Phenotype of single hepatocytes expressing an activated version of ß-catenin in liver of transgenic mice.

The gene CTNNB1 encoding ß-catenin is mutated in about 30% of hepatocellular carcinoma, generally often combined with other genetic alterations. In transgenic mice, it has been shown that activation of ß-catenin in more than 70% of all hepatocytes causes immediate proliferation leading to hepatomegaly. In this study we established a novel mouse model where ß-catenin is activated only in individual, dispersed hepatocytes. Hepatocyte-specific expression of activated point-mutated ß-catenin (human ß-catenin(S33Y)) was established using the Cre/loxP system. Expression of several downstream targets of ß-catenin signaling such as glutamine synthetase and several cytochrome P450 isoforms was confirmed by immunostaining. Only a minor portion of hepatocytes expressed the ß-catenin(S33Y) transgene, which were mainly positioned as dispersed individual cells within the normal liver parenchyma. The hepatocytes with activated ß-catenin did not show increased proliferation and the mice did not develop hepatomegaly. In conclusion, activated ß-catenin in single hepatocytes induces a gene expression pattern in hepatocytes which is similar to that of Ctnnb1-mutated mouse liver tumors, but is apparently not sufficient to induce increased cell proliferation. Therefore, onset of proliferation seems to require concomitant activation of ß-catenin in clusters of hepatocytes, suggesting a role of cell-cell communication in this process.

Coordinate regulation of cytochrome P450 1a1 expression in mouse liver by the aryl hydrocarbon receptor and the beta-catenin pathway.

The expression of cytochrome P450 (CYP) 1a1 and other drug-metabolizing enzymes is controlled by the aryl hydrocarbon receptor (AhR), which is activated by dioxin-type inducers leading to transcriptional induction of target genes. Here, we show that a second level of transcriptional control exists in hepatocytes, which is tightly linked to the Wnt/ß-catenin/T-cell factor (TCF) signaling pathway. In transgenic mice, hepatic expression of CYP1A (and other CYP isoforms) is stimulated by the expression of mutationally activated ß-catenin(S33Y) in the absence of AhR-activating compounds but repressed after knockout of ß-catenin. These effects were further analyzed in vitro, and the stimulatory role of ß-catenin was ascribed to a TCF-binding site within the CYP1A1 promoter. Moreover, ß-catenin signaling acted cooperatively with AhR agonists via AhR-binding sites on the DNA during the induction of Cyp1a1 in vivo and in vitro. Activation of ß-catenin enhanced the transactivation potential of ligand-activated AhR at its DNA-binding sites without altering the total amount of DNA-bound AhR. Coimmunoprecipitation demonstrated a physical interaction between AhR and ß-catenin. Furthermore, the present results suggest that transcriptional induction of the AhR by ß-catenin does not play a major role in ß-catenin-dependent regulation of Cyp1a1 expression and that inhibition of ß-catenin signaling by ligand-activated AhR, as recently observed in the intestine does not occur in mouse liver. In conclusion, signaling through ß-catenin activates basal CYP1A1 expression and augments CYP1A1 induction by AhR ligands through enhancement of the transactivation potential of the AhR.

Contributions of the Ah receptor to bilirubin homeostasis and its antioxidative and atheroprotective functions.

Abstract The homeostasis and atheroprotective function of bilirubin could be an appealing model to investigate one of the many physiologic functions of the human aryl hydrocarbon receptor (AhR). Several clinical and epidemiological studies have been carried out on key enzymes generating and eliminating bilirubin (heme oxygenase-1 and UDP-glucuronosyltransferase UGT1A1, respectively) and their regulation by the AhR. Studies with AhR-deficient mice strongly suggest a role of the AhR in vascular biology. Atherosclerosis, a major cause of premature death, is initiated by pro-oxidative insults of the vascular endothelium. The strong antioxidant and activator of AhR bilirubin is generated in vascular endothelial cells, smooth muscles and macrophages. It acts mostly in the lipid environment, thereby complementing other antioxidants such as glutathione which act mostly on water-soluble proteins. In conclusion, the atheroprotective functions of bilirubin might not only provide models to study physiologic functions of the human AhR but also provide opportunities to improve prevention and treatment of a major life-threatening disease.

Wnt/beta-catenin signaling activates and determines hepatic zonal expression of glutathione S-transferases in mouse liver.

Glutathione S-transferases (GSTs) play an essential role in the elimination of xenobiotic-derived electrophilic metabolites and also catalyze certain steps in the conversion of endogenous molecules. Their expression is controlled by different transcription factors, such as the antioxidant-activated Nrf2 or the constitutive androstane receptor. Here, we show that the Wnt/beta-catenin pathway is also involved in the transcriptional regulation of GSTs: GSTm2, GSTm3, and GSTm6 are overexpressed in mouse hepatomas with activating Ctnnb1 (encoding beta-catenin) mutations and in transgenic hepatocytes expressing activated beta-catenin. Inversely, GSTm expression is reduced in mice with hepatocyte-specific knock out of Ctnnb1. Activation of beta-catenin-dependent signaling stimulates GSTm expression in vitro. Activation of beta-catenin in mouse hepatoma cells activates GSTm3 promoter-driven reporter activity, independently of beta-catenin/T-cell factor sites, via a retinoid X receptor-binding site. By contrast, GSTm expression is inhibited upon Ras activation in mouse liver tumors and transgenic hepatocytes. Recent studies by different groups have shown that beta-catenin-dependent signaling is involved in the transcriptional control of "perivenous" expression of various cytochrome P450s in mouse liver, whereas Ras signaling was hypothesized to antagonize the perivenous hepatocyte phenotype. In synopsis with our present results, it now appears that the Wnt/beta-catenin pathway functions as a master regulator of the expression of both phase I and phase II drug-metabolizing enzymes in perivenous hepatocytes from mouse liver.

ReProGlo: a new stem cell-based reporter assay aimed to predict embryotoxic potential of drugs and chemicals.

A stem cell-based reporter assay was developed to detect drug-induced alterations in the canonical Wnt/beta-catenin signaling pathway, which is involved in the regulation of early embryonic development. The so-called ReProGlo assay allows simultaneous determination of cell viability and luciferase reporter activity in a high throughput 96-well microtiter format. A clone of mouse embryonic stem (mES) cells stably expressing the SuperTopFlash reporter was established. This allows Wnt pathway activity determinations in undifferentiated mES cells and their differentiated descendants. Several test chemicals were analyzed in the new assay system. Known embryotoxicants like retinoic acid or lithium chloride induced concentration-dependent increases in reporter activity. The potency of valproic acid and a series of structural analogs to activate the Wnt pathway correlated well with their reported teratogenic activity in the mouse. Cyclophosphamide was also active but only after metabolic activation by hepatocytes. The new test may help to predict embryotoxic potential of chemicals.

The mammalian aryl hydrocarbon (Ah) receptor: from mediator of dioxin toxicity toward physiological functions in skin and liver.

The mammalian Ah receptor (AhR) is a ligand-activated transcription factor with multiple functions in adaptive metabolism, development and dioxin toxicity in a variety of organs and cell systems. Phenotypes observed following sustained activation by dioxin or in AhR-null mice suggest organ-dependent physiological functions. These functions are probably deregulated following exposure to dioxin. We focus on skin and liver to facilitate discussion of mechanisms linking phenotypes and AhR-modulated genotypes. After a brief summary of currently discussed AhR ligand candidates, two groups of direct AhR target genes/proteins and associated functions are highlighted: (i) xenobiotic-metabolizing enzymes which are also involved in homeostasis of endogenous ligands and (ii) proteins controlling cell proliferation/apoptosis, differentiation and inflammation. Homeostatic feedback loops might not only include CYP1A1 but also Phase II enzymes such as UGT1A1 which controls the antioxidant AhR ligand bilirubin. The AhR is involved in extensive crosstalk with other transcription factors and multiple signaling pathways. Efforts elucidating the pathway toward identification of physiological functions of the AhR remain challenging and promising.

Topological aspects of oligomeric UDP-glucuronosyltransferases in endoplasmic reticulum membranes: advances and open questions.

UDP-glucuronosyltransferases (UGTs) represent major Phase II enzymes involved in detoxification of endo- and xenobiotics, including many drugs. The intraluminal orientation of the active site of UGTs in endoplasmic reticulum membranes necessitates a number of transporters in these membranes, for example, for UDP-glucuronic acid and glucuronides, the latter being insufficiently characterized. In addition, accumulating evidence suggests that UGTs are functional as homo- and heterodimers in monoglucuronide formation. They may form tetramers in diglucuronide formation. UGT oligomers probably serve to stabilize UGT monomers and fine-tune UGT activity. Glucuronide disposition may also be influenced by endoplasmic reticulum-localized beta-glucuronidase, possibly involved in hydrolysis of hormone and drug glucuronides in target cells. The present commentary reviews recent advances and addresses open questions. Resolution of these questions may help to understand many problems of glucuronide synthesis and disposition in vivo, for example, under-prediction of the in vivo clearance of drugs mostly eliminated by glucuronidation by in vitro enzyme kinetic parameters of UGTs.

Coordinate regulation of human drug-metabolizing enzymes, and conjugate transporters by the Ah receptor, pregnane X receptor and constitutive androstane receptor.

Coordinate regulation of Phase I and II drug-metabolizing enzymes and conjugate transporters by nuclear receptors suggests that these proteins evolved to an integrated biotransformation system. Two major groups of ligand-activated nuclear receptors/xenosensors evolved: the Ah receptor (activated by aryl hydrocarbons and drugs such as omeprazole) and type 2 steroid receptors such as PXR and CAR, activated by drugs such as rifampicin, carbamazepin and phenytoin. It is increasingly recognized that there is considerable cross-talk between these xenosensors. Therefore, an attempt was made to discuss biotransformation by the Ah receptor together with that of PXR and CAR. Due to considerable species differences the emphasis is on human biotransformation. Agonists coordinately induce biotransformation due to common xenosensor-binding response elements in the regulatory region of target genes. However, whereas different groups of xenobiotics appear to more selectively stimulate CYPs (Phase I), their regulatory control largely converged in modulating Phase II metabolism and transport. Biotransformation appears to be tightly controlled to achieve efficient homeostasis of endobiotics and detoxification of dietary phytochemicals, but nuclear receptor agonists may also lead to potentially harmful drug interactions.

Promotion of hepatocarcinogenesis in humans and animal models.

Risk assessment based on rodent carcinogenicity data depends on the assumption of similarity between rodents and humans. While this assumption is conceivable in the case of genotoxic initiating carcinogens, considerable species differences have been observed with nongenotoxic tumor promoters. This heterogeneous group of agents increases the probability of cancer by stimulating selection and clonal expansion of cells transformed during tumor initiation. Since tumor promoters differentially affect normal tissue and preneoplastic cell clones, their action cannot be discussed without knowledge of persistent genomic and epigenetic alterations occurring during initiation and formation of preneoplastic cells. Chemical carcinogenesis, and in particular, tumor promotion, is known to be tissue specific. We focus on hepatocarcinogenesis in humans and in animal models and emphasize two different modes of action: (1) chronic cytotoxicity leading to promotion of liver carcinogenesis in both humans and animal models; (2) sustained activation of orphan receptors such as CAR, PPARalpha and Ah receptor leading to promotion of rodent but probably not human hepatocarcinogenesis. Further studies on the different modes of action may help to avoid overestimation of the risk of liver tumor promotion.

Lack of effect of extracellular adenosine generation and signaling on renal erythropoietin secretion during hypoxia.

Previous studies have yielded conflicting results as to whether extracellular adenosine generation and signaling contributes to hypoxia-induced increases in renal erythropoietin (EPO) secretion. In this study, we combined pharmacological and genetic approaches to elucidate a potential contribution of extracellular adenosine to renal EPO release in mice. To stimulate EPO secretion, we used murine carbon monoxide exposure (400 and 750 parts per million CO, 4 h), ambient hypoxia (8% oxygen, 4 h), or arterial hemodilution. Because the ecto-5-nucleotidase (CD73, conversion of AMP to adenosine) is considered the pacemaker of extracellular adenosine generation, we first tested the effect of blocking extracellular adenosine generation with the specific CD73-inhibitor adenosine 5'-(alpha,beta-methylene) diphosphate (APCP) or by gene-targeted deletion of cd73. These studies showed that neither APCP-treatment nor targeted deletion of cd73 resulted in changes of stimulated EPO mRNA or serum levels, although the increases of adenosine levels in the kidney following CO exposure were attenuated in mice with APCP treatment or in cd73(-/-) mice. Moreover, pharmacological studies using specific inhibitors of individual adenosine receptors (A1 AR, DPCPX; A 2A AR, DMPX; A 2B AR, PSB 1115; A3AR, MRS 1191) showed no effect on stimulated increases of EPO mRNA or serum levels. Finally, stimulated EPO secretion was not attenuated in gene-targeted mice lacking A1A(-/-, A2A AR-/-, A2BAR(-/-), or A3AR-/-. Together, these studies combine genetic and pharmacological in vivo evidence that increases of EPO secretion during limited oxygen availability are not affected by extracellular adenosine generation or signaling.

Serum components and activated Ha-ras antagonize expression of perivenous marker genes stimulated by beta-catenin signaling in mouse hepatocytes.

Hepatocytes of the periportal and perivenous zones of the liver lobule show marked differences in the contents and activities of many enzymes and other proteins. Previous studies from our and other groups have pointed towards an important role of beta-catenin-dependent signaling in the regulation of expression of genes encoding proteins with preferential perivenous localization, whereas, in contrast, signaling through Ras-dependent pathway(s) may induce a 'periportal' phenotype. We have now conducted a series of experiments to further investigate this hypothesis. In transgenic mice with scattered expression of an activated Ha-ras (Ha-ras(G12V)) mutant in liver, expression of the perivenous markers glutamine synthetase and two cytochrome P450 isoforms was completely abolished in those hepatocytes demonstrating constitutively activated extracellular signal-regulated kinase activity, even though they were located directly adjacent to central veins. Similarly, incubation of primary hepatocytes or hepatoma cells with increasing amounts of serum caused a concentration-dependent attenuation of expression of perivenous marker mRNAs, whereas the expression of periportal markers was increased. The inhibitory effect of high amounts of serum on the expression of perivenous markers was also observed if their expression was stimulated by activation of beta-catenin signaling, and comparable inhibitory effects were seen in cells stably transfected with a T-cell factor/lymphoid-enhancing factor-driven luciferase reporter. Epidermal growth factor could partly mimic serum effects in hepatoma cells, and its effect could be blocked by an inhibitor of extracellular signal-regulated kinase activity. These data suggest that activation of the Ras/mitogen-activated protein kinase (extracellular signal-regulated kinase) pathway favors periportal gene expression while simultaneously antagonizing a perivenous phenotype of hepatocytes.

Global gene expression in Ha-ras and B-raf mutated mouse liver tumors.

Chemically-induced mouse liver tumors harbor mutations in different oncogenes. About 50% of tumors contain activating mutations in the Ha-ras gene contain and about 20% of tumors show point mutations in the B-raf oncogene. We have investigated the gene expression profiles in tumors of the 2 genotypes by microarray analysis. In total, approximately 500 genes or expressed sequences were aberrantly expressed in tumors relative to normal liver tissue. Around two/thirds of them were significantly altered in both Ha-ras and B-raf mutated liver tumors, and most of the remaining genes showed at least qualitatively comparable changes in both tumor types. Several functional clusters were hypothesized in tumors of the 2 genotypes which involve alterations in a battery of genes encoding enzymes of lipid metabolism. The similarity in the patterns of global gene expression of Ha-ras and B-raf mutated liver tumors suggests that mutational activation of the 2 oncogenes results in activation of a common set of transcriptional regulators.

Protective role of ecto-5'-nucleotidase (CD73) in renal ischemia.

Acute renal failure from ischemia significantly contributes to cardiovascular morbidity and mortality. Extracellular adenosine has been implicated as an anti-inflammatory metabolite particularly during conditions of limited oxygen availability (e.g., ischemia). Because ecto-5'-nucleotidase (CD73) is rate limiting for extracellular adenosine generation, this study examined the contribution of CD73-dependent adenosine production to ischemic preconditioning (IP) of the kidneys. After the initial observation that murine CD73 transcript, protein, and function are induced by renal IP, its role in IP-mediated kidney protection was studied. In fact, increases in renal adenosine concentration with IP are attenuated in cd73(-/-) mice. Moreover, pharmacologic inhibition of CD73 or its targeted gene deletion abolished renal protection by IP as measured by clearance studies, plasma electrolytes, and renal tubular destruction, and reconstitution of cd73(-/-) mice with soluble 5'-nucleotidase resulted in complete restoration of renal protection by IP. Finally, renal injury after ischemia was attenuated by intraperitoneal treatment of wild-type mice with soluble 5'-nucleotidase to a similar degree as by IP. Taken together, these data reveal what is believed to be a previously unrecognized role of CD73 in renal protection from ischemia and suggest treatment with soluble 5'-nucleotidase as a novel therapeutic approach in the treatment of renal diseases that are precipitated by limited oxygen availability.

Coordinate regulation of Phase I and II xenobiotic metabolisms by the Ah receptor and Nrf2.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with important roles in metabolic adaptation, normal physiology and dioxin toxicology. Metabolic adaptation is based on coordinate regulation of a set of xenobiotic-metabolizing enzymes (XMEs), termed AhR battery. Coordination is achieved by AhR/Arnt-binding to XREs (xenobiotic response elements), identified in the 5' upstream region of AhR target genes. The AhR battery encodes Phase I and II enzymes. Interestingly, these Phase II genes are linked to the Nrf2 gene battery that encodes enzymes that are essential in protection against oxidative/electrophile stress. Nrf2 binds to AREs (antioxidant response elements) in the regulatory region of a large and distinct set of target genes. Functionally characterized response elements such as XREs and AREs in the regulatory region of target genes may provide a genetic basis to understand AhR- and Nrf2-induced genes. Linkage between AhR and Nrf2 batteries is probably achieved by multiple mechanisms, including Nrf2 as a target gene of the AhR, indirect activation of Nrf2 via CYP1A1-generated reactive oxygen species, and direct cross-interaction of AhR/XRE and Nrf2/ARE signaling. Linkage appears to be species- and cell-dependent. However, mechanisms linking XRE- and ARE-controlled Phase II genes need further investigation. Tightened coupling between Phases I and II by AhR- and Nrf2-induced XMEs may greatly attenuate health risks posed by CYP1A1-generated toxic intermediates and reactive oxygen species. Better recognition of coordinate Phase I and II metabolisms may improve risk assessment of reactive toxic intermediates in the extrapolation to low level endo- and xenobiotic exposure.

Zonal gene expression in mouse liver resembles expression patterns of Ha-ras and beta-catenin mutated hepatomas.

Hepatocytes of the periportal and perivenous zones of the liver lobule differ in their levels and activities of various enzymes and other proteins. We have recently suggested that beta-catenin- and Ras-dependent signaling pathways play an important role in the regulation of perivenous and periportal gene expression profiles. This hypothesis was primarily based on similarities in zonal differences in gene expression of hepatocytes from normal liver with gene expression patterns of liver tumors: several proteins and mRNAs preferentially expressed in periportal hepatocytes were often overexpressed in Ha-ras mutated mouse liver tumors, whereas perivenous markers were overexpressed in Ctnnb1 (encoding beta-catenin) mutated tumors. We have now extended this work by use of data from two previously conducted microarray analyses aimed to analyze 1) global gene expression patterns of Ha-ras and Ctnnb1 mutated mouse liver tumors and 2) transcriptome differences between periportal and perivenous mouse hepatocytes. By comparison of the datasets, 134 genes or expressed sequences were identified that were present in both datasets. Gene expression patterns in perivenous hepatocytes and Ctnnb1 mutated hepatoma cells were strongly correlated: 96.5% of the genes present in both datasets were regulated in the same direction. In analogy, expression of 74.1% of the genes deregulated in Ha-ras mutated tumors was correlated with the respective expression patterns in periportal hepatocytes. These findings favor the hypothesis that gene expression patterns in periportal and perivenous hepatocytes are regulated, at least in part, by Ras- and beta-catenin-dependent signaling pathways.

Use of a hanging-weight system for isolated renal artery occlusion during ischemic preconditioning in mice.

Renal failure from ischemia contributes to morbidity and mortality. Ischemic preconditioning (IP) represents a powerful strategy for kidney protection, and recent advances in transgenic mice may help elucidate its molecular mechanisms. However, murine IP is technically challenging and experimental details significantly influence results. Thus we developed a novel model for renal IP using a hanging-weight system for isolated renal artery occlusion. In contrast to previous models, this technique eliminates the need for clamping the vascular pedicle (artery/vein). In fact, assessment of renal injury after different time periods of ischemia (10-60 min) revealed highly reproducible increases in plasma creatinine and potassium levels, while creatinine clearance, urinary flow and potassium/sodium excretion were significantly attenuated. Using different numbers of IP cycles, we found maximal protection with four cycles of 4 min of ischemia-reperfusion. In contrast, no significant renal protection was observed with IP of the vascular pedicle. To assess transcriptional responses in this model, we isolated RNA from preconditioned kidneys and found time-dependent induction of erythropoietin mRNA and plasma levels with IP. Taken together, this model provides highly reproducible renal injury and protection by IP, thus minimizing variability associated with previous techniques based on clamping of the renal pedicle. Further studies on renal ischemia/IP in mice may consider this technique.

Differential gene expression in periportal and perivenous mouse hepatocytes.

Hepatocytes located in the periportal and perivenous zones of the liver lobule show remarkable differences in the levels and activities of various enzymes and other proteins. To analyze global gene expression patterns of periportal and perivenous hepatocytes, enriched populations of the two cell types were isolated by combined collagenase/digitonin perfusion from mouse liver and used for microarray analysis. In total, 198 genes and expressed sequences were identified that demonstrated a >/= 2-fold difference in expression between hepatocytes from the two different zones of the liver. A subset of 20 genes was additionally analyzed by real-time RT-PCR, validating the results obtained by the microarray analysis. Several of the differentially expressed genes encoded key enzymes of intermediary metabolism, including those involved in glycolysis and gluconeogenesis, fatty acid degradation, cholesterol and bile acid metabolism, amino acid degradation and ammonia utilization. In addition, several enzymes of phase I and phase II of xenobiotic metabolism were differentially expressed in periportal and perivenous hepatocytes. Our results confirm previous findings on metabolic zonation in liver, and extend our knowledge of the regulatory mechanisms at the transcriptional level.

Rex3 (reduced in expression 3) as a new tumor marker in mouse hepatocarcinogenesis.

In a previous microarray expression analysis, Rex3, a gene formerly not linked to tumor formation, was found to be highly overexpressed in both Ctnnb1-(beta-Catenin) and Ha-ras-mutated mouse liver tumors. Subsequent analyses by in situ hybridization and real-time PCR confirmed a general liver tumor-specific overexpression of the gene (up to 400-fold). To investigate the role of Rex3 in liver tumors, hepatoma cells were transfected with FLAG- and Myc-tagged Rex3 expression vectors. Rex3 was shown to be exclusively localized to the cytoplasm, as determined by fluorescence microscopy and Western blotting. However, forced overexpression of Rex3 did not significantly affect proliferation or stress-induced apoptosis of transfected mouse hepatoma cells. Rex3 mRNA was determined in primary hepatocytes in culture by real-time PCR. In primary mouse hepatocytes, expression of Rex3 increased while cells dedifferentiated in culture. This effect was abolished when hepatocytes were maintained in a differentiated state. Furthermore, expression of Rex3 decreased in mouse liver with age of mice and the expression profile was highly correlated to that of the tumor markers alpha-fetoprotein and H19. The findings suggest a role of Rex3 as a marker for hepatocyte differentiation/dedifferentiation processes and tumor formation.

Role of renal nerves and salt intake on erythropoietin secretion in rats following carbon monoxide exposure.

Because the data from the literature contain conflicting results regarding the role of renal nerves and angiotensin II in hypoxia-induced erythropoietin (EPO) secretion, we evaluated the effect of renal nerves and salt intake in rats on EPO secretion stimulated by carbon monoxide (CO). Serum levels and renal mRNA content of EPO were similarly elevated by exposure to different CO concentrations in a dose-dependent manner in rats with bilateral renal denervation (DNX) and in sham-denervated controls (INN). However, at 600 ppm CO, serum concentrations and mRNA of EPO were significantly higher in DNX compared with INN rats (p < 0.05). This increase of EPO secretion in DNX rats could be blocked by administration of neuropeptide Y (NPY) (p < 0.05), whereas the NPY receptor antagonist did not enhance EPO secretion in INN rats after CO exposure. Agonists and antagonists of beta-adrenergic receptors had no effect on EPO secretion. High-salt (HS) diet reduced EPO secretory response at 600 ppm CO by 55% compared with INN rats on normal salt diet (p < 0.01). In addition, DNX increased EPO secretion in rats on low-salt and HS diet, whereas plasma renin activity did not correlate with EPO levels under these experimental conditions. In summary, our data suggest that renal nerves contribute to the half-maximal EPO secretory response to CO exposure, possibly via NPY receptors.

Activation of coupled Ah receptor and Nrf2 gene batteries by dietary phytochemicals in relation to chemoprevention.

The Ah receptor (AhR) is a ligand-activated transcription factor and member of the bHLH/PAS (basic helix-loop-helix/Per-Arnt-Sim) family of chemosensors and developmental regulators. It represents a multifunctional molecular switch involved in regulation of endo- and xenobiotic metabolism, in vascular development and in dioxin-mediated toxicities. Recently, the oxidative stress-protecting Nrf2 has been shown to be a downstream target of the AhR [Miao W, Hu L, Scrivens PJ, Batist G. Transcriptional regulation of NF-E2 p45-regulated factor (NRF2) expression by the aryl hydrocarbon receptor-xenobiotic response element signaling pathway. J Biol Chem 2005;280:20340-8]. This finding offers the possibility that distinct but partially overlapping AhR and Nrf2 gene batteries of Phase II xenobiotic-metabolizing enzymes can be synergistically activated by a number of phytochemicals, acting as selective or mixed activators of target genes. In addition, it is conceivable that AhR-mediated oxidative/electrophile stress may be attenuated by coupled Nrf2 activation. The commentary discusses potentials and limitations of (i) selective Nrf2 and of (ii) synergistic AhR plus Nrf2 activation by phytochemicals in efforts towards chemoprevention of cancer and degenerative diseases, and describes clinical trials providing the expectation that chemopreventive measures may favorably modulate unavoidable endo- and exogenous toxin exposures in high risk populations.