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Compromise of skeletal muscle metabolism and composition may underlie the etiology of cardiovascular and metabolic disease risk from environmental arsenic exposures. We reported that arsenic impairs muscle maintenance and regeneration by inducing maladaptive mitochondrial phenotypes in muscle stem cells (MuSC), connective tissue fibroblasts (CTF), and myofibers. We also found that arsenic imparts a dysfunctional memory in the extracellular matrix (ECM) that disrupts the MuSC niche and is sufficient to favor the expansion and differentiation of fibrogenic MuSC subpopulations. To investigate the signaling mechanisms involved in imparting a dysfunctional ECM, we isolated skeletal muscle tissue and CTF from mice exposed to 0 or 100 μg/l arsenic in their drinking water for 5 weeks. ECM elaborated by arsenic-exposed CTF decreased myogenesis and increased fibrogenic/adipogenic MuSC subpopulations and differentiation. However, treating arsenic-exposed mice with SS-31, a mitochondrially targeted peptide that repairs the respiratory chain, reversed the arsenic-promoted CTF phenotype to one that elaborated an ECM supporting normal myogenic differentiation. SS-31 treatment also reversed arsenic-induced Notch1 expression, resulting in an improved muscle regeneration after injury. We found that persistent arsenic-induced CTF Notch1 expression caused the elaboration of dysfunctional ECM with increased expression of the Notch ligand DLL4. This DLL4 in the ECM was responsible for misdirecting MuSC myogenic differentiation. These data indicate that arsenic impairs muscle maintenance and regenerative capacity by targeting CTF mitochondria and mitochondrially directed expression of dysfunctional regulators in the stem cell niche. Therapies that restore muscle cell mitochondria may effectively treat arsenic-induced skeletal muscle dysfunction and compositional decline.

The impact of foodborne metals on the burden of disease has been largely overlooked, in comparison to the attention on acute diseases associated with infectious foodborne agents. Four articles in this special section describe in detail the burden of disease from foodborne lead, methylmercury, arsenic, and cadmium. Ingested lead and methylmercury are causally associated with lifelong intellectual disability. Long term ingestion of arsenic is causally associated with an increased risk of cancer. Long term ingestion of cadmium is causally associated with an increased risk of late stage chronic kidney disease. This article presents an overview of the burden of disease from these four foodborne metals and discusses them in the context of the World Health Organization's initiative to estimate the global burden of foodborne disease. The results indicate that in 2015, ingestion of arsenic, methylmercury, lead, and cadmium resulted in more than 1 million illnesses, over 56,000 deaths, and more than 9 million disability-adjusted life years (DALYs) worldwide. The greatest impact on DALYs was in the Western Pacific B subregion. All of the metals were found to have high DALYs per case in comparison with other foodborne disease agents, including infectious and parasitic agents. In addition, lead, arsenic, and methylmercury were found to have high DALYs per 100,000 population in comparison to other foodborne disease agents.

Arsenic is a ubiquitous, naturally occurring metalloid that poses a significant risk for human cancer and non-cancer diseases. It is now evident that arsenic contamination in food, especially rice and grains, presents a significant exposure to hundreds of millions of individuals worldwide. However, the disease risk from chronic exposure to the low amounts of arsenic found in food remains to be established. Thus, this research estimates the global burdens of disease expressed as Disability-Adjusted Life Years (DALYs) for lung, skin and bladder cancers, as well as coronary heart disease (CHD) attributable to inorganic arsenic in food. To determine foodborne inorganic arsenic exposures worldwide, we used the World Health Organization (WHO) estimates of food consumption in 17 country clusters, in conjunction with the reported measurements of total and inorganic arsenic in different foods. We estimated cancer potency factors for arsenic related bladder and lung cancers, and from US Environmental Protection Agency risk estimates for skin cancer to calculate the cancer incidence in males and females within each of the WHO member states. Summary relative risk estimates and population attributable fractions were developed to estimate the YLD, YLL, and DALYs for arsenic-induced CHD. The findings indicate that, globally, each year the combined DALYs for all cancers attributable to inorganic arsenic in food are approximately 1.4 million with variation in global distribution based on population and food consumption patterns. The global burden of CHD attributable to foodborne inorganic arsenic also varied with WHO region and may contribute as much as 49 million DALYs. However, in contrast to cancer burden, there is a threshold effect for arsenic-associated CHD with no increased risk of heart disease at the expected lower bound of arsenic consumption in food. These estimates indicate that foodborne arsenic exposure causes a significant yet avoidable global burden of human disease.

Aging is accompanied by disrupted information flow, resulting from accumulation of molecular mistakes. These mistakes ultimately give rise to debilitating disorders including skeletal muscle wasting, or sarcopenia. To derive a global metric of growing ‘disorderliness’ of aging muscle, we employed a statistical physics approach to estimate the state parameter, entropy, as a function of genes associated with hallmarks of aging. Escalating network entropy reached an inflection point at old age, while structural and functional alterations progressed into oldest-old age. To probe the potential for restoration of molecular ‘order’ and reversal of the sarcopenic phenotype, we systemically overexpressed the longevity protein, Klotho, via AAV. Klotho overexpression modulated genes representing all hallmarks of aging in old and oldest-old mice, but pathway enrichment revealed directions of changes were, for many genes, age-dependent. Functional improvements were also age-dependent. Klotho improved strength in old mice, but failed to induce benefits beyond the entropic tipping point.

Aging is accompanied by a disrupted information flow, which results from accumulation of molecular mistakes. These mistakes ultimately give rise to debilitating disorders such as skeletal muscle wasting, or sarcopenia. To estimate the growing “disorderliness” of the aging muscle system, we employed a statistical physics approach to estimate the state parameter, entropy, as a function of genes associated with hallmarks of aging. Although the most prominent structural and functional alterations were observed in the oldest old mice (27-29 months), we found that the escalating network entropy reached an inflection point at old age (22-24 months). To probe the potential for restoration of molecular “order” and reversal of the sarcopenic phenotype, we overexpressed the longevity protein, α-Klotho. Klotho overexpression modulated genes representing all hallmarks of aging in both old and oldest-old mice. However, whereas Klotho improved strength in old mice, intervention failed to induce a benefit beyond the entropic tipping point.

Arsenic exposure impairs muscle metabolism, maintenance, progenitor cell differentiation, and regeneration following acute injury. Low to moderate arsenic exposures target muscle fiber and progenitor cell mitochondria to epigenetically decrease muscle quality and regeneration. However, the mechanisms for how low levels of arsenic signal for prolonged mitochondrial dysfunction are not known. In this study, arsenic attenuated murine C2C12 myoblasts differentiation and resulted in abnormal undifferentiated myoblast proliferation. Arsenic prolonged ligand-independent phosphorylation of mitochondrially localized epidermal growth factor receptor (EGFR), a major driver of proliferation. Treating cells with a selective EGFR kinase inhibitor, AG-1478, prevented arsenic inhibition of myoblast differentiation. AG-1478 decreased arsenic-induced colocalization of pY845EGFR with mitochondrial cytochrome C oxidase subunit II, as well as arsenic-enhanced mitochondrial membrane potential, reactive oxygen species generation, and cell cycling. All of the arsenic effects on mitochondrial signaling and cell fate were mitigated or reversed by addition of mitochondrially targeted agents that restored mitochondrial integrity and function. Thus, arsenic-driven pathogenesis in skeletal muscle requires sustained mitochondrial EGFR activation that promotes progenitor cell cycling and proliferation at the detriment of proper differentiation. Collectively, these findings suggest that the arsenic-activated mitochondrial EGFR pathway drives pathogenic signaling for impaired myoblast metabolism and function.

Skeletal muscle mass reduction has been implicated in insulin resistance (IR) that promotes cardiometabolic diseases. We have previously reported that arsenic exposure increases IR concomitantly with the reduction of skeletal muscle mass among individuals exposed to arsenic. The arsenic methylation capacity is linked to the susceptibility to some arsenic exposure-related diseases. However, it remains unknown whether the arsenic methylation capacity affects the arsenic-induced reduction of muscle mass and elevation of IR. Therefore, this study examined the associations between the arsenic methylation status and skeletal muscle mass measures with regard to IR by recruiting 437 participants from low- and high-arsenic exposure areas in Bangladesh. The subjects’ skeletal muscle mass was estimated by their lean body mass (LBM) and serum creatinine levels. Subjects’ drinking water arsenic concentrations were positively associated with total urinary arsenic concentrations and the percentages of MMA, as well as inversely associated with the percentages of DMA and the secondary methylation index (SMI). Subjects’ LBM and serum creatinine levels were positively associated with the percentage of DMA and SMI, as well as inversely associated with the percentage of MMA. HOMA-IR showed an inverse association with SMI, with a confounding effect of sex. Our results suggest that reduced secondary methylation capacity is involved in the arsenic-induced skeletal muscle loss that may be implicated in arsenic-induced IR and cardiometabolic diseases.

Summary Age‐related declines in skeletal muscle regeneration have been attributed to muscle stem cell (MuSC) dysfunction. Aged MuSCs display a fibrogenic conversion, leading to fibrosis and impaired recovery after injury. Although studies have demonstrated the influence of in vitro substrate characteristics on stem cell fate, whether and how aging of the extracellular matrix (ECM) affects stem cell behavior has not been investigated. Here, we investigated the direct effect of the aged muscle ECM on MuSC lineage specification. Quantification of ECM topology and muscle mechanical properties reveals decreased collagen tortuosity and muscle stiffening with increasing age. Age‐related ECM alterations directly disrupt MuSC responses, and MuSCs seeded ex vivo onto decellularized ECM constructs derived from aged muscle display increased expression of fibrogenic markers and decreased myogenicity, compared to MuSCs seeded onto young ECM. This fibrogenic conversion is recapitulated in vitro when MuSCs are seeded directly onto matrices elaborated by aged fibroblasts. When compared to young fibroblasts, fibroblasts isolated from aged muscle display increased nuclear levels of the mechanosensors, Yes‐associated protein (YAP)/transcriptional coactivator with PDZ‐binding motif (TAZ), consistent with exposure to a stiff microenvironment in vivo. Accordingly, preconditioning of young fibroblasts by seeding them onto a substrate engineered to mimic the stiffness of aged muscle increases YAP/TAZ nuclear translocation and promotes secretion of a matrix that favors MuSC fibrogenesis. The findings here suggest that an age‐related increase in muscle stiffness drives YAP/TAZ‐mediated pathogenic expression of matricellular proteins by fibroblasts, ultimately disrupting MuSC fate.

The year 2017 marked the 20th anniversary of the first publication describing Klotho. This single protein was and is remarkable in that its absence in mice conferred an accelerated aging, or progeroid, phenotype with a dramatically shortened life span. On the other hand, genetic overexpression extended both health span and life span by an impressive 30%. Not only has Klotho deficiency been linked to a number of debilitating age-related illnesses but many subsequent reports have lent credence to the idea that Klotho can compress the period of morbidity and extend the life span of both model organisms and humans. This suggests that Klotho functions as an integrator of organ systems, making it both a promising tool for advancing our understanding of the biology of aging and an intriguing target for interventional studies. In this review, we highlight advances in our understanding of Klotho as well as key challenges that have somewhat limited our view, and thus translational potential, of this potent protein.

Metals are both essential for cardiovascular health and among the oldest known cardiovascular toxicants. Recent findings, such as large prospective epidemiological studies or finding that metal chelation therapy reducing cardiovascular events in cardiovascular disease (CVD) patients, strengthen the link between metal exposures and disease promotion. However, pathogenic mechanisms for metals promoting human CVD remain poorly understood and are often controversial due to difficulty in interpreting and translating results from high dose animal toxicity studies to causal disease etiologies in humans. There is increasing appreciation that lower, chronic environmental exposures, exposures occurring in critical developmental windows, or exposures at occupational and therapeutic levels may have subtler effects in causing or modifying CVD. Recent epidemiological studies and studies in genetic rodent models that focus on environmental exposures indicate that pathogenic effects of metals on cell signaling and epigenetic regulation are not random and cannot be attributed to competition with endogenous ions or excessive oxidant-mediated injury. Resolution of the molecular understanding of pathogenic metal effects remains a challenge complicated by complex differential concentration and temporal exposure relationships, as well as genetic determinants. This chapter presents pathogenic effects of metals in the heart and vasculature with an emphasis on the cellular and molecular actions underlying CVD promotion and clinically significant toxicities.

Background : Amyotrophic lateral sclerosis (ALS) is a serious and rapidly fatal neurodegenerative disorder with an annual incidence of 1–2.6/100,000 persons. Few known risk factors exist although gene–environment interaction is suspected. We investigated the relationship between suspected neurotoxicant hazardous air pollutants (HAPs) exposure and ALS. Methods : A case–control study involving sporadic ALS cases ( n = 51) and matched controls ( n = 51) was conducted from 2008 to 2011. Geocoded residential addresses were linked to U.S. EPA NATA data (1999, 2002, and 2005) by census tract. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using conditional logistic regression. Results : Residential exposure to aromatic solvents significantly elevated the risk of ALS among cases compared to controls in 2002 (OR = 5.03, 95% CI: 1.29, 19.53) and 1999 (OR = 4.27, 95% CI: 1.09, 16.79) following adjustment for education, smoking, and other exposure groups. Metals, pesticides, and other HAPs were not associated with ALS. Conclusions : A potential relationship is suggested between residential ambient air aromatic solvent exposure and risk of ALS in this study.

Arsenic is a global health hazard that impacts over 140 million individuals worldwide. Epidemiological studies reveal prominent muscle dysfunction and mobility declines following arsenic exposure; yet, mechanisms underlying such declines are unknown. The objective of this study was to test the novel hypothesis that arsenic drives a maladaptive fibroblast phenotype to promote pathogenic myomatrix remodeling and compromise the muscle stem (satellite) cell (MuSC) niche. Mice were exposed to environmentally relevant levels of arsenic in drinking water before receiving a local muscle injury. Arsenic-exposed muscles displayed pathogenic matrix remodeling, defective myofiber regeneration and impaired functional recovery, relative to controls. When naïve human MuSCs were seeded onto three-dimensional decellularized muscle constructs derived from arsenic-exposed muscles, cells displayed an increased fibrogenic conversion and decreased myogenicity, compared with cells seeded onto control constructs. Consistent with myomatrix alterations, fibroblasts isolated from arsenic-exposed muscle displayed sustained expression of matrix remodeling genes, the majority of which were mediated by NF-κB. Inhibition of NF-κB during arsenic exposure preserved normal myofiber structure and functional recovery after injury, suggesting that NF-κB signaling serves as an important mechanism of action for the deleterious effects of arsenic on tissue healing. Taken together, the results from this study implicate myomatrix biophysical and/or biochemical characteristics as culprits in arsenic-induced MuSC dysfunction and impaired muscle regeneration. It is anticipated that these findings may aid in the development of strategies to prevent or revert the effects of arsenic on tissue healing and, more broadly, provide insight into the influence of the native myomatrix on stem cell behavior. Video Highlight: https://youtu.be/v1E7yGKdCLM

Over 4 million individuals in the United States, and over 140 million individuals worldwide, are exposed daily to arsenic-contaminated drinking water. Human exposures can range from below the current limit of 10 μg/L to over 1mg/L, with 100 μg/L promoting disease in a large portion of those exposed. Although increased attention has recently been paid to myopathy following arsenic exposure, the pathogenic mechanisms underlying clinical symptoms remain poorly understood. This study tested the hypothesis that arsenic induces lasting muscle mitochondrial dysfunction and impairs metabolism. Compared to nonexposed controls, mice exposed to drinking water containing 100 μg/L arsenite for 5 weeks demonstrated impaired muscle function, mitochondrial myopathy, and altered oxygen consumption that were concomitant with increased mitochondrial fusion gene transcription. There were no differences in the levels of inorganic arsenic or its monomethyl and dimethyl metabolites between controls and exposed muscles, confirming that arsenic does not accumulate in muscle. Nevertheless, muscle progenitor cells isolated from exposed mice recapitulated the aberrant myofiber phenotype and were more resistant to oxidative stress, generated more reactive oxygen species, and displayed autophagic mitochondrial morphology, compared to cells isolated from nonexposed mice. These pathological changes from a possible maladaptive oxidative stress response provide insight into declines in muscle functioning caused by exposure to this common environmental contaminant.

Dysfunctional lipid and glucose metabolism contribute to metabolic syndrome-a major public health concern that enhances cardiovascular disease risk. Arsenic (As(III)) exposure may increase metabolic syndrome and cardiovascular disease risk by impairing adipose tissue differentiation, function, and insulin sensitivity through pathogenic mechanisms that remain unclear. We hypothesized that As(III) signals through the Pertussis toxin (Ptx) sensitive, Gi protein-coupled receptor (GPCR) to impair adipogenesis, as previously demonstrated for its stimulation of vascular oxidant generation, angiogenesis, and remodeling. Because both As(III) and GPCR ligands inhibit progenitor cell differentiation into adipocytes, we investigated the hypothesis in a model of low-passage human mesenchymal stem cells (hMSC). As(III) (0.1-1.0 µM) suppressed dexamethasone/insulin-induced hMSC adipogenesis, as indicated by decreased transcriptional promoters of differentiation, decreased fat droplet formation, and decreased expression of differentiated adipocyte markers, such as adiponectin and perilipin. Preincubating hMSC with Ptx prevented 90% of the suppressive effect of As(III). Selective competitive antagonists of Gi-coupled endothelin-1 type A and B receptors were ~60% effective in blocking As(III) inhibition and combination of antagonists to both receptors were 85% effective. In contrast, antagonists to the sphingosine-1-phosphate type 1 receptor (previously shown to mediate As(III) vascular effects) or the angiotensin II type 1 receptor were ineffective in blocking As(III) effects. These studies suggest a majority of arsenic-inhibited adipocyte differentiation, and metabolism requires endothelin-1 GPCRs and that As(III) effects on GPCR signaling are tissue and context specific. This may represent a significant mechanism for the contribution of arsenic exposure to increased metabolic and cardiovascular diseases.

Chronic arsenic exposure is a worldwide health problem. Although arsenic-induced cancer has been widely studied, comparatively little attention has been paid to arsenic-induced vascular disease. Epidemiological studies have shown that chronic arsenic exposure is associated with increased morbidity and mortality from cardiovascular disease. In addition, studies suggest that susceptibility to arsenic-induced vascular disease may be modified by nutritional factors in addition to genetic factors. Recently, animal models for arsenic-induced atherosclerosis and liver sinusoidal endothelial cell dysfunction have been developed. Initial studies in these models show that arsenic exposure accelerates and exacerbates atherosclerosis in apolipoprotein E–knockout mice. Microarray studies of liver mRNA and micro-RNA abundance in mice exposed in utero suggest that a permanent state of stress is induced by the arsenic exposure. Furthermore, the livers of the arsenic-exposed mice have activated pathways involved in immune responses suggesting a pro-hyperinflammatory state. Arsenic exposure of mice after weaning shows a clear dose-response in the extent of disease exacerbation. In addition, increased inflammation in arterial wall is evident. In response to arsenic-stimulated oxidative signaling, liver sinusoidal endothelium differentiates into a continuous endothelium that limits nutrient exchange and waste elimination. Data suggest that nicotinamide adenine dinucleotide phosphate oxidase–derived superoxide or its derivatives are essential second messengers in the signaling pathway for arsenic-stimulated vessel remodeling. The recent findings provide future directions for research into the cardiovascular effects of arsenic exposure.

Carbon nanotubes (CNT), with their applications in industry and medicine, may lead to new risks to human health. CNT induce a robust pulmonary inflammation and oxidative stress in rodents. Realistic exposures to CNT may occur in conjunction with other pathogenic impacts (microbial infections) and trigger enhanced responses. We evaluated interactions between pharyngeal aspiration of single-walled CNT (SWCNT) and bacterial pulmonary infection of C57BL/6 mice with Listeria monocytogenes (LM). Mice were given SWCNT (0, 10, and 40 mug/mouse) and 3 days later were exposed to LM (10(3) bacteria/mouse). Sequential exposure to SWCNT/LM amplified lung inflammation and collagen formation. Despite this robust inflammatory response, SWCNT pre-exposure significantly decreased the pulmonary clearance of LM-exposed mice measured 3 to 7 days after microbial infection versus PBS/LM-treated mice. Decreased bacterial clearance in SWCNT-pre-exposed mice was associated with decreased phagocytosis of bacteria by macrophages and a decrease in nitric oxide production by these phagocytes. Pre-incubation of naïve alveolar macrophages with SWCNT in vitro also resulted in decreased nitric oxide generation and suppressed phagocytizing activity toward LM. Failure of SWCNT-exposed mice to clear LM led to a continued elevation in nearly all major chemokines and acute phase cytokines into the later course of infection. In SWCNT/LM-exposed mice, bronchoalveolar lavage neutrophils, alveolar macrophages, and lymphocytes, as well as lactate dehydrogenase level, were increased compared with mice exposed to SWCNT or LM alone. In conclusion, enhanced acute inflammation and pulmonary injury with delayed bacterial clearance after SWCNT exposure may lead to increased susceptibility to lung infection in exposed populations.

Vascular system function involves complex interactions among the vascular endothelium, smooth muscle, the immune system, and the nervous system. The toxic metals cadmium (Cd), arsenic (As), and lead (Pb) can target the vascular system in a variety of ways, ranging from hemorrhagic injury to subtle pathogenic remodeling and metabolic changes. Acute Cd exposure results in hemorrhagic injury to the testis, although some strains of animals are resistant to this effect. A comparison of Cd-sensitive with Cd-resistant mouse strains showed that expression of the Slc39a8 gene, encoding the ZIP8 transporter, in the testis vasculature endothelium is responsible for this difference. Endogenously, ZIP8 is a Mn(2+)/HCO(3)(-)symporter that may also contribute to Cd damage in the kidney. Chronic Cd exposure is associated with various cardiovascular disorders such as hypertension and cardiomyopathy and it is reported to have both carcinogenic and anticarcinogenic activities. At noncytotoxic concentrations of 10-100nM, Cd can inhibit chemotaxis and tube formation of vascular endothelial cells. These angiostatic effects may be mediated through disruption of vascular endothelial cadherin, a Ca(2+)-dependent cell adhesion molecule. With regard to As, ingestion of water containing disease-promoting concentrations of As promotes capillarization of the liver sinusoidal endothelium. Because capillarization is a hallmark precursor for liver fibrosis and contributes to an imbalance of lipid metabolism, this As effect on hepatic endothelial cells may be a pathogenic mechanism underlying As-related vascular diseases. With regard to Pb, perinatal exposure may cause sustained elevations in adult blood pressure, and genetically susceptible animals may show enhanced sensitivity to this effect. Taken together, these data indicate that the vascular system is a critical target of metal toxicity and that actions of metals on the vascular system may play important roles in mediating the pathophysiologic effects of metals in specific target organs.

The vascular effects of arsenic in drinking water are global health concerns contributing to human disease worldwide. Arsenic targets the endothelial cells lining blood vessels, and endothelial cell activation or dysfunction may underlie the pathogenesis of both arsenic-induced vascular diseases and arsenic-enhanced tumorigenesis. The purpose of the current studies was to demonstrate that exposing mice to drinking water containing environmentally relevant levels of arsenic promoted endothelial cell dysfunction and pathologic vascular remodeling. Increased angiogenesis, neovascularization, and inflammatory cell infiltration were observed in Matrigel plugs implanted in C57BL/6 mice following 5-week exposures to 5-500 ppb arsenic [Soucy, N.V., Mayka, D., Klei, L.R., Nemec, A.A., Bauer, J.A., Barchowsky, A., 2005. Neovascularization and angiogenic gene expression following chronic arsenic exposure in mice. Cardiovasc.Toxicol 5, 29-42]. Therefore, functional in vivo effects of arsenic on endothelial cell function and vessel remodeling in an endogenous vascular bed were investigated in the liver. Liver sinusoidal endothelial cells (LSEC) became progressively defenestrated and underwent capillarization to decrease vessel porosity following exposure to 250 ppb arsenic for 2 weeks. Sinusoidal expression of PECAM-1 and laminin-1 proteins, a hallmark of capillarization, was also increased by 2 weeks of exposure. LSEC caveolin-1 protein and caveolae expression were induced after 2 weeks of exposure indicating a compensatory change. Likewise, CD45/CD68-positive inflammatory cells did not accumulate in the livers until after LSEC porosity was decreased, indicating that inflammation is a consequence and not a cause of the arsenic-induced LSEC phenotype. The data demonstrate that the liver vasculature is an early target of pathogenic arsenic effects and that the mouse liver vasculature is a sensitive model for investigating vascular health effects of arsenic.

The vascular effects of arsenic are a global public health concern that contribute to disease in tens of millions of people worldwide.1 Whereas the role of environmental contaminants in the etiology of vascular diseases and in the vascular contributions to organ dysfunction remains poorly defined, epidemiological studies have associated As(III) exposures to increased risk of cardiovascular diseases1 and vascular contributions to liver disease.2 Liver effects associated with arsenic in drinking water include noncirrhotic portal fibrosis and, to a lesser extent, portal hypertension.2,3 These pathologic conditions involve increased vascular channels in the portal regions of the liver. Higher levels of chronic As(III) consumption increase urinary levels of porphyrins, a biomarker for liver injury, which are more pronounced in people under 20 years of age.4 In addition, cardiac and liver disorders are the major side effects of therapeutic As(III) regimes that treat leukemias.5 Despite epidemiological evidence that the liver vasculature is a pathogenic target of chronic As(III) ingestion,2 the direct effects of As(III) on liver vascular cells are unknown. In other vascular beds and isolated cell cultures, As(III) affects both endothelial and smooth muscle cell physiology. Arsenic(III) stimulates angiogenic processes in cultured endothelial cells and neovascularization in intact mouse and avian models.6–9 These angiogenic effects promote tumorigenesis in mice, as well as vascular remodeling.10,11 Stimulation of endothelial cell proliferation occurs at concentrations of As(III) that are not cytotoxic, whereas higher concentrations are cytotoxic and inhibit angiogenesis in tumors.10,11 Arsenic(III) stimulates cultured smooth muscle cells to proliferate and express vascular endothelial cell growth factor (VEGF), a primary mediator of angiogenesis.12 Although these effects of As(III) on cells from systemic blood vessels are well recognized, there is little data on the effects of As(III) exposures on liver vascular remodeling or specifically on the fenestrated sinusoidal endothelium. The liver sinusoidal endothelial cells (SECs) are highly specialized fenestrated cells that are unique in the extensive heterogeneity of vascular endothelium.13 Early in development, these cells differentiate to lose markers of a continuous endothelium, such as junctional expression of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) and a basement membrane containing the matrix protein laminin-1.14 In the differentiation process, the SECs become fenestrated to allow sieving of circulating nutrients, lipids, and lipoproteins for normal liver metabolism.13 The SEC angiogenic process is different from angiogenesis in endothelial cells of systemic vessels, because there is no increase in sinusoidal vessel number or density. Instead, SEC angiogenesis is a dedifferentiation and maturation process called capillarization with diagnostic hallmarks of SEC defenestration and renewed surface expression of PECAM-1 and laminin-1 proteins.13,15–18 SEC defenestration and formation of tight intercellular junctions limits transendothelial cell transport.13,16,19,20 Capillarization precedes vascular remodeling of other liver vessels, such as hepatic arterioles and PBVP, causing blood flow shunting, vascular channel formation, and eventually liver fibrosis.15,16,21 Liver angiogenesis in general is recognized as an important pathogenic process not only in portal fibrosis, but also in portal hypertension and progression of HCCs.18,22–25 Finally, animal models have been used to demonstrate that liver capillarization affects the systemic vasculature by decreasing liver metabolism of lipids, lipoproteins, and glucose to promote atherogenesis in response to environmental stresses and aging.13,26,27 In mouse tumors or surrogate in vivo neovascularization assays, low to moderate levels of As(III) (5–250 ppb) in drinking water are angiogenic.7,28 However, As(III) effects on the liver endothelium or pathogenic angiogenic processes in the liver are unknown. The aim of this study was to investigate whether a chronic, human-relevant, environmental exposure to As(III) causes SEC dedifferentiation and dysfunctional capillarization in intact mice. The data indicate that prolonged As(III) exposure causes SEC maturation and sustained functional changes in SEC cell signaling.

Hyperoxia is cytotoxic and depresses many cellular metabolic functions including protein synthesis. Translational control is exerted primarily during initiation by two mechanisms: 1) through inhibition of translation initiation complex formation via sequestration of the cap-binding protein, eukaryotic initiation factor (eIF) 4E, with inhibitory 4E-binding proteins (4E-BP); and 2) by prevention of eIF2-GTP-tRNA[Formula: see text] formation and eIF2B activity by phosphorylated eIF2α. In this report, exposure of human lung fibroblasts to 95% O2decreased the incorporation of thymidine into DNA at 6 h and the incorporation of leucine into protein beginning at 12 h. The reductions in DNA and protein synthesis were accompanied by increased phosphorylation of eIF4E protein and reduced phosphorylation of 4E-BP1. At 24 h, hyperoxia shifted 4E-BP1 phosphorylation to lesser-phosphorylated isoforms, increased eIF4E expression, and increased the association of eIF4E with 4E-BP1. Although hyperoxia did not change eIF2α expression, it increased its phosphorylation at Ser51, but not until 48 h. In addition, the activation of eIF2α was not accompanied by the formation of stress granules. These findings suggest that hyperoxia diminishes protein synthesis by increasing eIF4E phosphorylation and enhancing the affinity of 4E-BP1 for eIF4E.

Chronic environmental and occupational exposures to low levels of metals are associated with increased incidence of pulmonary and cardiopulmonary diseases. The cellular and molecular mechanisms of action of metals in the lung are unresolved and involve complex pleiotrophic effects. These effects are mediated by direct reaction of the metals with cellular macromolecules and indirect effects of reactive oxygen species generated when cells are exposed to metals. This review focuses on cell signaling pathways activated by two metals, chromium and nickel, that are known to promote a variety of lung diseases, including fibrosis, obstructive disease, and cancer. These signaling pathways are discussed in relation to the inclusion or exclusion of reactive oxygen in mediating cellular activation following metal exposures.

Biodegradable magnesium (Mg) alloys have been investigated for craniofacial and orthopedic bone fracture fixation due to their initial mechanical strength and high biocompatibility. Although Mg alloys have been reported to enhance bone regeneration in vivo, and enhanced osteogenic marker expression in human bone marrow stromal cells (hBMSCs) cultured in Mg alloy extract was reported, however, the biological mechanism is not fully understood. Thus, it is important to elucidate which signaling pathway in the hBMSCs are activated by Mg(2+) that enhances bone formation. We investigated possible mechanisms underlying effects of Mg(2+) on bone regeneration by culturing differentiated and undifferentiated hBMSCs in the presence of culture medium containing 10 mM MgSO4 both with or without osteogenic factors. mRNA expression of osteogenic genes was analyzed using quantitative PCR arrays. Quantitative PCR array data indicated increased mRNA expression of collagen type X and insulin-like growth factor 2, and decreased expression of integrin alpha 3 in the presence of 10 mM MgSO4. Moreover, Western blotting analysis showed enhanced expression of collagen type X, vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-2α, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in the presence of 10 mM MgSO4. In conclusion, 10 mM of MgSO4 enhanced the production of collagen type X and VEGF by hBMSCs. These results also suggest that Mg(2+) released from bone fixation devices may promote bone regeneration by enhancing the production of collagen type X and VEGF of osteogenic cells in bone tissue.

Background: Arsenic is a ubiquitous, naturally occurring metalloid that poses a significant human cancer risk. While water consumption provides the majority of human exposure, millions of individuals worldwide are significantly exposed to arsenic through naturally occurring levels of arsenic in grains, vegetables, meats and fish, as well as through food processed with water containing arsenic. Thus, we estimated the global burdens of disease for bladder, lung, and skin cancers attributable to inorganic arsenic in food. Methods: To determine foodborne inorganic arsenic exposures worldwide, we used World Health Organization estimates of food consumption in thirteen country clusters, in conjunction with reported measurements of total and inorganic arsenic in different foods. We estimated slope factors for arsenic-related bladder and lung cancers, and used the U.S. Environmental Protection Agency skin cancer slope factor, to calculate the annual risk of the cancer incidence in males and females within each country cluster. Results: We estimated that each year 9,129 to 119,176 additional cases of bladder cancer, 11,844 to 121,442 of lung cancer, and 10,729 to 110,015 of skin cancer worldwide are attributable to inorganic arsenic in food. Conclusions: These estimates indicate that foodborne arsenic exposure causes a significant global burden of human disease. Impact: Estimating the global cancer burden caused by arsenic exposure in food will support policies that reduce exposure to disease-promoting environmental hazards. Cancer Epidemiol Biomarkers Prev; 23(7); 1187–94. ©2014 AACR.

MK-852, a cyclic heptapeptide, is a potent platelet fibrinogen receptor antagonist. When administered to normal healthy male subjects by 1- and 4-hour constant rate intravenous infusions, it provides a generally well-tolerated and reversible means of inhibition of platelet function. At infusion rates of 1 microgram/kg/min for 1 hour and 0.44 microgram/kg/min for 4 hours, respectively, MK-852 extended baseline bleeding time by greater than 2.2-fold and 2.6-fold, inhibited ADP-induced platelet aggregation by 76% and 69%, and inhibited collagen-induced platelet aggregation by 65% and 67%, respectively. The pharmacokinetics of MK-852 include an elimination half-life of approximately 2 hours, total clearance of about 150 ml/min, and volume of distribution of about 18 liters. Examination of the relationship between MK-852 whole-blood concentration in vitro and inhibition of platelet aggregation showed an EC50 of about 55 ng/ml and a Hill coefficient of 1.55. The infusions were generally well tolerated, with no study drug-related changes in blood counts or biochemical profiles.

The inflammatory cytokine, TNF-alpha, induces IL-8 gene transcription via a mechanism involving proteasome-mediated IkappaBalpha degradation and NF-kappaB activation. Here, we investigated whether arsenic, which has been shown to inhibit the ubiquitin-proteasome pathway, could inhibit TNF-alpha-mediated increases in IL-8 expression. Using RT-PCR, we show that the addition of TNF-alpha to human bronchial epithelial (BEAS 2B) or embryonic kidney (HEK293) cells resulted in increased steady-state levels of IL-8 mRNA. This was preceded by a rapid decrease in cellular IkappaBalpha levels, as demonstrated by Western analysis, and an increase in nuclear levels of NF-kappaB, as demonstrated by gel shift analysis. Further demonstrating the activation of NF-kappaB, TNF-alpha induced the transcription of a NF-kappaB-dependent reporter gene. Exposing the cells to 500 microM arsenite, prior to adding TNF-alpha, completely inhibited IkappaBalpha degradation, NF-kappaB translocation, NF-kappaB-dependent gene transcription, and transcription of the endogenous gene for IL-8. In comparison with the proteasome inhibitor MG-132, which does not affect the phosphorylation and ubiquitination of IkappaBalpha, arsenite inhibited the phosphorylation of IkappaBalpha. Furthermore, arsenite directly blocked the activity of IKK, the kinase responsible for IkappaBalpha phosphorylation. These studies demonstrate that high levels of arsenic may inhibit NF-kappaB-mediated gene transcription by specifically blocking IKK activity, thereby limiting the phosphorylation and subsequent degradation of the NF-kappaB inhibitor, IkappaBalpha.

Magnesium alloys are being investigated for load-bearing bone fixation devices due to their initial mechanical strength, modulus similar to native bone, biocompatibility and ability to degrade in vivo. Previous studies have found Mg alloys to support bone regeneration in vivo, but the mechanisms have not been investigated in detail. In this study, we analyzed the effects of Mg(2+) stimulation on intracellular signaling mechanisms of human bone marrow stromal cells (hBMSCs). hBMSCs were cultured in medium containing 0.8, 5, 10, 20 and 100mM MgSO4, either with or without osteogenic induction factors. After 3weeks, mineralization of extracellular matrix (ECM) was analyzed by Alizarin red staining, and gene expression was analyzed by quantitative polymerase chain reaction array. Mineralization of ECM was enhanced at 5 and 10mM MgSO4, and collagen type X mRNA (COL10A1, an ECM protein deposited during bone healing) expression was increased at 10mM MgSO4 both with and without osteogenic factors. We also confirmed the increased production of collagen type X protein by Western blotting. Next, we investigated the mechanisms of intracellular signaling by analyzing the protein production of hypoxia-inducible factor (HIF)-1α and 2α (transcription factors of COL10A1), vascular endothelial growth factor (VEGF) (activated by HIF-2α) and peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α (transcription coactivator of VEGF). We observed that 10mM MgSO4 stimulation enhanced COL10A1 and VEGF expression, possibly via HIF-2α in undifferentiated hBMSCs and via PGC-1α in osteogenic cells. These data suggest possible ECM proteins and transcription factors affected by Mg(2+) that are responsible for the enhanced bone regeneration observed around degradable Mg orthopedic/craniofacial devices.

Arsenic in drinking water promotes a number of diseases that may stem from dysfunctional adipose lipid and glucose metabolism. Arsenic inhibits adipocyte differentiation and promotes insulin resistance; however, little is known of the impacts of and mechanisms for arsenic effects on adipose lipid storage and lipolysis. Based on our earlier studies of arsenic-signaling mechanisms for vascular remodeling and inhibition of adipogenesis, we investigated the hypothesis that arsenic acts through specific adipocyte G-protein-coupled receptors (GPCRs) to promote lipolysis and decrease lipid storage. We first demonstrated that 5-week exposure of mice to 100 μg/l of arsenic in drinking water stimulated epididymal adipocyte hypertrophy, reduced the adipose tissue expression of perilipin (PLIN1, a lipid droplet coat protein), and increased perivascular ectopic fat deposition in skeletal muscle. Incubating adipocytes, differentiated from adipose-derived human mesenchymal stem cell, with arsenic stimulated lipolysis and decreased both Nile Red positive lipid droplets and PLIN1 expression. Arsenic-stimulated lipolysis was not associated with increased cAMP levels. However, preincubation of adipocytes with the Gi inhibitor, Pertussis toxin, attenuated As(III)-stimulated lipolysis and lipid droplet loss. Antagonizing Gi-coupled endothelin-1 type A and B receptors (EDNRA/EDNRB) also attenuated arsenic effects, but antagonizing other adipose Gi-coupled receptors that regulate fat metabolism was ineffective. The endothelin receptors have different roles in arsenic responses because only EDNRA inhibition prevented arsenic-stimulated lipolysis, but antagonists to either receptor protected lipid droplets and PLIN1 expression. These data support a role for specific GPCRs in arsenic signaling for aberrant lipid storage and metabolism that may contribute to the pathogenesis of metabolic disease caused by environmental arsenic exposures.

Background/Aims: Environmental and occupational exposures are implicated as risk factors for amyotrophic lateral sclerosis (ALS), the etiology of which is largely unknown, although no causal relationships have been established. Objective: The aim of the study was to evaluate the associations of personal risk factors and self-reported environmental and occupational exposures with risk of ALS. Methods: The cases involved ALS patients (n = 66) identified from major neurological centers in Pittsburgh and Philadelphia, Pa., USA, from 2008 to 2010. The age-, race- and sex-matched controls included outpatient hospital and population-based controls (n = 66). A detailed questionnaire obtaining data on occupation, vocational and avocational exposure as well as personal lifestyle factors was administered. Results: Occupational exposure to metals (odds ratio, OR = 3.65; 95% CI: 1.15, 11.60) and pesticides (OR = 6.50; 95% CI: 1.78, 23.77) was related to increased risk of ALS after controlling for smoking and education. No associations were found for occupational exposure to organic or aromatic solvents. Conclusion: Workers exposed to metals and pesticides may be at greater risk of ALS. Future research should involve more accurate exposure assessment through the use of job exposure matrices, confirmation of occupation and biomarkers.

Nuclear factor kappa B (NF-kappa B) is a transcription factor regulating expression of genes intrinsic to inflammation and cell proliferation--features of asbestos-associated diseases. In studies here, crocidolite asbestos caused protracted and dose-responsive increases in proteins binding to nuclear NF-kappa B-binding DNA elements in hamster tracheal epithelial (HTE) cells. This binding was modulated by cellular glutathione levels. Antibodies recognizing p65 and p50 protein members of the NF-kappa B family revealed these proteins in two of the DNA complexes. Transient transfection assays with a construct containing six NF-kappa B-binding DNA consensus sites linked to a luciferase reporter gene indicated that asbestos induced transcriptional activation of NF-kappa B-dependent genes, an observation that was confirmed by northern blot analyses for c-myc mRNA levels in HTE cells. Studies suggest that NF-kappa B induction by asbestos is a key event in regulation of multiple genes involved in the pathogenesis of asbestos-related lung cancers.

Objective To determine the pharmacokinetics, pharmacodynamics and safety of the acetylcholinesterase inhibitor zifrosilone in healthy male volunteers. Methods Pharmacokinetics, pharmacodynamics, and tolerance of zifrosilone were studied in a double-blind, sequential, single-escalating-dose, randomized panel design. Each panel consisted of six subjects, with four subjects receiving zifrosilone (10, 30, 60, 90, 120, 150, 200, 250, and 300 mg orally) and two subjects receiving matching placebo. Serial blood samples were obtained for zifrosilone plasma concentrations and red blood cell acetylcholinesterase and butyrylcholinesterase activities. Participating subjects (n = 54) were men between the ages of 18 and 45 years. Each subject had a normal physical examination, electrocardiogram, serum chemistries, hematology, urinalysis, and test for human immunodeficiency virus at screening. Results A greater than proportionate increase in mean plasma concentration values for area under the curve from time zero to infinity was observed over the 200 to 300 mg dose range groups. Red blood cell acetylcholinesterase showed a dose-inhibition relationship, with a mean maximum inhibition of 20.9% at 10 mg that increased to 62.1% at 300 mg. Butyrylcholinesterase activity was relatively unaffected by zifrosilone (

Toxicology and careers in toxicology, as well as many other scientific disciplines, are undergoing rapid and dramatic changes as new discoveries, technologies, and hazards advance at a blinding rate. There are new and ever increasing demands on toxicologists to keep pace with expanding global economies, highly fluid policy debates, and increasingly complex global threats to public health. These demands must be met with new paradigms for multidisciplinary, technologically complex, and collaborative approaches that require advanced and continuing education in toxicology and associated disciplines. This requires paradigm shifts in educational programs that support recruitment, development, and training of the modern toxicologist, as well as continued education and retraining of the midcareer professional to keep pace and sustain careers in industry, government, and academia. The Society of Toxicology convened the Toxicology Educational Summit to discuss the state of toxicology education and to strategically address educational needs and the sustained advancement of toxicology as a profession. The Summit focused on core issues of: building for the future of toxicology through educational programs; defining education and training needs; developing the "Total Toxicologist"; continued training and retraining toxicologists to sustain their careers; and, finally, supporting toxicology education and professional development. This report summarizes the outcomes of the Summit, presents examples of successful programs that advance toxicology education, and concludes with strategies that will insure the future of toxicology through advanced educational initiatives.

Lung carcinomas and pulmonary fibrosis (asbestosis) occur in asbestos workers. Understanding the pathogenesis of these diseases is complicated because of potential confounding factors, such as smoking, which is not a risk factor in mesothelioma. The modes of action (MOA) of various types of asbestos in the development of lung cancers, asbestosis, and mesotheliomas appear to be different. Moreover, asbestos fibers may act differentially at various stages of these diseases, and have different potencies as compared to other naturally occurring and synthetic fibers. This literature review describes patterns of deposition and retention of various types of asbestos and other fibers after inhalation, methods of translocation within the lung, and dissolution of various fiber types in lung compartments and cells in vitro. Comprehensive dose-response studies at fiber concentrations inhaled by humans as well as bivariate size distributions (lengths and widths), types, and sources of fibers are rarely defined in published studies and are needed. Species-specific responses may occur. Mechanistic studies have some of these limitations, but have suggested that changes in gene expression (either fiber-catalyzed directly or by cell elaboration of oxidants), epigenetic changes, and receptor-mediated or other intracellular signaling cascades may play roles in various stages of the development of lung cancers or asbestosis.

Losartan is an orally active, nonpeptide angiotensin II (Ang II) (site-1) receptor antagonist. We conducted a multiple-dose study in healthy male volunteers to investigate the tolerability, blood pressure effects, and changes in plasma renin activity (PRA) and plasma Ang II concentration associated with once-daily administration of 100 mg losartan for a week. Subjects were studied on a standardized sodium diet (24-hour urinary sodium excretion, 98 +/- 37 [SD] mEq per 24 hours on the placebo run-in day). Measurements of blood pressure, heart rate, PRA, Ang II, and aldosterone were taken during a placebo run-in day and after single and multiple (7 days) daily doses of losartan (100 mg, n = 10) or placebo (n = 4). Ang II was measured specifically by high performance liquid chromatography coupled with radioimmunoassay. In subjects given losartan, respective decreases (systolic/diastolic) from run-in in supine blood pressure 6 hours after dosing were (mean +/- SD), compared with the placebo run-in day, first dose: -8.8 +/- 9.6/-6.8 +/- 5.0, last dose: -11.6 +/- 8.9/-7.0 +/- 4.8 mm Hg (p < 0.05 for all changes). At this 6-hour time point, corresponding increases from run-in in PRA were from 1.2 +/- 0.6 to 12.0 +/- 6.3 (first dose) and 9.6 +/- 4.9 (last dose) ng angiotensin I per milliliter per hour and in Ang II were from 4.3 +/- 1.7 to 72.4 +/- 33.3 and 45.7 +/- 14.1 pg/mL. All changes in PRA and Ang II were statistically significant within the losartan-treated group, and the biochemical changes were significantly greater than those in the placebo-treated group. The increment in Ang II was less after the last dose than after the first (p < 0.05). The drug was well tolerated by all subjects. These data indicate that, under the conditions of this study, losartan administration (100 mg/day for eight doses over 9 days) results in treatment-related decreases in blood pressure and increases in PRA and Ang II octapeptide.

An integral membrane protein, Claudin 5 (CLDN5), is a critical component of endothelial tight junctions that control pericellular permeability. Breaching of endothelial barriers is a key event in the development of pulmonary edema during acute lung injury (ALI). A major irritant in smoke, acrolein can induce ALI possibly by altering CLDN5 expression. This study sought to determine the cell signaling mechanism controlling endothelial CLDN5 expression during ALI. To assess susceptibility, 12 mouse strains were exposed to acrolein (10 ppm, 24 h), and survival monitored. Histology, lavage protein, and CLDN5 transcripts were measured in the lung of the most sensitive and resistant strains. CLDN5 transcripts and phosphorylation status of forkhead box O1 (FOXO1) and catenin (cadherin-associated protein) beta 1 (CTNNB1) proteins were determined in control and acrolein-treated human endothelial cells. Mean survival time (MST) varied more than 2-fold among strains with the susceptible (BALB/cByJ) and resistant (129X1/SvJ) strains (MST, 17.3 ± 1.9 h vs. 41.4 ± 5.1 h, respectively). Histological analysis revealed earlier perivascular enlargement in the BALB/cByJ than in 129X1/SvJ mouse lung. Lung CLDN5 transcript and protein increased more in the resistant strain than in the susceptible strain. In human endothelial cells, 30 nM acrolein increased CLDN5 transcripts and increased p-FOXO1 protein levels. The phosphatidylinositol 3-kinase inhibitor LY294002 diminished the acrolein-induced increased CLDN5 transcript. Acrolein (300 nM) decreased CLDN5 transcripts, which were accompanied by increased FOXO1 and CTNNB1. The phosphorylation status of these transcription factors was consistent with the observed CLDN5 alteration. Preservation of endothelial CLDN5 may be a novel clinical approach for ALI therapy.

Telomeres protect the chromosome ends and consist of guanine-rich repeats coated by specialized proteins. Critically short telomeres are associated with disease, aging and cancer. Defects in telomere replication can lead to telomere loss, which can be prevented by telomerase-mediated telomere elongation or activities of the Werner syndrome helicase/exonuclease protein (WRN). Both telomerase and WRN attenuate cytotoxicity induced by the environmental carcinogen hexavalent chromium (Cr(VI)), which promotes replication stress and DNA polymerase arrest. However, it is not known whether Cr(VI)-induced replication stress impacts telomere integrity. Here we report that Cr(VI) exposure of human fibroblasts induced telomeric damage as indicated by phosphorylated H2AX (gammaH2AX) at telomeric foci. The induced gammaH2AX foci occurred in S-phase cells, which is indicative of replication fork stalling or collapse. Telomere fluorescence in situ hybridization (FISH) of metaphase chromosomes revealed that Cr(VI) exposure induced an increase in telomere loss and sister chromatid fusions that were rescued by telomerase activity. Human cells depleted for WRN protein exhibited a delayed reduction in telomeric and non-telomeric damage, indicated by gammaH2AX foci, during recovery from Cr(VI) exposure, consistent with WRN roles in repairing damaged replication forks. Telomere FISH of chromosome spreads revealed that WRN protects against Cr(VI)-induced telomere loss and downstream chromosome fusions, but does not prevent chromosome fusions that retain telomere sequence at the fusion point. Our studies indicate that environmentally induced replication stress leads to telomere loss and aberrations that are suppressed by telomerase-mediated telomere elongation or WRN functions in replication fork restoration.

Metals are both essential for cardiovascular health and among the oldest known cardiovascular toxicants. Recent findings, such as large prospective epidemiological studies or finding that metal chelation therapy reducing cardiovascular events in cardiovascular disease (CVD) patients, strengthen the link between metal exposures and disease promotion. However, pathogenic mechanisms for metals promoting human CVD remain poorly understood and are often controversial due to difficulty in interpreting and translating results from high dose animal toxicity studies to causal disease etiologies in humans. There is increasing appreciation that lower, chronic environmental exposures, exposures occurring in critical developmental windows, or exposures at occupational and therapeutic levels may have subtler effects in causing or modifying CVD. Recent epidemiological studies and studies in genetic rodent models that focus on environmental exposures indicate that pathogenic effects of metals on cell signaling and epigenetic regulation are not random and cannot be attributed to competition with endogenous ions or excessive oxidant-mediated injury. Resolution of the molecular understanding of pathogenic metal effects remains a challenge complicated by complex differential concentration and temporal exposure relationships, as well as genetic determinants. This chapter presents pathogenic effects of metals in the heart and vasculature with an emphasis on the cellular and molecular actions underlying CVD promotion and clinically significant toxicities.

Werner syndrome is a premature aging disorder characterized by cancer predisposition that is caused by loss of the Werner syndrome protein (WRN) helicase/exonuclease DNA repair protein. Hexavalent chromium is an environmental carcinogen and genotoxicant that is associated with respiratory cancers and induces several forms of DNA damage, including lesions that interfere with DNA replication. Based on the evidence that WRN protein facilitates repair of stalled and collapsed replication forks, we hypothesized that WRN functions in the cellular response to and recovery from Cr(VI)-induced genotoxicity and genomic instability. Here we report that human cells deficient in WRN protein are hypersensitive to Cr(VI) toxicity, and exhibit a delayed reduction in DNA breaks and stalled replication forks, indicated by gammaH2AX foci, during recovery from Cr(VI) exposure. Cr(VI)-induced WRN protein translocation from the nucleoli into nucleoplasmic foci in S-phase cells, and these foci colocalized with gammaH2AX foci indicating WRN responds to replication-associated DNA damage. As further evidence that Cr(VI) triggers stalled DNA replication, we observed Cr(VI) treatment induced an accumulation of cells in S-phase that exhibited high levels of gammaH2AX foci. Therefore, these data demonstrate a novel role for WRN protein in cellular protection against the environmental genotoxicant Cr(VI) and further provide evidence that Cr(VI) induces DNA replicative stress which has implications for aging and cancer.

Arsenic in drinking water is a major public health concern as it increases risk and incidence of cardiovascular disease and cancer. Arsenic exposure affects multiple vascular beds, promoting liver sinusoidal capillarization and portal hypertension, ischemic heart disease, peripheral vascular disease, and tumor angiogenesis. While Rac1-GTPase and NADPH oxidase activities are essential for arsenic-stimulated endothelial cell signaling for angiogenesis or liver sinusoid capillarization, the mechanism for initiating these effects is unknown. We found that arsenic-stimulated cell signaling and angiogenic gene expression in human microvascular endothelial cells were Pertussis toxin sensitive, indicating a G-protein coupled signaling pathway. Incubating human microvascular endothelial cells with the sphingosine-1-phosphate type 1 receptor (S1P(1)) inhibitor VPC23019 or performing small interfering RNA knockdown of S1P(1) blocked arsenic-stimulated HMVEC angiogenic gene expression and tube formation, but did not affect induction of either HMOX1 or IL8. Liver sinusoidal endothelial cells (LSECs) defenestrate and capillarize in response to aging and environmental oxidant stresses. We found that S1P(1) was enriched on LSECs in vivo and in primary cell culture and that VPC23019 inhibited both sphingosine-1-phosphate-stimulated and arsenic-stimulated LSEC oxidant generation and defenestration. These studies identified novel roles for S1P(1) in mediating arsenic stimulation of both angiogenesis and pathogenic LSEC capillarization, as well as demonstrating a role for S1P(1) in mediating environmental responses in the liver vasculature, providing possible mechanistic insight into arsenic-induced vascular pathogenesis and disease.

Ca2+ metabolism and its relationship to arachidonic acid release were studied in cultured pig aortic endothelial cells. When cells were treated with bradykinin, a rapid rise in intracellular Ca2+ concentration ([Ca2+]i) occurred. Arachidonic acid release from cells prelabelled with [3H]arachidonic acid and subjected to flow-through conditions closely followed the changes in [Ca2+]i. Attenuation of the Ca2+ response by chelating extracellular and intracellular Ca2+ or by desensitization of receptors led to comparable attenuation of arachidonate release. Activation of protein kinase C inhibited Ca2+ mobilization in response to bradykinin and stimulated arachidonic acid release. Inhibition of protein kinase C had no effect on bradykinin-stimulated arachidonic acid release, suggesting that protein kinase C does not mediate the bradykinin response. The role of GTP-binding regulatory proteins (G-proteins) in mediating the bradykinin response was also investigated. Bradykinin-stimulated arachidonic acid release was not diminished by preincubation with pertussis toxin. Treatment with the G-protein activator AlF4- resulted in the release of a large pool of arachidonic acid and the formation of lysophospholipids. Combined treatment with AlF4- and bradykinin resulted in a greater than additive effect on arachidonic acid release. In contrast with bradykinin, AlF(4-)-stimulated arachidonic acid release was not dependent on the presence of extracellular Ca2+ or the mobilization of intracellular Ca2+. These results demonstrate Ca(2+)-dependent (bradykinin) and Ca(2+)-independent (AlF4-) pathways of phospholipase A2 activation.

Isomers (−,+) of the antitumor agent gossypol (G) were studied for their ability to reduce tumor ATP and blood flow in rats bearing subcutaneously implanted pancreatic tumors. A 50% reduction in tumor ATP/Pi within lh of a single injection of −G was associated with a 60% decline in tumor blood flow To determine if these changes in tumor physiology could be due to a direct drug effect on tumor endothelium, G isomers were compared for their ability to alter protein ( 125 l-BSA) permeability and metabolic ( 32 P) labeling of cultured endothelial cells. Treatments for lh produced no endothelial cells leakage, but 24h exposures to either −G(5μM) or +G(50μM) produced complete permeability of the monolayers to 125 l-BSA. In contrast, 0.5-l.0h exposures to −G(4μM) or +G(10μM) produced 2 to 3-fold increases in phosphorylated 27kDa heat-shock protein, hsp-27. Hsp-27 phosphoprotein isoforms were differentially labelled following −G and +G exposures with the phosphorylation profile of −G appearing most similar to that of oxyradical producing agents known to induce hsp-27 and injure endothelial cells. we postulate that the tumor ischemic effects of −G are mediated by endothelial response to oxyradical production in a mechanism similar to that of tissue ischemia-reperfusion injury.

Environmental arsenic exposure, through drinking contaminated water, is a significant risk factor for developing vascular diseases and is associated with liver portal hypertension, vascular shunting, and portal fibrosis through unknown mechanisms. We found that the addition of low doses of arsenite to the drinking water of mice resulted in marked pathologic remodeling in liver sinusoidal endothelial cells (SECs), including SEC defenestration, capillarization, increased junctional PECAM-1 expression, protein nitration, and decreased liver clearance of modified albumin. Furthermore, the pathologic changes observed after in vivo exposure were recapitulated in isolated mouse SECs exposed to arsenic in culture. To investigate the role of NADPH oxidase-generated ROS in this remodeling, we examined the effect of arsenite in the drinking water of mice deficient for the p47 subunit of the NADPH oxidase and found that knockout mice were protected from arsenite-induced capillarization and protein nitration. Furthermore, ex vivo arsenic exposure increased SEC superoxide generation, and this effect was inhibited by addition of a Nox2 inhibitor and quenched by the cell-permeant superoxide scavenger. In addition, inhibiting either oxidant generation or Rac1-GTPase blocked ex vivo arsenic-stimulated SEC differentiation and dysfunction. Our data indicate that a Nox2-based oxidase is required for SEC capillarization and that it may play a central role in vessel remodeling following environmentally relevant arsenic exposures.

Exposure of animals to hyperoxia decreases lung VEGF mRNA expression concomitant with an acute increase in VEGF protein within the epithelial lining fluid (ELF). The VEGF concentration in ELF is in excess of that found in the plasma, leading to the hypothesis that hyperoxia stimulates the release of VEGF protein from stores within the extracellular matrix. To test this hypothesis in a cell culture system, we exposed A549 cells to 95% O(2) (Ox) for 48 h followed by recovery in room air (RA) for 24 h. We found that Ox increased VEGF protein two- to threefold within the medium at 48 h of exposure and during recovery. Heparin clearing revealed the medium to contain a 50/50 mixture of the heparin-binding (VEGF(165)) and heparin-nonbinding (VEGF(121)) proteins and that Ox increased both proteins equally. Transcriptional activation of VEGF seems unlikely to explain the increase in VEGF protein, as expression of full-length and splice variant VEGF mRNA was unchanged by hyperoxia. Analysis of cell-associated VEGF proteins found that Ox increased the expression of VEGF(121) and VEGF(165) proteins. Blocking binding sites with exogenous heparin enhanced VEGF protein in the medium from RA-grown cells, whereas heparinase digestion of bound VEGF revealed a greater reserve of VEGF protein in RA cells. Collectively these findings indicate that hyperoxia enhances the expression of VEGF(121/165) proteins and facilitates the release of VEGF(165) from cell-associated stores. Increases in VEGF in ELF may represent an adaptive response fostering cell survival and type II cell proliferation in O(2)-induced lung injury.