MicroRNA Expression Influences Methylmercury-Induced Lipid Accumulation and Mitochondrial Toxicity in Caenorhabditis elegans.

Metabolic effects of methylmercury (MeHg) are gaining wider attention. We have previously shown that MeHg causes lipid dysregulation in Caenorhabditis elegans (C. elegans), leading to altered gene expression, increased triglyceride levels and lipid storage, and altered feeding behaviors. Transcriptional regulators, such as transcription factors and microRNAs (miRNAs), have been shown to regulate lipid storage, serum triglycerides, and adipogenic gene expression in human and rodent models of metabolic diseases. As we recently investigated adipogenic transcription factors induced by MeHg, we were, therefore, interested in whether MeHg may also regulate miRNA sequences to cause metabolic dysfunction. Lipid dysregulation, as measured by triglyceride levels, lipid storage sites, and feeding behaviors, was assessed in wild-type (N2) worms and in transgenic worms that either were sensitive to miRNA expression or were unable to process miRNAs. Worms that were sensitive to the miRNA expression were protected from MeHg-induced lipid dysregulation. In contrast, the mutant worms that were unable to process miRNAs had exacerbated MeHg-induced lipid dysregulation. Concurrent with differential lipid homeostasis, miRNA-expression mutants had altered MeHg-induced mitochondrial toxicity as compared to N2, with the miRNA-sensitive mutants showing mitochondrial protection and the miRNA-processing mutants showing increased mitotoxicity. Taken together, our data demonstrate that the expression of miRNAs is an important determinant in MeHg toxicity and MeHg-induced metabolic dysfunction in C. elegans.

Manganese Is Essential for Neuronal Health.

The understanding of manganese (Mn) biology, in particular its cellular regulation and role in neurological disease, is an area of expanding interest. Mn is an essential micronutrient that is required for the activity of a diverse set of enzymatic proteins (e.g., arginase and glutamine synthase). Although necessary for life, Mn is toxic in excess. Thus, maintaining appropriate levels of intracellular Mn is critical. Unlike other essential metals, cell-level homeostatic mechanisms of Mn have not been identified. In this review, we discuss common forms of Mn exposure, absorption, and transport via regulated uptake/exchange at the gut and blood-brain barrier and via biliary excretion. We present the current understanding of cellular uptake and efflux as well as subcellular storage and transport of Mn. In addition, we highlight the Mn-dependent and Mn-responsive pathways implicated in the growing evidence of its role in Parkinson's disease and Huntington's disease. We conclude with suggestions for future focuses of Mn health-related research.

Dopaminergic neurotoxicity of S-ethyl N,N-dipropylthiocarbamate (EPTC), molinate, and S-methyl-N,N-diethylthiocarbamate (MeDETC) in Caenorhabditis elegans.

Epidemiological studies corroborate a correlation between pesticide use and Parkinson's disease (PD). Thiocarbamate and dithiocarbamate pesticides are widely used and produce neurotoxicity in the peripheral nervous system. Recent evidence from rodent studies suggests that these compounds also cause dopaminergic (DAergic) dysfunction and altered protein processing, two hallmarks of PD. However, DAergic neurotoxicity has yet to be documented. We assessed DAergic dysfunction in Caenorhabditis elegans (C. elegans) to investigate the ability of thiocarbamate pesticides to induce DAergic neurodegeneration. Acute treatment with either S-ethyl N,N-dipropylthiocarbamate (EPTC), molinate, or a common reactive intermediate of dithiocarbamate and thiocarbamate metabolism, S-methyl-N,N-diethylthiocarbamate (MeDETC), to gradual loss of DAergic cell morphology and structure over the course of 6 days in worms expressing green fluorescent protein (GFP) under a DAergic cell specific promoter. HPLC analysis revealed decreased DA content in the worms immediately following exposure to MeDETC, EPTC, and molinate. In addition, worms treated with the three test compounds showed a drastic loss of DAergic-dependent behavior over a time course similar to changes in DAergic cell morphology. Alterations in the DAergic system were specific, as loss of cell structure and neurotransmitter content was not observed in cholinergic, glutamatergic, or GABAergic systems. Overall, our data suggest that thiocarbamate pesticides promote neurodegeneration and DAergic cell dysfunction in C. elegans, and may be an environmental risk factor for PD.

Involvement of AAT transporters in methylmercury toxicity in Caenorhabditis elegans.

Methylmercury (MeHg) is a potent neurotoxin that enters mammalian cells as a conjugate with L-cysteine through L-type large neutral amino acid transporter, LAT1, by a molecular mimicry mechanism by structurally resembling L-methionine. Caenorhabditis elegans (C. elegans) has been increasingly used to study the neurotoxic effects of MeHg, but little is known about uptake and transport of MeHg in the worm. This study examined whether MeHg uptake through LAT1 is evolutionarily conserved in nematodes. MeHg toxicity in C. elegans was blocked by pre-treatment of worms with l-methionine, suggesting a role for amino acid transporters in MeHg transport. Knockdown of aat-1, aat-2, and aat-3, worm homologues to LAT1, increased the survival of C. elegans following MeHg treatment and significantly attenuated MeHg content following exposure. These results indicate that MeHg is transported in the worm by a conserved mechanism dependent on functioning amino acid transporters.

The Role of skn-1 in methylmercury-induced latent dopaminergic neurodegeneration.

Mercury (Hg) is a persistent environmental bioaccumulative metal, with developmental exposure to methylmercury (MeHg) resulting in long-term health effects. We examined the impact of early-life exposure to MeHg and knockdown of skn-1 on dopaminergic (DAergic) neurodegeneration in the nematode Caenorhabditis elegans. SKN-1, a the major stress-activated cytoprotective transcription factors, promotes the transcription of enzymes that scavenge free radicals, synthesizes glutathione and catalyzes reactions that increase xenobiotic excretion. Deletions or mutations in this gene suppress stress resistance. Thus, we hypothesized that the extent of MeHg's toxicity is dependent on intact skn-1 response; therefore skn-1 knockout (KO) worms would show heightened sensitivity to MeHg-induced toxicity compared to wildtype worms. In this study we identified the impact of early-life MeHg exposure on Hg content, stress reactivity and DAergic neurodegeneration in wildtype, and skn-1KO C. elegans. Hg content, measured by Inductively Coupled Plasma Mass Spectrometry, showed no strain-dependent differences. Reactive oxygen species generation was dramatically increased in skn-1KO compared to wildtype worms. Structural integrity of DAergic neurons was microscopically assessed by visualization of fluorescently-labeled neurons, and revealed loss of neurons in skn-1KO and MeHg exposed worms compared to wildtype controls. Dopamine levels detected by High-performance liquid chromatography, were decreased in response to MeHg exposure and decreased in skn-1KO worms, and functional behavioral assays showed similar findings. Combined, these studies suggest that knockdown of skn-1 in the nematode increases DAergic sensitivity to MeHg exposure following a period of latency.

Expression Profiling of Adipogenic and Anti-Adipogenic MicroRNA Sequences following Methylmercury Exposure in Caenorhabditis elegans.

MicroRNA (miRNA) are important regulators of gene expression that respond not only to developmental and pathological cues, but also to environmental stimuli. Dyslipidemia is a hallmark of metabolic conditions and has been shown to significantly affect the expression of circulating miRNA sequences. Recently, our lab has shown that the environmental toxicant methylmercury (MeHg) causes dyslipidemia in the Caenorhabditis elegans model organism. While 10 and 20 µM MeHg increases the expression of adipogenic transcription factors and lipid-binding proteins in worms, there is limited information on how the toxicant affects the miRNA regulators of these genes. We hypothesized that MeHg would increase the expression of adipogenic miRNA sequences and/or decrease the expression of anti-adipogenic miRNA sequences. We further hypothesized that the target mRNA sequences for the miRNAs affected by MeHg would be consequently altered. We selected three potentially adipogenic (mir-34, mir-124, and mir-355) and three potentially anti-adipogenic (mir-240, mir-786, and let-7) miRNA sequences homologous to known human miRNA sequences altered in obesity, and quantified their levels 24 h and 48 h post MeHg treatment. At 24 h post exposure, MeHg significantly increased expression of both the adipogenic and anti-adipogenic miRNA sequences 1.5-3x above untreated control. By 48 h post exposure, only the adipogenic miRNA sequences were elevated, while the anti-adipogenic miRNA sequences were decreased by 50% compared to untreated control. These data suggest that there are developmental changes in miRNA expression over time following MeHg exposure. We next selected one target mRNA sequence for each miRNA sequence based on miRNA-mRNA relationships observed in humans. MeHg altered the gene expression of all the target genes assayed. Except for mir-34, all the tested miRNA-mRNA sequences showed a conserved relationship between nematode and humans. To determine whether the selected miRNA sequences were involved in lipid accumulation in response to MeHg, lipid storage was investigated in transgenic worm strains that lacked the specific miRNA strains. Of the six strains investigated, only the mir-124 and let-7 mutant worms had lipid storage levels that were statistically different from wild type, suggesting that these two sequences can be potential mediators of MeHg-induced lipid dysregulation.

Association between Heavy Metals, Metalloids and Metabolic Syndrome: New Insights and Approaches.

Metabolic syndrome (MetS) is an important public health issue that affects millions of people around the world and is growing to pandemic-like proportions. This syndrome is defined by the World Health Organization (WHO) as a pathologic condition characterized by abdominal obesity, insulin resistance, hypertension, and hyperlipidemia. Moreover, the etiology of MetS is multifactorial, involving many environmental factors, including toxicant exposures. Several studies have associated MetS with heavy metals exposure, which is the focus of this review. Environmental and/or occupational exposure to heavy metals are a major risk, contributing to the development of chronic diseases. Of particular note, toxic metals such as mercury, lead, and cadmium may contribute to the development of MetS by altering oxidative stress, IL-6 signaling, apoptosis, altered lipoprotein metabolism, fluid shear stress and atherosclerosis, and other mechanisms. In this review, we discuss the known and potential roles of heavy metals in MetS etiology as well as potential targeted pathways that are associated with MetS. Furthermore, we describe how new approaches involving proteomic and transcriptome analysis, as well as bioinformatic tools, may help bring about an understanding of the involvement of heavy metals and metalloids in MetS.

Electrophilic adduction of ubiquitin activating enzyme E1 by N,N-diethyldithiocarbamate inhibits ubiquitin activation and is accompanied by striatal injury in the rat.

Previous studies have shown ubiquitin activating enzyme E1 to be sensitive to adduction through both Michael addition and SN(2) chemistry in vitro. E1 presents a biologically important putative protein target for adduction due to its role in initiating ubiquitin based protein processing and the involvement of impaired ubiquitin protein processing in two types of familial Parkinson's disease. We tested whether E1 is susceptible to xenobiotic-mediated electrophilic adduction in vivo and explored the potential contribution of E1 adduction to neurodegenerative events in an animal model. N,N-Diethyldithiocarbamate (DEDC) was administered to rats using a protocol that produces covalent cysteine modifications in vivo, and brain E1 protein adducts were characterized and mapped using shotgun LC-MS/MS. E1 activity, global and specific protein expression, and protein carbonyls were used to characterize cellular responses and injury in whole brain and dorsal striatal samples. The data demonstrate that DEDC treatment produced S-(ethylaminocarbonyl) adducts on Cys234 and Cys179 residues of E1 and decreased the levels of activated E1 and total ubiquitinated proteins. Proteomic analysis of whole brain samples identified expression changes for proteins involved in myelin structure, antioxidant response, and catechol metabolism, systems often disrupted in neurodegenerative disease. Our studies also delineated localized injury within the striatum as indicated by decreased levels of tyrosine hydroxylase, elevated protein carbonyl content, increased antioxidant enzyme and α-synuclein expression, and enhanced phosphorylation of tau and tyrosine hydroxylase. These data are consistent with E1 having similar susceptibility to adduction in vivo as previously reported in vitro and support further investigation into environmental agent adduction of E1 as a potential contributing factor to neurodegenerative disease. Additionally, this study supports the predictive value of in vitro screens for identifying sensitive protein targets that can be used to guide subsequent in vivo experiments.

SIRT1 is a redox-sensitive deacetylase that is post-translationally modified by oxidants and carbonyl stress.

Sirtuin1 (SIRT1) deacetylase levels are decreased in chronic inflammatory conditions and aging where oxidative stress occurs. We determined the mechanism of SIRT1 redox post-translational modifications leading to its degradation. Human lung epithelial cells exposed to hydrogen peroxide (150-250 microM), aldehyde-acrolein (10-30 microM), and cigarette smoke extract (CSE; 0.1-1.5%) in the presence of intracellular glutathione-modulating agents at 1-24 h, and oxidative post-translational modifications were assayed in cells, as well as in lungs of mice lacking and overexpressing glutaredoxin-1 (Glrx1), and wild-type (WT) mice in response to cigarette smoke (CS). CSE and aldehydes dose and time dependently decreased SIRT1 protein levels, with EC(50) of 1% for CSE and 30 microM for acrolein at 6 h, and >80% inhibition at 24 h with CSE, which was regulated by modulation of intracellular thiol status of the cells. CS decreased the lung levels of SIRT1 in WT mice, which was enhanced by deficiency of Glrx1 and prevented by overexpression of Glrx1. Oxidants, aldehydes, and CS induced carbonyl modifications on SIRT1 on cysteine residues concomitant with decreased SIRT1 activity. Proteomics studies revealed alkylation of cysteine residue on SIRT1. Our data suggest that oxidants/aldehydes covalently modify SIRT1, decreasing enzymatic activity and marking the protein for proteasomal degradation, which has implications in inflammatory conditions.

Methylmercury-Induced Metabolic Alterations in Caenorhabditis elegans Are Diet-Dependent.

Methylmercury (MeHg) is a well-known neurotoxicant; however, its role in metabolic diseases has been gaining wider attention. Chronic exposure to MeHg in human populations shows an association with diabetes mellitus and metabolic syndrome (MS). As the incidences of both obesity and MS are on the rise globally, it is important to understand the potential role of MeHg in the development of the disease. There is a dearth of information on dietary interactions between MeHg and lipids, which play an important role in developing MS. We have previously shown that MeHg increases food seeking behaviors, lipid levels, fat storage, and pro-adipogenic gene expression in C. elegans fed the standard OP50 Escherichia coli diet. However, we hypothesized that these metabolic changes could be prevented if the worms were fed a bacterial diet lower in lipid content. We tested whether C. elegans developed metabolic alterations in response to MeHg if they were fed two alternative E. coli strains (HT115 and HB101) that are known absorb significantly less lipids from their media. Additionally, to explore the effect of a high-lipid and high-cholesterol diet on MeHg-induced metabolic dysfunction, we supplemented the OP50 strain with twice the standard concentration of cholesterol in the nematode growth media. Wild-type worms fed either the HB101 or HT115 diet were more resistant to MeHg than the worms fed the OP50 diet, showing a significant right-hand shift in the dose-response survival curve. Worms fed the OP50 diet supplemented with cholesterol were more sensitive to MeHg, showing a significant left-hand shift in the dose-response survival curve. Changes in sensitivity to MeHg by differential diet were not due to altered MeHg intake in the worms as measured by inductively coupled mass spectrometry. Worms fed the low-fat diets showed protection from MeHg-induced metabolic changes, including decreased food consumption, lower triglyceride content, and lower fat storage than the worms fed either of the higher-fat diets. Oxidative stress is a common characteristic of both MeHg exposure and high-fat diets. Worms fed either OP50 or OP50 supplemented with cholesterol and treated with MeHg had significantly higher levels of reactive oxygen species, carbonylated proteins, and loss of glutathione than the worms fed the HT115 or HB101 low-lipid diets. Taken together, our data suggest a synergistic effect of MeHg and dietary lipid levels on MeHg toxicity and fat metabolism in C. elegans, which may affect the ability of MeHg to cause metabolic dysfunction.

SIRT1 regulates oxidant- and cigarette smoke-induced eNOS acetylation in endothelial cells: Role of resveratrol.

Endothelial nitric oxide synthase (eNOS) plays a crucial role in endothelial cell functions. SIRT1, a NAD(+)-dependent deacetylase, is shown to regulate endothelial function and hence any alteration in endothelial SIRT1 will affect normal vascular physiology. Cigarette smoke (CS)-mediated oxidative stress is implicated in endothelial dysfunction. However, the role of SIRT1 in regulation of eNOS by CS and oxidants are not known. We hypothesized that CS-mediated oxidative stress downregulates SIRT1 leading to acetylation of eNOS which results in reduced nitric oxide (NO)-mediated signaling and endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) exposed to cigarette smoke extract (CSE) and H(2)O(2) showed decreased SIRT1 levels, activity, but increased phosphorylation concomitant with increased eNOS acetylation. Pre-treatment of endothelial cells with resveratrol significantly attenuated the CSE- and oxidant-mediated SIRT1 levels and eNOS acetylation. These findings suggest that CS- and oxidant-mediated reduction of SIRT1 is associated with acetylation of eNOS which have implications in endothelial dysfunction.

PARP-1 inhibition does not restore oxidant-mediated reduction in SIRT1 activity.

Sirtuin1 (SIRT1) deacetylase and poly(ADP-ribose)-polymerase-1 (PARP-1) respond to environmental cues, and both require NAD(+) cofactor for their enzymatic activities. However, the functional link between environmental/oxidative stress-mediated activation of PARP-1 and SIRT1 through NAD(+) cofactor availability is not known. We investigated whether NAD(+) depletion by PARP-1 activation plays a role in environmental stimuli/oxidant-induced reduction in SIRT1 activity. Both H(2)O(2) and cigarette smoke (CS) decreased intracellular NAD(+) levels in vitro in lung epithelial cells and in vivo in lungs of mice exposed to CS. Pharmacological PARP-1 inhibition prevented oxidant-induced NAD(+) loss and attenuated loss of SIRT1 activity. Oxidants decreased SIRT1 activity in lung epithelial cells; however increasing cellular NAD(+) cofactor levels by PARP-1 inhibition or NAD(+) precursors was unable to restore SIRT1 activity. SIRT1 was found to be carbonylated by CS, which was not reversed by PARP-1 inhibition or selective SIRT1 activator. Overall, these data suggest that environmental/oxidant stress-induced SIRT1 down-regulation and PARP-1 activation are independent events despite both enzymes sharing the same cofactor.

Nrf2 deficiency influences susceptibility to steroid resistance via HDAC2 reduction.

Abnormal lung inflammation and oxidant burden are associated with a significant reduction in histone deacetylase 2 (HDAC2) abundance and steroid resistance. We hypothesized that Nrf2 regulates steroid sensitivity via HDAC2 in response to inflammation in mouse lung. Furthermore, HDAC2 deficiency leads to steroid resistance in attenuating lung inflammatory response, which may be due to oxidant/antioxidant imbalance. Loss of antioxidant transcription factor Nrf2 resulted in decreased HDAC2 level in lung, and increased inflammatory lung response which was not reversed by steroid. Thus, steroid resistance or inability of steroids to control lung inflammatory response is dependent on Nrf2-HDAC2 axis. These findings have implications in steroid resistance, particularly during the conditions of oxidative stress when the lungs are more susceptible to inflammatory response, which is seen in patients with chronic obstructive pulmonary disease, asthma, rheumatoid arthritis, and inflammatory bowel disease.

Regulation of SIRT1 in cellular functions: role of polyphenols.

Sirtuin 1 (SIRT1) is known to deacetylate histones and non-histone proteins including transcription factors thereby regulating metabolism, stress resistance, cellular survival, cellular senescence/aging, inflammation-immune function, endothelial functions, and circadian rhythms. Naturally occurring dietary polyphenols, such as resveratrol, curcumin, quercetin, and catechins, have antioxidant and anti-inflammatory properties via modulating different pathways, such as NF-kappaB- and mitogen activated protein kinase-dependent signaling pathways. In addition, these polyphenols have also been shown to activate SIRT1 directly or indirectly in a variety of models. Therefore, activation of SIRT1 by polyphenols is beneficial for regulation of calorie restriction, oxidative stress, inflammation, cellular senescence, autophagy/apoptosis, autoimmunity, metabolism, adipogenesis, circadian rhythm, skeletal muscle function, mitochondria biogenesis and endothelial dysfunction. In this review, we describe the regulation of SIRT1 by dietary polyphenols in various cellular functions in response to environmental and pro-inflammatory stimuli.

Methylmercury Induces Metabolic Alterations in Caenorhabditis elegans: Role for C/EBP Transcription Factor.

Methylmercury (MeHg) is a well-known neurotoxicant; however, its role in metabolic diseases has been gaining wider attention. We have previously shown that MeHg causes metabolic alterations in Caenorhabditis elegans, leading to decreased nicotinamide adenine dinucleotide cofactor, mitochondrial dysfunction, and oxidative stress. We were, therefore, interested in whether MeHg also affects nutrient metabolism, particularly lipid homeostasis, which may contribute to the development of metabolic conditions such as obesity or metabolic syndrome (MS). RNA from wild-type worms exposed to MeHg was collected immediately after treatment and used for gene expression analysis by DNA microarray. MeHg differentially regulated 215 genes, 17 genes involved in lipid homeostasis, and 12 genes involved in carbohydrate homeostasis. Of particular interest was cebp-1, the worm ortholog to human C/EBP, a pro-adipogenic transcription factor implicated in MS. MeHg increased the expression of cebp-1 as well as pro-adipogenic transcription factors sbp-1 and nhr-49, triglyceride synthesis enzyme acl-6, and lipid transport proteins vit-2 and vit-6. Concurrent with the altered gene expression, MeHg increased triglyceride levels, lipid storage, and feeding behaviors. Worms expressing mutant cebp-1 were protected from MeHg-induced alterations in lipid content, feeding behaviors, and gene expression, highlighting the importance of this transcription factor in the worm's response to MeHg. Taken together, our data demonstrate that MeHg induces biochemical, metabolic, and behavioral changes in C. elegans that can lead to metabolic dysfunction.

Genetic ablation of NADPH oxidase enhances susceptibility to cigarette smoke-induced lung inflammation and emphysema in mice.

Cigarette smoke (CS) induces recruitment of inflammatory cells in the lungs leading to the generation of reactive oxygen species (ROS), which are involved in lung inflammation and injury. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a multimeric system that is responsible for ROS production in mammalian cells. We hypothesized that NADPH oxidase-derived ROS play an important role in lung inflammation and injury and that targeted ablation of components of NADPH oxidase (p47(phox) and gp91(phox)) would protect lungs against the detrimental effects of CS. To test this hypothesis, we exposed p47(phox-/-) and gp91(phox-/-) mice to CS and examined inflammatory response and injury in the lung. Surprisingly, although CS-induced ROS production was decreased in the lungs of p47(phox-/-) and gp91(phox-/-) mice compared with wild-type mice, the inflammatory response was significantly increased and was accompanied by development of distal airspace enlargement and alveolar destruction. This pathological abnormality was associated with enhanced activation of the TLR4-nuclear factor-kappaB pathway in response to CS exposure in p47(phox-/-) and gp91(phox-/-) mice. This phenomenon was confirmed by in vitro studies in which treatment of peritoneal macrophages with a nuclear factor-kappaB inhibitor reversed the CS-induced release of proinflammatory mediators. Thus, these data suggest that genetic ablation of components of NADPH oxidase enhances susceptibility to the proinflammatory effects of CS leading to airspace enlargement and alveolar damage.

VEGFR-2 inhibition augments cigarette smoke-induced oxidative stress and inflammatory responses leading to endothelial dysfunction.

Vascular endothelial growth factor (VEGF) induces phosphorylation of VEGF receptor-2 (VEGFR-2) and activates the downstream signaling pathway resulting in endothelial cell migration, proliferation, and survival. Cigarette smoking is associated with abnormal vascular and endothelial function, leading to airspace enlargement. Herein, we investigated the mechanism of cigarette smoke (CS) -induced endothelial dysfunction by studying the VEGF-VEGFR-2 signaling in mouse lung and human endothelial cells. CS exposure caused oxidative stress, as shown by increased levels of 4-hydroxy-2-nonenal-adducts in mouse lung and reactive oxygen species generation in human lung microvascular endothelial cells (HMVEC-Ls). Inhibition of VEGFR-2 by a specific kinase inhibitor (NVP-AAD777) enhanced the CS-induced oxidative stress, causing augmented inflammatory cell influx and proinflammatory mediators release in mouse lung. The levels of endothelial nitric oxide synthase (eNOS) and phosphorylated (p) -eNOS in the lungs of mice exposed to CS and/or treated with VEGFR-2 inhibitor were decreased. CS down-regulated VEGFR-2 expression, eNOS levels, and VEGF-induced VEGFR-2 phosphorylation in HMVEC-Ls, resulting in impaired VEGF-induced endothelial cell migration and angiogenesis. Overall, these data show that inhibition of VEGFR-2 augmented CS-induced oxidative stress and inflammatory responses leading to endothelial dysfunction. This explains the mechanism of endothelial dysfunction in smokers and has implications in understanding the pathogenesis of pulmonary and cardiovascular diseases.

Variation in the biological half-life of methylmercury in humans: Methods, measurements and meaning.

BACKGROUND: Understanding methylmercury (MeHg) toxicity requires a complete understanding of its fundamental toxicokinetic and toxicodynamic characteristics in the human body. The biological half-life (t1/2) of MeHg is a kinetic property that directly influences the body burden of Hg that results from repeated exposures such as can occur with fish and seafood consumption. The t1/2 of MeHg in humans is approximately 50 days, equivalent to an elimination rate (kel) of 0.014 day-1. However, numerous studies report a wide range of half-life values (t1/2 < 30 to >120 days), demonstrating that significant variation in the biological process of MeHg elimination exists. This variation is a source of considerable uncertainty in deriving a meaningful reference dose for MeHg applicable to all individuals in a population. SCOPE OF REVIEW: First, we summarize fundamentals of MeHg toxicokinetics, emphasizing the central role that biological half-life plays in MeHg dosimetry. We next present important considerations for how kinetic analyses are performed. We provide an example of how MeHg half-life variation directly influences the body burden and, in certain contexts, can result in MeHg levels exceeding the US EPA Reference Dose. We then survey existing studies that report MeHg half-life determinations in people. MAJOR CONCLUSIONS: Recent advances in methods of determining MeHg kinetics in people have made individualized assessment of MeHg elimination rates more accurate and readily obtainable. GENERAL SIGNIFICANCE: Characterization of MeHg half-life, particularly in vulnerable individuals, such as pregnant women and children, will diminish the remaining toxicokinetic uncertainty surrounding MeHg exposures and will better inform the risk assessment process.

Disruption of p21 attenuates lung inflammation induced by cigarette smoke, LPS, and fMLP in mice.

The cyclin-dependent kinase inhibitor p21(CIP1/WAF1/SDI1) (p21) is an important inhibitory checkpoint regulator of cell cycle progression in response to oxidative and genotoxic stresses. It is known that p21 potentiates inflammatory response and inhibits apoptosis and proliferation, leading to cellular senescence. However, the role of endogenous p21 in regulation of lung inflammatory and injurious responses by cigarette smoke (CS) or other pro-inflammatory stimuli is not known. We hypothesized that p21 is an important modifier of lung inflammation and injury, and genetic ablation of p21 will confer protection against CS and other pro-inflammatory stimuli (lipopolysacchride [LPS] and N-formyl-methionyl-leucyl-phenylalanine [fMLP])-mediated lung inflammation and injury. To test this hypothesis, p21-deficient (p21-/-) and wild-type mice were exposed to CS, LPS, or fMLP, and the lung oxidative stress and inflammatory responses as well as airspace enlargement were assessed. We found that targeted disruption of p21 attenuated CS-, LPS-, or fMLP-mediated lung inflammatory responses in mice. CS-mediated oxidative stress and fMLP-induced airspace enlargement were also decreased in lungs of p21-/- mice compared with wild-type mice. The mechanism underlying this finding was associated with decreased NF-kappaB activation, and reactive oxygen species generation by decreased phosphorylation of p47(phox) and down-modulating the activation of p21-activated kinase. Our data provide insight into the mechanism of pro-inflammatory effect of p21, and the loss of p21 protects against lung oxidative and inflammatory responses, and airspace enlargement in response to multiple pro-inflammatory stimuli. These data may have ramifications in CS-induced senescence in the pathogenesis of chronic obstructive pulmonary disease/emphysema.

IKK alpha causes chromatin modification on pro-inflammatory genes by cigarette smoke in mouse lung.

Cigarette smoke (CS) induces abnormal and sustained lung inflammation; however, the molecular mechanism underlying sustained inflammation is not known. It is well known that activation of I kappaB kinase beta (IKK beta) leads to transient translocation of active NF-kappaB (RelA/p65-p50) in the nucleus and transcription of pro-inflammatory genes, whereas the role of IKK alpha in perpetuation of sustained inflammatory response is not known. We hypothesized that CS activates IKK alpha and causes histone acetylation on the promoters of pro-inflammatory genes, leading to sustained transcription of pro-inflammatory mediators in mouse lung in vivo and in human monocyte/macrophage cell line (MonoMac6) in vitro. CS exposure to C57BL/6J mice resulted in activation of IKK alpha, leading to phosphorylation of ser10 and acetylation of lys9 on histone H3 on the promoters of IL-6 and MIP-2 genes in mouse lung. The increased level of IKK alpha was associated with increased acetylation of lys310 RelA/p65 on pro-inflammatory gene promoters. The role of IKK alpha in CS-induced chromatin modification was confirmed by gain and loss of IKK alpha in MonoMac6 cells. Overexpression of IKK alpha was associated with augmentation of CS-induced pro-inflammatory effects, and phosphorylation of ser10 and acetylation of lys9 on histone H3, whereas transfection of IKK alpha dominant-negative mutants reduced CS-induced chromatin modification and pro-inflammatory cytokine release. Moreover, phosphorylation of ser276 and acetylation of lys310 of RelA/p65 was augmented in response to CS extract in MonoMac6 cells transfected with IKK alpha. Taken together, these data suggest that IKK alpha plays a key role in CS-induced pro-inflammatory gene transcription through phospho-acetylation of both RelA/p65 and histone H3.