). In contrast, hZIP1 gene expression and transporter protein were markedly down-regulated and zinc was depleted in adenocarcinomatous glands and in prostate intra-epithelial neoplastic foci (PIN). These changes occur early in malignancy and are sustained during its progression in the peripheral zone. hZIP1 is also expressed in the malignant cell lines LNCaP, PC-3, DU-145; and in the nonmalignant cell lines HPr-1 and BPH-1., Conclusion: The studies clearly establish that hZIP1 gene expression is down regulated and zinc is depleted in adenocarcinomatous glands. The fact that all the malignant cell lines express hZIP1 indicates that the down-regulation in adenocarcinomatous glands is likely due to in situ gene silencing. These observations, coupled with the numerous and consistent reports of loss of zinc accumulation in malignant cells in prostate cancer, lead to the plausible proposal that down regulation of hZIP1 is a critical early event in the development prostate cancer.

Zinc is an essential metal for all cells. It plays a role in a wide variety of physiological and biochemical processes. In the prostate epithelial cell the accumulation of high cellular zinc is a specialized function that is necessary for these cells to carry out the major physiological functions of production and secretion of citrate. The production of citrate and its secretion into prostatic fluid is a differentiated function of the prostate epithelial cells that is apparently important for reproduction. The loss of citrate and zinc accumulation is the most consistent and persistent characteristic of prostate malignancy. This characteristic of prostate cancer indicates that the lost ability of the malignant cells to accumulate zinc and citrate is an important factor in the development and progression of malignancy. The lost ability of the epithelial cells to accumulate zinc and thus to also accumulate citrate is the result of decreased expression of specific zinc uptake transporters. The purpose of this presentation is to review the current understanding of zinc and zinc homeostasis in the prostate and the role of zinc and zinc transporters in the normal function of the prostate and the pathogenesis of prostate cancer.

Previous studies have shown that in a cell-free system, metallothionein (MT) releases zinc when the environment becomes oxidized and the released zinc is transferred to a zinc-binding protein if such a protein is present. However, it is unknown whether and how zinc transfers from MT to other proteins in vivo. The present study was undertaken to test the hypothesis that if zinc transfer from MT to other proteins occurs in vivo, the transfer would proceed through a direct interaction between MT and a specific group of proteins. The heart extract obtained from MT-null mice was incubated with 65Zn-MT or 65ZnCl2 and the proteins receiving 65Zn were separated by blue-native PAGE (BN-PAGE) or sodium dodecyl sulfate-PAGE (SDS-PAGE), and detected by autoradiography. A unique 65Zn-binding band was observed from the 65Zn-MT-incubated, but not the 65ZnCl2-incubated preparation. The analysis using matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry revealed that mitochondrial aconitase (m-aconitase) was among the proteins accepting Zn directly from Zn-MT. The m-aconitase, not the cytosolic aconitase (c-aconitase), was co-immunoprecipitated with MT. This study demonstrates that MT transfers zinc to m-aconitase through a direct interaction.

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.

Thiazolidinedione (TZD) derivatives have been reported to undergo metabolic activation of the TZD ring to produce reactive intermediates. In the case of troglitazone, it was proposed that a P450-mediated S-oxidation leads to TZD ring scission and the formation of a sulfenic acid intermediate, which may be trapped as a GSH conjugate. In the present study, we employed a model compound {denoted MRL-A, (+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethoxy)phenyl]methyl]benzamide} to investigate the mechanism of TZD ring scission. When MRL-A was incubated with monkey liver microsomes (or recombinant P450 3A4 and NADPH-P450 reductase) in the presence of NADPH and oxygen, the major products of TZD ring scission were the free thiol metabolite (M2) and its dimer (M3). Furthermore, a GSH conjugate of M2 (M4) also was formed when the incubation mixture was supplemented with GSH. Experiments with isolated M2 suggested that this metabolite was unstable and underwent spontaneous autooxidation to M3. A qualitatively similar metabolite profile was observed when MRL-A was incubated with recombinant P450 3A4 and cumene hydroperoxide. Because an oxygen atom is transferred to MRL-A under these conditions, these data suggested that S-oxidation alone may result in TZD ring scission and formation of M2 via a sulfenic acid intermediate. Also, because the latter incubation mixture did not contain any reducing agents, the formation of M2 may have occurred due to disproportionation of the sulfenic acid. When NADPH was added to the incubation mixture containing P450 3A4 and cumene hydroperoxide, the formation of M3 increased, suggesting that the sulfenic acid was reduced to M2 by NADPH and subsequently underwent dimerization to yield M3 (vide supra). When NADPH was replaced by GSH, the formation of M4 increased, consistent with reduction of the sulfenic acid by GSH. In summary, these results suggest that the TZD ring in MRL-A is activated by an initial P450-mediated S-oxidation step followed by spontaneous scission of the TZD ring to a putative sulfenic acid intermediate; the latter species then undergoes reduction to the free thiol by GSH, NADPH, and/or disproportionation. Finally, the thiol may dimerize to the corresponding disulfide or, in the presence of S-adenosylmethionine, form the stable S-methyl derivative.

An interference leached from polypropylene tubes was identified to be a sulfoxide oxidative product of didodecyl 3,3'-thiodipropionate (DDTDP) that is used to prevent oxidative degradation of synthetic polymers. A sulfone oxidative product of DDTDP leached from the polypropylene tubes was also observed. The interfering compounds were isolated by LC and characterized using time-of-flight mass spectrometry and NMR. Authentic sulfoxide and sulfone products of DDTDP were also prepared by reacting DDTDP with hydrogen peroxide reaching an unequivocal structural assignment. In conclusion, when analytes of interest are solubilized in predominantly organic solvents and kept in polypropylene containers, the possibility of contamination from leached chemicals should be taken into account.

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.

MK-0767, (+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide, is a thiazolidinedione-containing dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist that has been studied as a potential treatment for patients with type 2 diabetes. MK-0767 contains a chiral center at the C-5 position of the thiazolidinedione ring and was being developed as the racemate, due to the rapid interconversion of its enantiomers in biological samples. In the present work the in vitro and in vivo concentration ratios of the (+)-(R) to (-)-(S) enantiomers of MK-0767 were determined in plasma from humans (in vitro only) and nonclinical species used in the toxicological evaluation of rac-MK-0767, namely CD-1 mice, Sprague-Dawley rats, beagle dogs, New Zealand white rabbits, and rhesus monkeys. The R/S ratio was determined by chiral liquid chromatography/tandem mass spectrometry. Species differences were observed in the in vitro and in vivo enantiomeric ratios, as well as differences between in vitro and in vivo in some species. The in vitro R/S ratio was similar in dogs and humans (approximately 1.5-1.7). In rats and monkeys, the ratio was approximately unity, both in vitro and in vivo. In mice, the ratio was higher in vitro (approximately 1) than in vivo (approximately 0.6), while in rabbits it was higher in vivo (approximately 1) than in vitro (approximately 0.5). These results suggested that differential binding of the MK-0767 enantiomers to plasma and tissue proteins and other macromolecules may be affecting the R/S ratio both in vitro and in vivo, since in protein-free systems MK-0767 exists as the racemate., (Copyright 2005 John Wiley & Sons, Ltd.)

The effect of incubation of coupled liver mitochondria on varying concentration of zinc ion was determined. A low concentration of 6 microM zinc ion was found to inhibit the rate of oxygen consumption of the liver mitochondria significantly [P < 0.01]. There was uncoupling of the liver mitochondria when subjected to varying incubation periods. There was no change observed in the control experiment. Zinc-citrate inhibited the rate of oxygen consumption significantly [P < 0.01] when compare with the control. The changes observed in the Zn-aspartate were insignificant. Zn-EDTA had no inhibitory or stimulatory effect on the rate of liver mitochondrial oxygen consumption.

The role of specific cytochrome P450 (P450) isoforms in the metabolism of ethinylestradiol (EE) was evaluated. The recombinant human P450 isozymes CYP1A1, CYP1A2, CYP2C9, CYP2C19, and CYP3A4 were found to be capable of catalyzing the metabolism of EE (1 microM). Without exception, the major metabolite was 2-hydroxy-EE. The highest catalytic efficiency (Vmax/Km) was observed with rCYP1A1, followed by rCYP3A4, rCYP2C9, and rCYP1A2. The P450 isoforms 3A4 and 2C9 were shown to play a significant role in the formation of 2-hydroxy-EE in a pool of human liver microsomes by using isoform-specific monoclonal antibodies, in which the inhibition of formation was approximately 54 and 24%, respectively. The involvement of CYP3A4 and CYP2C9 was further confirmed by using selective chemical inhibitors (i.e., ketoconazole and sulfaphenazole). The relative contribution of each P450 isoform to the 2-hydroxylation pathway was obtained from the catalytic efficiency of each isoform normalized by its relative abundance in the same pool of human liver microsomes, as determined by quantitative Western blot analysis. Collectively, these results suggested that multiple P450 isoforms were involved in the oxidative metabolism of EE in human liver microsomes, with CYP3A4 and CYP2C9 as the major contributing enzymes., (Copyright 2004 The American Society for Pharmacology and Experimental Therapeutics)

MK-0767 [(+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide] is a novel thiazolidinedione-containing peroxisome proliferator-activated receptor alpha/gamma agonist. In rats dosed orally with [14C]MK-0767, a dihydrohydroxy-S-glutathionyl conjugate of the parent compound was identified in the bile using liquid chromatography-mass spectometry and 1H NMR techniques. The formation of the conjugate likely proceeded via an arene oxide intermediate. The corresponding cysteinylglycine and cysteinyl conjugates likely formed from the further metabolism of the dihydrohydroxy-S-glutathionyl conjugate also were detected in rat bile. The dihydrohydroxy-S-glutathionyl conjugate was formed in vitro following the incubation of MK-0767 and glutathione with rat, dog, or monkey liver microsomes, and its formation was NADPH-dependent; however, this conjugate was not detected in human liver microsomal incubations. When incubated with rat intestinal contents, the dihydrohydroxy-S-glutathionyl conjugate was reduced to the parent compound (MK-0767), suggesting the involvement of intestinal microflora in its metabolism. There was no reduction of the conjugate by rat intestinal cytosol.