Controversy over the role of antioxidants in cancer has persisted for decades. Here, we demonstrate that synthesis of the antioxidant glutathione (GSH), driven by GCLM, is required for cancer ...initiation. Genetic loss of Gclm prevents a tumor’s ability to drive malignant transformation. Intriguingly, these findings can be replicated using an inhibitor of GSH synthesis, but only if delivered prior to cancer onset, suggesting that at later stages of tumor progression GSH becomes dispensable potentially due to compensation from alternative antioxidant pathways. Remarkably, combined inhibition of GSH and thioredoxin antioxidant pathways leads to a synergistic cancer cell death in vitro and in vivo, demonstrating the importance of these two antioxidants to tumor progression and as potential targets for therapeutic intervention.
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•The GSH antioxidant pathway is required for cancer initiation•After cancer initiation, GSH is dispensable due to alternative antioxidant pathways•The TXN antioxidant pathway is upregulated in tumors•Inhibition of both GSH and TXN pathways causes synergistic cancer cell death
Harris et al. show that the antioxidant glutathione (GSH) is required for cancer initiation but not for established tumors partly due to upregulation of the thioredoxin (TXN) antioxidant pathway in the latter. Consequently, blocking both GSH and TXN pathways synergistically inhibits tumor growth.
MicroRNAs (miRNAs) are important regulators of gene expression and define part of the epigenetic signature. Their influence on every realm of biomedicine is established and progressively increasing. ...The impact of environment on human health is enormous. Among environmental risk factors impinging on quality of life are those of chemical nature (toxic chemicals, heavy metals, pollutants, and pesticides) as well as those related to everyday life such as exposure to noise or mental and psychosocial stress. Recent Advances: This review elaborates on the relationship between miRNAs and these environmental risk factors.
The most relevant facts underlying the role of miRNAs in the response to these environmental stressors, including redox regulatory changes and oxidative stress, are highlighted and discussed. In the cases wherein miRNA mutations are relevant for this response, the pertinent literature is also reviewed.
We conclude that, even though in some cases important advances have been made regarding close correlations between specific miRNAs and biological responses to environmental risk factors, a need for prospective large-cohort studies is likely necessary to establish causative roles. Antioxid. Redox Signal. 28, 773-796.
Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. The first and rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL, previously known as ...γ-glutamylcysteine synthetase). GCL is a heterodimeric protein composed of catalytic (GCLC) and modifier (GCLM) subunits that are expressed from different genes. GCLC catalyzes a unique γ-carboxyl linkage from glutamate to cysteine and requires ATP and Mg
++ as cofactors in this reaction. GCLM increases the
V
max and
K
cat of GCLC, decreases the
K
m for glutamate and ATP, and increases the
K
i for GSH-mediated feedback inhibition of GCL. While post-translational modifications of GCLC (e.g. phosphorylation, myristoylation, caspase-mediated cleavage) have modest effects on GCL activity, oxidative stress dramatically affects GCL holoenzyme formation and activity. Pyridine nucleotides can also modulate GCL activity in some species. Variability in GCL expression is associated with several disease phenotypes and transgenic mouse and rat models promise to be highly useful for investigating the relationships between GCL activity, GSH synthesis, and disease in humans.
Engineered nanomaterials (ENMs) have potential benefits, but they also present safety concerns for human health. Interlaboratory studies in rodents using standardized protocols are needed to assess ...ENM toxicity.
Four laboratories evaluated lung responses in C57BL/6 mice to ENMs delivered by oropharyngeal aspiration (OPA), and three labs evaluated Sprague-Dawley (SD) or Fisher 344 (F344) rats following intratracheal instillation (IT). ENMs tested included three forms of titanium dioxide (TiO2) anatase/rutile spheres (TiO2-P25), anatase spheres (TiO2-A), and anatase nanobelts (TiO2-NBs) and three forms of multiwalled carbon nanotubes (MWCNTs) original (O), purified (P), and carboxylic acid "functionalized" (F). One day after treatment, bronchoalveolar lavage fluid was collected to determine differential cell counts, lactate dehydrogenase (LDH), and protein. Lungs were fixed for histopathology. Responses were also examined at 7 days (TiO2 forms) and 21 days (MWCNTs) after treatment.
TiO2-A, TiO2-P25, and TiO2-NB caused significant neutrophilia in mice at 1 day in three of four labs. TiO2-NB caused neutrophilia in rats at 1 day in two of three labs, and TiO2-P25 and TiO2-A had no significant effect in any of the labs. Inflammation induced by TiO2 in mice and rats resolved by day 7. All MWCNT types caused neutrophilia at 1 day in three of four mouse labs and in all rat labs. Three of four labs observed similar histopathology to O-MWCNTs and TiO2-NBs in mice.
ENMs produced similar patterns of neutrophilia and pathology in rats and mice. Although interlaboratory variability was found in the degree of neutrophilia caused by the three types of TiO2 nanoparticles, similar findings of relative potency for the three types of MWCNTs were found across all laboratories, thus providing greater confidence in these interlaboratory comparisons.
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Dostopno za:
CEKLJ, DOBA, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
•Doxorubicin is a substrate of murine Cbr3.•Gclm−/− hepatocytes express higher levels of Cbr3 than Gclm+/+ hepatocytes.•More doxorubicinol is made by Gclm−/− hepatocytes than Gclm+/+ ...hepatocytes.•Gclm−/− hepatocytes sensitize co-cultured C2C12 cells to doxorubicin toxicity.
Doxorubicin is highly effective at inducing DNA double-strand breaks in rapidly dividing cells, which has led to it being a widely used cancer chemotherapeutic. However, clinical administration of doxorubicin is limited by off-target cardiotoxicity, which is thought to be mediated by doxorubicinol, the primary alcohol metabolite of doxorubicin. Carbonyl reductase 1 (CBR1), a well-characterized monomeric enzyme present at high basal levels in the liver, is known to exhibit activity toward doxorubicin. Little is known about a closely related enzyme, carbonyl reductase 3 (CBR3), which is present in the liver at low basal levels but is highly inducible by the transcription factor Nrf2. Genetic polymorphisms in CBR3, but not CBR1, are associated with differential cardiac outcomes in doxorubicin treated pediatric patients. Cbr3 mRNA and CBR3 protein are highly expressed in the livers of Gclm−/− mice (a mouse model of glutathione deficiency) relative to wild type mice. In the present study, we first investigated the ability of CBR3 to metabolize doxorubicin. Incubations of doxorubicin and purified recombinant murine CBR3 (mCBR3) were analyzed for doxorubicinol formation using HPLC, revealing for the first time that doxorubicin is a substrate of mCBR3. Moreover, hepatocytes from Gclm−/− mice produced more doxorubicinol than Gclm+/+ hepatocytes. In addition, differentiated rat myoblasts (C2C12 cells) co-cultured with primary Gclm−/− murine hepatocytes were more sensitive to doxorubicin-induced cytostasis/cytotoxicity than incubations with Gclm+/+ hepatocytes. Our results indicate a potentially important role for CBR3 in doxorubicin-induced cardiotoxicity. Because there is likely to be variability in hepatic CBR3 activity in humans (due to either genetic or epigenetic influences on its expression), these data also suggest that inhibition of CBR3 may provide protection from doxorubicinol cardiotoxicity.
Semiconductor quantum dots (Qdots) are a promising new technology with benefits in the areas of medical diagnostics and therapeutics. Qdots generally consist of a semiconductor core, capping shell, ...and surface coating. The semiconductor core of Qdots is often composed of group II and VI metals (e.g., Cd, Se, Te, Hg) that are known to have toxic properties. Various surface coatings have been shown to stabilize Qdots and thus shield cells from the toxic properties of their core elements. In this study, HepG2 cells and primary human liver (PHL) cells were chosen as in vitro tissue culture models of human liver to examine the possible adverse effects of tri-n-octylphosphine oxide, poly(maleic anhydride-alt-1-tetradecene) copolymer (TOPO-PMAT)-coated CdSe/ZnS Qdots (TOPO-PMAT Qdots). The TOPO-PMAT coating is desirable for increasing aqueous solubility and ease of conjugation to targeting moieties (e.g., aptamers and peptides). HepG2 cells avidly incorporated these TOPO-PMAT Qdots into subcellular vesicles. However, PHL cells did not efficiently take up TOPO-PMAT Qdots, but nonparenchymal cells did (especially Kupffer cells). No acute toxicity or morphological changes were noted in either system at the exposure levels used (up to 40 nM). However, cellular stress markers and pro-inflammatory cytokines/chemokines were increased in the PHL cell cultures, suggesting that TOPO-PMAT Qdots are not likely to cause acute cytotoxicity in the liver but may elicit inflammation/hepatitis, demonstrating the importance of relevant preclinical safety models. Thus, further in vivo studies are warranted to ensure that TOPO-PMAT-coated Qdots used in biomedical applications do not induce inflammatory responses as a consequence of hepatic uptake.
Over the past several years evidence has been accumulating from in vivo animal studies, observations in humans, and in vitro studies, that organophosphorus (OP) insecticides may induce oxidative ...stress. Such effects may contribute to some of the toxic manifestations of OPs, particularly upon chronic or developmental exposures. The aim of this study was to investigate the role of oxidative stress in the neurotoxicity of two commonly used OPs, chlorpyrifos (CPF) and diazinon (DZ), their oxygen analogs (CPO and DZO), and their “inactive” metabolites (TCP and IMP), in neuronal cells from a genetic model of glutathione deficiency. Cerebellar granule neurons from wild type mice (
Gclm +/+) and mice lacking the modifier subunit of glutamate cysteine ligase (
Gclm −/−), the first and limiting step in the synthesis of glutathione (GSH), were utilized. The latter display very low levels of GSH and are more susceptible to the toxicity of agents that increase oxidative stress. CPO and DZO were the most cytotoxic compounds, followed by CPF and DZ, while TCP and IMP displayed lower toxicity. Toxicity was significantly higher (10- to 25-fold) in neurons from
Gclm (−/−) mice, and was antagonized by various antioxidants. Depletion of GSH from
Gclm (+/+) neurons significantly increased their sensitivity to OP toxicity. OPs increased intracellular levels of reactive oxygen species and lipid peroxidation and in both cases the effects were greater in neurons from
Gclm (−/−) mice. OPs did not alter intracellular levels of GSH, but significantly increased those of oxidized glutathione (GSSG). Cytotoxicity was not antagonized by cholinergic antagonists, but was decreased by the calcium chelator BAPTA-AM. These studies indicate that cytotoxicity of OPs involves generation of reactive oxygen species and is modulated by intracellular GSH, and suggest that it may involve disturbances in intracellular homeostasis of calcium.
Antioxidant signaling/communication is among the most important cellular defense and survival pathways, and the importance of redox signaling and homeostasis in aging has been well-documented. ...Intracellular levels of glutathione (GSH), a very important endogenous antioxidant, both govern and are governed by the Nrf2 pathway through expression of genes involved in its biosynthesis, including the subunits of the rate-limiting enzyme (glutamate cysteine ligase, GCL) in GSH production, GCLC and GCLM. Mice homozygous null for the Gclm gene are severely deficient in GSH compared to wild-type controls, expressing approximately 10% of normal GSH levels. To compensate for GSH deficiency, Gclm null mice have upregulated redox-regulated genes, and, surprisingly, are less susceptible to certain types of oxidative damage. Furthermore, young Gclm null mice display an interesting lean phenotype, resistance to high fat diet-induced diabetes and obesity, improved insulin and glucose tolerance, and decreased expression of genes involved in lipogenesis. However, the persistence of this phenotype has not been investigated into old age, which is important in light of studies which suggest aging attenuates antioxidant signaling, particularly in response to exogenous stimuli. In this work, we addressed whether aging compromises the favorable phenotype of increased antioxidant activity and improved glucose homeostasis observed in younger Gclm null mice. We present data showing that under basal conditions and in response to cadmium exposure (2 mg/kg, dosed once via intraperitoneal injection), the phenotype previously described in young (<6 months) Gclm null mice persists into old age (24+ months). We also provide evidence that transcriptional activation of the Nrf2, AMPK, and PPARγ pathways underlie the favorable metabolic phenotype observed previously in young Gclm null mice.
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•Young Gclm-/- mice have lower body weight, more insulin sensitivity, and glucose tolerance relative to young Gclm+/+ mice.•The improved glucose homeostasis in young Gclm-/- mice under basal conditions and cadmium treatment are maintained in old age.•Gclm-/- mice have increased Nrf2-regulated genes and beta oxidation; and decreased gluconeogenesis and fatty acid synthesis.
Polycyclic aromatic hydrocarbons (PAH) such as benzoapyrene (BaP) are ubiquitous environmental pollutants found in tobacco smoke, air pollution, and grilled foods. Prenatal exposure to BaP causes ...premature reproductive senescence in mice, and other PAHs are transplacental ovarian carcinogens. Glutathione (GSH) is critical for detoxification of the reactive metabolites of PAHs. Therefore, we hypothesized that mice that are genetically deficient in GSH synthesis, due to deletion of the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have increased destruction of oogonia, premature ovarian failure, and ovarian tumorigenesis after transplacental BaP exposure compared with Gclm(+/+) females. Gclm(+/-) female and male mice were mated, and dams were treated with 0, 2, or 10 mg/kg/d BaP in sesame oil by gavage from gestational days 7 to 16. Compared with oil-treated F1 females of the same genotype, Gclm(-/-) prenatally BaP-treated females had significantly greater decrements in offspring production than Gclm(+/+) BaP-treated females. Similarly, we observed significant BaP dose × Gclm genotype interactions on ovarian follicle counts and ovarian tumor multiplicity at 7.5 months of age, with Gclm(-/-) females having greater decrements in follicle numbers and more ovarian tumors in response to prenatal BaP exposure than Gclm(+/+) females. The ovarian tumors were positive for the epithelial marker cytokeratin. Our results show that prenatal exposure of females to BaP causes premature ovarian failure and ovarian tumorigenesis and that embryonic GSH deficiency due to deletion of Gclm increases sensitivity to these transplacental ovarian effects of BaP.
The transcription factor Nrf2a induces a cellular antioxidant response and provides protection against chemical-induced oxidative stress, as well as playing a critical role in development and ...disease. Zebrafish are a powerful model to study the role of Nrf2a in these processes but have been limited by reliance on transient gene knockdown techniques or mutants with only partial functional alteration. We developed several lines of zebrafish carrying different null (loss of function, LOF) or hyperactive (gain of function, GOF) mutations to facilitate our understanding of the Nrf2a pathway in protecting against oxidative stress. The mutants confirmed Nrf2a dependence for induction of the antioxidant genes gclc, gstp, prdx1, and gpx1a and identified a role for Nrf2a in the baseline expression of these genes, as well as for sod1. Specifically, the 4-fold induction of gstp by tert-butyl hydroperoxide (tBHP) in wild type fish was abolished in LOF mutants. In addition, baseline gstp expression in GOF mutants increased by 12.6-fold and in LOF mutants was 0.8-fold relative to wild type. Nrf2a LOF mutants showed increased sensitivity to the acute toxicity of cumene hydroperoxide (CHP) and tBHP throughout the first 4 days of development. Conversely, GOF mutants were less sensitive to CHP toxicity during the first 4 days of development and were protected against the toxicity of both hydroperoxides after 4 dpf. Neither gain nor loss of Nrf2a modulated the toxicity of R-(−)-carvone (CAR), despite the ability of this compound to potently induce Nrf2a-dependent antioxidant genes. Similar to other species, GOF zebrafish mutants exhibited significant growth and survival defects. In summary, these new genetic tools can be used to facilitate the identification of downstream gene targets of Nrf2a, better define the role of Nrf2a in the toxicity of environmental chemicals, and further the study of diseases involving altered Nrf2a function.
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