Timothy P. Dalton

Aromatic Hydrocarbon Receptor Interaction with the Retinoblastoma Protein Potentiates Repression of E2F-dependent Transcription and Cell Cycle Arrest

Polyhalogenated aromatic hydrocarbons, of which 2,3,7,8-tetrachloro-p-dioxin (TCDD) is the prototype compound, elicit a variety of toxic, teratogenic, and carcinogenic responses in exposed animals and in humans. In cultured cells, TCDD shows marked effects on the regulation of cell cycle progression, including thymocyte apoptosis, induction of keratinocyte proliferation and terminal differentiation, and inhibition of estrogen-dependent proliferation in breast cancer cells. The presence of an LXCXE domain in the dioxin aromatic hydrocarbon receptor (AHR), suggested that the effects of TCDD on cell cycle regulation might be mediated by protein-protein interactions between AHR and the retinoblastoma protein (RB). Using the yeast two-hybrid system, AHR and RB were in fact shown to bind to each other. In vitro pull-down experiments with truncated AHR peptides indicated that at least two separate AHR domains form independent complexes with hypophosphorylated RB. Coimmunoprecipitation of whole cell lysates from human breast carcinoma MCF-7 cells, which express both proteins endogenously, revealed that AHR associates with RB in vivo only after receptor transformation and nuclear translocation. However, the AHR nuclear translocator and transcriptional heterodimerization partner, is not required for (nor is it a part of) the AHR·RB complexes detected in vitro. Ectopic expression of AHR and RB in human osteosarcoma SAOS-2 cells, which lack endogenous expression of both proteins, showed that AHR synergizes with RB to repress E2F-dependent transcription and to induce cell cycle arrest. Furthermore, AHR partly blocked T-antigen-mediated reversal of RB-dependent transcriptional repression. These results uncover a potential function for the AHR in cell cycle regulation and suggest that this function may be that of serving as an environmental sensor that signals cell cycle arrest when cells are exposed to certain environmental toxicants.

Oxidative and electrophilic stress induces multidrug resistance–associated protein transporters via the nuclear factor‐E2–related factor‐2 transcriptional pathway

Multidrug resistance–associated proteins (Mrps) are adenosine triphosphate–dependent transporters that efflux chemicals out of cells. In the liver, Mrp2 transports bilirubin‐glucuronide, glutathione (GSH), and drug conjugates into bile, whereas Mrp3 and Mrp4 efflux these entities into blood. The purpose of this study was to determine whether oxidative conditions (that is, the disruption of hepatic GSH synthesis) or the administration of nuclear factor‐E2–related factor‐2 (Nrf2) activators (oltipraz and butylated hydroxyanisole) can induce hepatic Mrp transporters and whether that induction is through the Nrf2 transcriptional pathway. Livers from hepatocyte‐specific glutamate‐cysteine ligase catalytic subunit–null mice had increased nuclear Nrf2 levels, marked gene and protein induction of the Nrf2 target gene NAD(P)H:quinone oxidoreductase 1, as well as Mrp2, Mrp3, and Mrp4 expression. The treatment of wild‐type and Nrf2‐null mice with oltipraz and butylated hydroxyanisole demonstrated that the induction of Mrp2, Mrp3, and Mrp4 is Nrf2‐dependent. In Hepa1c1c7 cells treated with the Nrf2 activator tert‐butyl hydroquinone, chromatin immunoprecipitation with Nrf2 antibodies revealed the binding of Nrf2 to antioxidant response elements in the promoter regions of mouse Mrp2 [−185 base pairs (bp)], Mrp3 (−9919 bp), and Mrp4 (−3767 bp). Conclusion: The activation of the Nrf2 regulatory pathway stimulates the coordinated induction of hepatic Mrps. (HEPATOLOGY 2007.)

Novel n-3 Fatty Acid Oxidation Products Activate Nrf2 by Destabilizing the Association between Keap1 and Cullin3

Consumption of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can mitigate the progression of diseases in which oxidative stress represents a common underlying biochemical process. Nrf2-regulated gene expression regulates detoxification of reactive oxygen species. EPA and DHA were subjected to an in vitro free radical oxidation process that models in vivo conditions. Oxidized n-3 fatty acids reacted directly with the negative regulator of Nrf2, Keap1, initiating Keap1 dissociation with Cullin3, thereby inducing Nrf2-directed gene expression. Liquid chromatography-tandem mass spectrometry analyses of oxidized EPA demonstrated the presence of novel cyclopentenone-containing molecules termed J3-isoprostanes in vitro and in vivo and were shown to induce Nrf2-directed gene expression. These experiments provide a biochemical basis for the hypothesis that formation of J-ring compounds generated from oxidation of EPA and DHA in vivo can reach concentrations high enough to induce Nrf2-based cellular defense systems.

ZIP8, Member of the Solute-Carrier-39 (SLC39) Metal-Transporter Family: Characterization of Transporter Properties

Cadmium is a dangerous metal distributed widely in the environment. Members of our laboratory recently identified the ZIP8 transporter protein, encoded by the mouse Slc39a8 gene, to be responsible for genetic differences in response to cadmium damage of the testis. Stable retroviral infection of the ZIP8 cDNA in mouse fetal fibroblast cultures (rvZIP8 cells) leads to as much as a 10-fold increase in the rate of intracellular cadmium influx and accumulation. In the present study, we showed that cadmium uptake operated maximally at pH 7.5 and a temperature of 37°C and was inhibited by cyanide. Of more than a dozen cations tested, manganese(II) was the best competitive cation for cadmium uptake. The Km for Cd2+ uptake was 0.62 μM, and the Km for Mn2+ uptake was 2.2 μM; thus, manganese is probably the physiological substrate for ZIP8. Cadmium uptake was independent of sodium, potassium or chloride ions, but strongly dependent on the presence of bicarbonate. By Western blot analysis of rvZIP8 cells, we showed that ZIP8 protein was glycosylated. Using Z-stack confocal microscopy in Madin-Darby canine kidney polarized epithelial cells, we found that ZIP8 was localized on the apical side—implying an important role for manganese or cadmium uptake and disposition. It is likely that ZIP8 is a \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Mn}^{2+}{/}\mathrm{HCO}_{3}^{-}\) \end{document} symporter, that a \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} gradient across the plasma membrane acts as the driving force for manganese uptake, and that cadmium is a rogue hitchhiker displacing manganese to cause cadmium-associated disease.

Slc39a14 Gene Encodes ZIP14, A Metal/Bicarbonate Symporter: Similarities to the ZIP8 Transporter

The mouse and human genomes contain 14 highly conserved SLC39 genes. Viewed from an evolutionary perspective, SLC39A14 and SLC39A8 are the most closely related, each having three noncoding exons 1. However, SLC39A14 has two exons 4, giving rise to Zrt- and Irt-related protein (ZIP)ZIP14A and ZIP14B alternatively spliced products. C57BL/6J mouse ZIP14A expression is highest in liver, duodenum, kidney, and testis; ZIP14B expression is highest in liver, duodenum, brain, and testis; and ZIP8 is highest in lung, testis, and kidney. We studied ZIP14 stably retroviral-infected mouse fetal fibroblast cultures and transiently transfected Madin-Darby canine kidney (MDCK) polarized epithelial cells. Our findings include: 1) ZIP14-mediated cadmium uptake is proportional to cell toxicity, but manganese is not; 2) ZIP14B has a higher affinity than ZIP14A toward Cd2+ (Km = 0.14 versus 1.1 μM) and Mn2+ uptake (Km = 4.4 versus 18.2 μM); 3) ZIP14A- and ZIP14B-mediated Cd2+ uptake is most inhibited by Zn2+, and next by Mn2+ and Cu2+; 4) like ZIP8, ZIP14A- and ZIP14B-mediated Cd2+ uptake is dependent on extracellular \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document}; 5) like ZIP8, ZIP14 transporters are localized on the apical surface of MDCK-ZIP cells; and 6) like ZIP8, ZIP14 proteins are glycosylated. Tissues such as intestine and liver, located between the environment and the animal, show high levels of ZIP14; given the high affinity for ZIP14, Cd2+ is likely to act as a rogue hitchhiker—displacing Zn2+ or Mn2+ and entering the body to cause unwanted cell damage and disease.

Initial Characterization of the Glutamate-Cysteine Ligase Modifier Subunit Gclm(−/−) Knockout Mouse NOVEL MODEL SYSTEM FOR A SEVERELY COMPROMISED OXIDATIVE STRESS RESPONSE

Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the GSH biosynthesis pathway. In higher eukaryotes, this enzyme is a heterodimer comprising a catalytic subunit (GCLC) and a modifier subunit (GCLM), which change the catalytic characteristics of the holoenzyme. To define the cellular function of GCLM, we disrupted the mouse Gclm gene to create a null allele. Gclm(−/−) mice are viable and fertile and have no overt phenotype. In liver, lung, pancreas, erythrocytes, and plasma, however, GSH levels in Gclm(−/−) mice were 9–16% of that in Gclm(+/+) littermates. Cysteine levels inGclm(−/−) mice were 9, 35, and 40% of that inGclm(+/+) mice in kidney, pancreas, and plasma, respectively, but remained unchanged in the liver and erythrocytes. Comparing the hepatic GCL holoenzyme with GCLC in the genetic absence of GCLM, we found the latter had an ∼2-fold increase inK m for glutamate and a dramatically enhanced sensitivity to GSH inhibition. The major decrease in GSH, combined with diminished GCL activity, rendered Gclm(−/−) fetal fibroblasts strikingly more sensitive to chemical oxidants such as H2O2. We conclude that theGclm(−/−) mouse represents a model of chronic GSH depletion that will be very useful in evaluating the role of the GCLM subunit and GSH in numerous pathophysiological conditions as well as in environmental toxicity associated with oxidant insult.

Glutamate cysteine ligase catalysis: dependence on ATP and modifier subunit for regulation of tissue glutathione levels.

Glutamate cysteine ligase (GCL), which synthesizes γ-glutamyl-cysteine (γ-GC), is the rate-limiting enzyme in GSH biosynthesis. γ-GC may be produced by the catalytic subunit GCLC or by the holoenzyme (GCLholo), which comprises GCLC and the modifier subunit GCLM. The Gclm(–/–) knock-out mouse shows tissue levels of GSH that are between 9 and 40% of the Gclm(+/+) wild-type mouse. In the present study, we used recombinant GCLC and GCLM and Gclm(–/–) mice to examine the role of GCLM on γ-GC synthesis by GCLholo. GCLM decreased the Km for ATP by ∼6-fold and, similar to other species, decreased the Km for glutamate and increased the Ki for feedback inhibition by GSH. Furthermore, GCLM increased by 4.4-fold the Kcat for γ-GC synthesis; this difference in catalytic efficiency of GCLholo versus GCLC allowed us to derive a mathematical relationship for γ-GC production and to determine the relative levels of GCLholo and GCLC; in homogenates of brain, liver, and lung, the ratio of GCLC to GCLholo was 7.0, 2.0, and 3.5, respectively. In kidney, however, the relationship between GCLC and GCLholo was complicated. Kidney contains GCLholo, free GCLC, and free GCLM, and free GCLC in kidney cannot interact with GCLM. Taken together, we conclude that, in most tissues, GCLM is limiting, suggesting that an increase in GCLM alone would increase γ-GC synthesis. On the other hand, our results from kidney suggest that γ-GC synthesis may be controlled post-translationally.

Redox Dysregulation Affects the Ventral But Not Dorsal Hippocampus: Impairment of Parvalbumin Neurons, Gamma Oscillations, and Related Behaviors

Elevated oxidative stress and alteration in antioxidant systems, including glutathione (GSH) decrease, are observed in schizophrenia. Genetic and functional data indicate that impaired GSH synthesis represents a susceptibility factor for the disorder. Here, we show that a genetically compromised GSH synthesis affects the morphological and functional integrity of hippocampal parvalbumin-immunoreactive (PV-IR) interneurons, known to be affected in schizophrenia. A GSH deficit causes a selective decrease of PV-IR interneurons in CA3 and dendate gyrus (DG) of the ventral but not dorsal hippocampus and a concomitant reduction of β/γ oscillations. Impairment of PV-IR interneurons emerges at the end of adolescence/early adulthood as oxidative stress increases or cumulates selectively in CA3 and DG of the ventral hippocampus. Such redox dysregulation alters stress and emotion-related behaviors but leaves spatial abilities intact, indicating functional disruption of the ventral but not dorsal hippocampus. Thus, a GSH deficit affects PV-IR interneurons integrity and neuronal synchrony in a region- and time-specific manner, leading to behavioral phenotypes related to psychiatric disorders.

Induction of cellular oxidative stress by aryl hydrocarbon receptor activation

The aryl hydrocarbon receptor (AHR) has long been associated with the induction of a battery of genes involved in the metabolism of foreign and endogenous compounds. Depending on experimental conditions, AHR can mediate either activation or amelioration of chemical toxicity. For the past decade, evidence has mounted that AHR is associated with a cellular oxidative stress response that must be considered when evaluating the mechanism of action of xenobiotics capable of activating AHR, or capable of metabolic activation by enzymes encoded by genes under control of AHR. In this review, we have evaluated the diverse mechanisms by which AHR generates an oxidative stress response, including inflammation, antioxidant and prooxidant enzymes and cytochrome P450. A review of the regulation of Ahr transcription and functional polymorphisms especially related to oxidative stress is also included. We have carefully avoided placing a value judgment on the degree of toxicity produced by such a response, in view of the realization that an oxidative response is involved in many normal physiological processes. Since the interface between physiological, adaptive and toxicological responses elicited by the AHR-mediated oxidative stress response is not clearly defined, it behooves the researcher to evaluate both toxicological and physiological features of the response.

Mitochondrial reactive oxygen production is dependent on the aromatic hydrocarbon receptor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin; TCDD) is a pervasive environmental contaminant that induces hepatic and extrahepatic oxidative stress. We have previously shown that dioxin increases mitochondrial respiration-dependent reactive oxygen production. In the present study we examined the dependence of mitochondrial reactive oxygen production on the aromatic hydrocarbon receptor (AHR), cytochrome P450 1A1 (CYP1A1), and cytochrome P450 1A2 (CYP1A2), proteins believed to be important in dioxin-induced liver toxicity. Congenic Ahr(-/-), Cyp1a1(-/-) and Cyp1a2(-/-) knockout mice, and C57BL/6J inbred mice as their Ahr/Cyp1a1/Cyp1a2(+/+) wild-type (wt) counterparts, were injected intraperitoneally with dioxin (15 microg/kg body weight) or corn-oil vehicle on 3 consecutive days. Liver mitochondria were examined 1 week following the first treatment. The level of mitochondrial H(2)O(2) production in vehicle-treated Ahr(-/-) mice was one fifth that found in vehicle-treated wt mice. Whereas dioxin caused a rise in succinate-stimulated mitochondrial H(2)O(2) production in the wt, Cyp1a1(-/-), and Cyp1a2(-/-) mice, this increase did not occur with the Ahr(-/-) knockout. The lack of H(2)O(2) production in Ahr(-/-) mice was not due to low levels of Mn(2+)-superoxide dismutase (SOD2) as shown by Western immunoblot analysis, nor was it due to high levels of mitochondrial glutathione peroxidase (GPX1) activity. Dioxin decreased mitochondrial aconitase (an enzyme inactivated by superoxide) by 44% in wt mice, by 26% in Cyp1a2(-/-) mice, and by 24% in Cyp1a1(-/-) mice; no change was observed in Ahr(-/-) mice. Dioxin treatment increased mitochondrial glutathione levels in the wt, Cyp1a1(-/-), and Cyp1a2(-/-) mice, but not in Ahr(-/-) mice. These results suggest that both constitutive and dioxin-induced mitochondrial reactive oxygen production is associated with a function of the AHR, and these effects are independent of either CYP1A1 or CYP1A2.