Fatal Epileptic Seizures in Mice Having Compromised Glutathione and Ascorbic Acid Biosynthesis.

Reduced glutathione (GSH) and ascorbic acid (AA) are the two most abundant low-molecular-weight antioxidants in mammalian tissues. knockout mice lack the gene encoding the modifier subunit of the rate-limiting enzyme in GSH biosynthesis; mice exhibit 10-40% of normal tissue GSH levels and show no overt phenotype. knockout mice, lacking a functional gene encoding L-gulono-γ-lactone oxidase, cannot synthesize AA and depend on dietary ascorbic acid for survival.

Generation of a Slc39a8 hypomorph mouse: markedly decreased ZIP8 Zn²⁺/(HCO₃⁻)₂ transporter expression.

Previously this laboratory has identified the mouse Slc39a8 gene encoding the ZIP8 transporter, important in cadmium uptake. ZIP8 functions endogenously as a electroneutral Zn(2+)/(HCO(3)(-))(2) symporter, moving both ions into the cell. The overall physiological importance of ZIP8 remains unclear.

Oral N-acetylcysteine rescues lethality of hepatocyte-specific Gclc-knockout mice, providing a model for hepatic cirrhosis.

Background & Aims: Certain liver diseases have been associated with depletion of glutathione (GSH), the major antioxidant in the liver. A recent report about Gclc(h/h) mice with a hepatocyte-specific ablation of Gclc (the gene encoding the catalytic subunit of the rate-limiting enzyme in GSH synthesis) has shown an essential role of GSH in hepatic function. Gclc(h/h) mice develop severe steatosis and die of liver failure within one month, due to ~95% depletion of hepatic GSH; mitochondria are the major affected organelles, displaying abnormal ultrastructure and impaired functioning.

Glutathione deficient C57BL/6J mice are not sensitized to ozone-induced lung injury.

In this study we examined the role of the antioxidant glutathione (GSH) in pulmonary susceptibility to ozone toxicity, utilizing GSH deficient C57BL/6J mice that lack the expression of glutamate-cysteine ligase modifier subunit (GCLM). Gclm(-/-) knockout mice had 70% GSH depletion in the lung. Gclm(+/+) wild-type and Gclm(-/-) mice were exposed to either 0.

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.

Early onset senescence occurs when fibroblasts lack the glutamate-cysteine ligase modifier subunit.

Cellular senescence is the irreversible entry of cells into growth arrest. Senescence of primary cells in culture has long been used as an in vitro model for aging. Glutamate-cysteine ligase (GCL) controls the synthetic rate of the important cellular antioxidant glutathione (GSH).

Curcumin, quercetin, and tBHQ modulate glutathione levels in astrocytes and neurons: importance of the glutamate cysteine ligase modifier subunit.

A decrease in GSH levels, the main redox regulator, can be observed in neurodegenerative diseases as well as in schizophrenia. In search for substances able to increase GSH, we evaluated the ability of curcumin (polyphenol), quercetin (flavonoid), and tert-butylhydroquinone (tBHQ) to up-regulate GSH-synthesizing enzymes. The gene expression, activity, and product levels of these enzymes were measured in cultured neurons and astrocytes.

Knock-in mouse lines expressing either mitochondrial or microsomal CYP1A1: differing responses to dietary benzo[a]pyrene as proof of principle.

In the past, CYP1A1 protein was known to be located in the endoplasmic reticulum (ER; microsomes). More recently, CYP1A1 was shown also to be targeted to the inner mitochondrial membrane; mitochondrial import is dependent on NH(2)-terminal processing that exposes a cryptic targeting signal. It is interesting that microsomal and mitochondrial CYP1A1 enzymes exhibit different substrate specificities, electron donors, and inducer properties.

Induction of oxidative stress responses by dioxin and other ligands of the aryl hydrocarbon receptor.

TCDD and other polyhalogenated aromatic hydrocarbon ligands of the aryl hydrocarbon receptor (AHR) have been classically considered as non-genotoxic compounds because they fail to be directly mutagenic in either bacteria or most in vitro assay systems. They do so in spite of having repeatedly been linked to oxidative stress and to mutagenic and carcinogenic outcomes. Oxidative stress, on the other hand, has been used as a marker for the toxicity of dioxin and its congeners.

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.

Cd2+ versus Zn2+ uptake by the ZIP8 HCO3--dependent symporter: kinetics, electrogenicity and trafficking.

The mouse Slc39a8 gene encodes the ZIP8 transporter, which has been shown to be a divalent cation/HCO3- symporter. Using ZIP8 cRNA-injected Xenopus oocyte cultures, we show herein that: [a] ZIP8-mediated cadmium (Cd(2+)) and zinc (Zn(2+)) uptake have V(max) values of 1.8+/-0.

Basal and inducible CYP1 mRNA quantitation and protein localization throughout the mouse gastrointestinal tract.

The CYP1A1, CYP1A2, and CYP1B1 enzymes are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); metabolism of BaP by these enzymes leads to electrophilic intermediates and genotoxicity. Throughout the gastrointestinal (GI) tract, we systematically compared basal and inducible levels of the CYP1 mRNAs by Q-PCR, and localized the CYP1 proteins by immunohistochemistry. Cyp1(+/+) wild-type were compared with the Cyp1a1(-/-), Cyp1a2(-/-), and Cyp1b1(-/-) single-knockout and Cyp1a1/1b1(-/-) and Cyp1a2/1b1(-/-) double-knockout mice.

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

Unlabelled: 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.

Generation of 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(-/-) mouse line.

Human/rodent CYP1A1 and CYP1A2 orthologs are well known to exhibit species-specific differences in substrate preferences and rates of metabolism. This lab previously characterized a BAC-transgenic mouse carrying the human CYP1A1_CYP1A2 locus; in this line, human dioxin-inducible CYP1A1 and basal vs dioxin-inducible CYP1A2 have been shown to be expressed normally (with regard to mRNAs, proteins and three enzyme activities) in every one of nine mouse tissues studied. The mouse Cyp1a1 and Cyp1a2 genes are oriented head-to-head and share a bidirectional promoter region of 13,954 bp.

Interaction between the catalytic and modifier subunits of glutamate-cysteine ligase.

Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione (GSH) biosynthesis pathway. This enzyme is a heterodimer, comprising a catalytic subunit (GCLC) and a regulatory subunit (GCLM). Although GCLC alone can catalyze the formation of l-gamma-glutamyl-l-cysteine, its binding with GCLM enhances the enzyme activity by lowering the K(m) for glutamate and ATP, and increasing the K(i) for GSH inhibition.

Hepatocyte-specific Gclc deletion leads to rapid onset of steatosis with mitochondrial injury and liver failure.

Unlabelled: Oxidative stress is considered to be a critical mediator in liver injury of various etiologies. Depletion of glutathione (GSH), the major antioxidant in liver, has been associated with numerous liver diseases. To explore the specific role of hepatic GSH in vivo, we targeted Gclc, a gene essential for GSH synthesis, so that it was flanked by loxP sites and used the albumin-cyclization recombination (Alb-Cre) transgene to disrupt the Gclc gene specifically in hepatocytes.

7H-dibenzo[c,g]carbazole metabolism by the mouse and human CYP1 family of enzymes.

Found in tobacco smoke, fossil fuel and other organic combustion products, 7H-dibenzo[c,g]carbazole (DBC) is a potent mouse lung carcinogen and potential human carcinogen. Although the first hydroxylation is critical for determining activation versus detoxication, the enzymes responsible for site-specific hydroxylation of DBC are not known. We found that DBC-DNA adduct levels are significantly higher in aromatic hydrocarbon receptor null Ahr(-/-) mice, suggesting that the induction of Aromatic hydrocarbon receptor (AHR)-regulated genes, such as those in the CYP1 family, decrease DBC genotoxicity.

The role of cytochrome P450 enzymes in endogenous signalling pathways and environmental carcinogenesis.

Some cytochrome P450 (CYP) heme-thiolate enzymes participate in the detoxication and, paradoxically, the formation of reactive intermediates of thousands of chemicals that can damage DNA, as well as lipids and proteins. CYP expression can also affect the production of molecules derived from arachidonic acid, and alters various downstream signal-transduction pathways. Such changes can be precursors to malignancy.

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.

TCDD decreases ATP levels and increases reactive oxygen production through changes in mitochondrial F(0)F(1)-ATP synthase and ubiquinone.

Mitochondria generate ATP and participate in signal transduction and cellular pathology and/or cell death. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) decreases hepatic ATP levels and generates mitochondrial oxidative DNA damage, which is exacerbated by increasing mitochondrial glutathione redox state and by inner membrane hyperpolarization. This study identifies mitochondrial targets of TCDD that initiate and sustain reactive oxygen production and decreased ATP levels.

Enhanced cadmium-induced testicular necrosis and renal proximal tubule damage caused by gene-dose increase in a Slc39a8-transgenic mouse line.

Resistance to cadmium (Cd)-induced testicular necrosis is an autosomal recessive trait defined as the Cdm locus. Using positional cloning, we previously identified the Slc39a8 (encoding an apical-surface ZIP8 transporter protein) as the gene most likely responsible for the phenotype. In situ hybridization revealed that endothelial cells of the testis vasculature express high ZIP8 levels in two sensitive inbred mouse strains and negligible amounts in two resistant strains.

Naphthalene toxicity in mice and aryl hydrocarbon receptor-mediated CYPs.

Naphthalene (NP) has been designated a "reasonably anticipated human carcinogen" because of positive responses in carcinogenicity bioassays in rodents. Whereas CYP2F enzymes are widely regarded as responsible for NP bioactivation, other metabolic enzymes--including CYP1A1 and CYP1A2--produce NP-1,2-oxide in vitro. We investigated the role of these aryl hydrocarbon receptor (AHR)-mediated enzymes in NP toxicity in two ways.

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.

For dioxin-induced birth defects, mouse or human CYP1A2 in maternal liver protects whereas mouse CYP1A1 and CYP1B1 are inconsequential.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) induces cleft palate and hydronephrosis in mice, when exposed in utero; these effects are mediated by the aryl hydrocarbon receptor. The Cyp1a1, Cyp1a2, and Cyp1b1 genes are up-regulated by the aryl hydrocarbon receptor. To elucidate their roles in dioxin-induced teratogenesis, we compared Cyp1a1(-/-), Cyp1a2(-/-), and Cyp1b1(-/-) knock-out mice with Cyp1(+/+) wild-type mice.

Comparison of mouse hepatic mitochondrial versus microsomal cytochromes P450 following TCDD treatment.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) induces cytochromes P450 (CYPs) such as CYP1A1 and CYP1A2 via activation of the aromatic hydrocarbon receptor (AHR). Herein we describe the TCDD-dependent enrichment of CYPs in liver microsomes and mitoplasts from C57BL/6J mice. TCDD-induced accumulation of CYP1A1 and CYP1A2 was observed in microsomes and mitoplasts after treatment with 15 microg TCDD/kg/d for 3d.