Multi-omics profiling reveals cellular pathways and functions regulated by ALDH1B1 in colon cancer cells.

Colon cancer is the third leading cause of cancer death globally. Although early screenings and advances in treatments have reduced mortality since 1970, identification of novel targets for therapeutic intervention is needed to address tumor heterogeneity and recurrence. Previous work identified aldehyde dehydrogenase 1B1 (ALDH1B1) as a critical factor in colon tumorigenesis. To investigate further, we utilized a human colon adenocarcinoma cell line (SW480) in which the ALDH1B1 protein expression has been knocked down by 80% via shRNA. Through multi-omics (transcriptomics, proteomics, and untargeted metabolomics) analysis, we identified the impact of ALDH1B1 knocking down (KD) on molecular signatures in colon cancer cells. Suppression of ALDH1B1 expression resulted in 357 differentially expressed genes (DEGs), 191 differentially expressed proteins (DEPs) and 891 differentially altered metabolites (DAMs). Functional annotation and enrichment analyses revealed that: (1) DEGs were enriched in integrin-linked kinase (ILK) signaling and growth and development pathways; (2) DEPs were mainly involved in apoptosis signaling and cellular stress response pathways; and (3) DAMs were associated with biosynthesis, intercellular and second messenger signaling. Collectively, the present study provides new molecular information associated with the cellular functions of ALDH1B1, which helps to direct future investigation of colon cancer.

Endocrine pancreas-specific Gclc gene deletion causes a severe diabetes phenotype.

Reduced glutathione (GSH) is an abundant antioxidant that regulates intracellular redox homeostasis by scavenging reactive oxygen species (ROS). Glutamate-cysteine ligase catalytic (GCLC) subunit is the rate-limiting step in GSH biosynthesis. Using the Pax6-Cre driver mouse line, we deleted expression of the Gclc gene in all pancreatic endocrine progenitor cells. Intriguingly, Gclc knockout (KO) mice, following weaning, exhibited an age-related, progressive diabetes phenotype, manifested as strikingly increased blood glucose and decreased plasma insulin levels. This severe diabetes trait is preceded by pathologic changes in islet of weanling mice. Gclc KO weanlings showed progressive abnormalities in pancreatic morphology including: islet-specific cellular vacuolization, decreased islet-cell mass, and alterations in islet hormone expression. Islets from newly-weaned mice displayed impaired glucose-stimulated insulin secretion, decreased insulin hormone gene expression, oxidative stress, and increased markers of cellular senescence. Our results suggest that GSH biosynthesis is essential for normal development of the mouse pancreatic islet, and that protection from oxidative stress-induced cellular senescence might prevent abnormal islet-cell damage during embryogenesis.

Alzheimer's disease: using gene/protein network machine learning for molecule discovery in olive oil.

Alzheimer's disease (AD) poses a profound human, social, and economic burden. Previous studies suggest that extra virgin olive oil (EVOO) may be helpful in preventing cognitive decline. Here, we present a network machine learning method for identifying bioactive phytochemicals in EVOO with the highest potential to impact the protein network linked to the development and progression of the AD. A balanced classification accuracy of 70.3 ± 2.6% was achieved in fivefold cross-validation settings for predicting late-stage experimental drugs targeting AD from other clinically approved drugs. The calibrated machine learning algorithm was then used to predict the likelihood of existing drugs and known EVOO phytochemicals to be similar in action to the drugs impacting AD protein networks. These analyses identified the following ten EVOO phytochemicals with the highest likelihood of being active against AD: quercetin, genistein, luteolin, palmitoleate, stearic acid, apigenin, epicatechin, kaempferol, squalene, and daidzein (in the order from the highest to the lowest likelihood). This in silico study presents a framework that brings together artificial intelligence, analytical chemistry, and omics studies to identify unique therapeutic agents. It provides new insights into how EVOO constituents may help treat or prevent AD and potentially provide a basis for consideration in future clinical studies.

Proteomic profiling reveals an association between ALDH and oxidative phosphorylation and DNA damage repair pathways in human colon adenocarcinoma stem cells.

Several members of the aldehyde dehydrogenase (ALDH) family, especially ALDH1 isoenzymes, have been identified as biomarkers of cancer stem cells (CSCs), a small subpopulation of oncogenic cells with self-renewal and multipotency capability. Consistent with this contention, cell populations with high ALDH enzymatic activity exhibit greater carcinogenic potential. It has been reported that ALDH1, especially ALDH1A1, serves as a valuable biomarker for colon CSCs. However, the functional roles of ALDHs in CSCs and solid tumors of the colon tissue is not fully understood. The aim of the present study was to identify molecular signature associated with high ALDH activity in human colorectal adenocarcinoma (COLO320DM) cells by proteomics profiling. Aldefluor™ assay was performed to sort COLO320DM cells exhibiting high (ALDHhigh) and low (ALDHlow) ALDH activity. Label-free quantitative proteomics analyses were conducted on these two cell populations. Proteomics profiling revealed a total of 229 differentially expressed proteins (DEPs) in ALDHhigh relative to ALDHlow cells, of which 182 were down-regulated and 47 were up-regulated. In agreement with previous studies, ALDH1A1 appeared to be the principal ALDH isozyme contributing to the Aldefluor™ assay activity in COLO320DM cells. Ingenuity pathway analysis of the proteomic datasets indicated that DEPs were associated with mitochondrial dysfunction, sirtuin signaling, oxidative phosphorylation and nucleotide excision repair. Our proteomics study predicts that high ALDH1A1 activity may be involved in these cellular pathways to promote a metabolic switch and cellular survival of CSCs.

Oxidative stress induces inflammation of lens cells and triggers immune surveillance of ocular tissues.

Recent reports have challenged the notion that the lens is immune-privileged. However, these studies have not fully identified the molecular mechanism(s) that promote immune surveillance of the lens. Using a mouse model of targeted glutathione (GSH) deficiency in ocular surface tissues, we have investigated the role of oxidative stress in upregulating cytokine expression and promoting immune surveillance of the eye. RNA-sequencing of lenses from postnatal day (P) 1-aged Gclcf/f;Le-CreTg/- (KO) and Gclcf/f;Le-Cre-/- control (CON) mice revealed upregulation of many cytokines (e.g., CCL4, GDF15, CSF1) and immune response genes in the lenses of KO mice. The eyes of KO mice had a greater number of cells in the aqueous and vitreous humors at P1, P20 and P50 than age-matched CON and Gclcw/w;Le-CreTg/- (CRE) mice. Histological analyses revealed the presence of innate immune cells (i.e., macrophages, leukocytes) in ocular structures of the KO mice. At P20, the expression of cytokines and ROS content was higher in the lenses of KO mice than in those from age-matched CRE and CON mice, suggesting that oxidative stress may induce cytokine expression. In vitro administration of the oxidant, hydrogen peroxide, and the depletion of GSH (using buthionine sulfoximine (BSO)) in 21EM15 lens epithelial cells induced cytokine expression, an effect that was prevented by co-treatment of the cells with N-acetyl-l-cysteine (NAC), a antioxidant. The in vivo and ex vivo induction of cytokine expression by oxidative stress was associated with the expression of markers of epithelial-to-mesenchymal transition (EMT), α-SMA, in lens cells. Given that EMT of lens epithelial cells causes posterior capsule opacification (PCO), we propose that oxidative stress induces cytokine expression, EMT and the development of PCO in a positive feedback loop. Collectively these data indicate that oxidative stress induces inflammation of lens cells which promotes immune surveillance of ocular structures.

Oxidative stress, glutathione, and CYP2E1 in 1,4-dioxane liver cytotoxicity and genotoxicity: insights from animal models.

1,4-Dioxane (DX) is an emerging drinking water contaminant worldwide, which poses a threat to public health due to its demonstrated liver carcinogenicity and potential for human exposure. The lack of drinking water standards for DX is attributed to undetermined mechanisms of DX carcinogenicity. This mini-review provides a brief discussion of a series of mechanistic studies, wherein unique mouse models were exposed to DX in drinking water to elucidate redox changes associated with DX cytotoxicity and genotoxicity. The overall conclusions from these studies support a direct genotoxic effect by high dose DX and imply that oxidative stress involving CYP2E1 activation may play a causal role in DX liver genotoxicity and potentially carcinogenicity. The mechanistic data derived from these studies can serve as important references to refine the assessment of carcinogenic pathways that may be triggered at environmentally relevant low doses of DX in future animal and human studies.

Lipidomics and Redox Lipidomics Indicate Early Stage Alcohol-Induced Liver Damage.

Alcoholic fatty liver disease (AFLD) is characterized by lipid accumulation and inflammation and can progress to cirrhosis and cancer in the liver. AFLD diagnosis currently relies on histological analysis of liver biopsies. Early detection permits interventions that would prevent progression to cirrhosis or later stages of the disease. Herein, we have conducted the first comprehensive time-course study of lipids using novel state-of-the art lipidomics methods in plasma and liver in the early stages of a mouse model of AFLD, i.e., Lieber-DeCarli diet model. In ethanol-treated mice, changes in liver tissue included up-regulation of triglycerides (TGs) and oxidized TGs and down-regulation of phosphatidylcholine, lysophosphatidylcholine, and 20-22-carbon-containing lipid-mediator precursors. An increase in oxidized TGs preceded histological signs of early AFLD, i.e., steatosis, with these changes observed in both the liver and plasma. The major lipid classes dysregulated by ethanol play important roles in hepatic inflammation, steatosis, and oxidative damage. Conclusion: Alcohol consumption alters the liver lipidome before overt histological markers of early AFLD. This introduces the exciting possibility that specific lipids may serve as earlier biomarkers of AFLD than those currently being used.

Oxidative stress and genotoxicity in 1,4-dioxane liver toxicity as evidenced in a mouse model of glutathione deficiency.

1,4-Dioxane (DX) is a synthetic chemical used as a stabilizer for industrial solvents. Recent occurrence data show widespread and significant contamination of drinking water with DX in the US. DX is classified by the International Agency for Research on Cancer as a group 2B carcinogen with the primary target organ being the liver in animal studies. Despite the exposure and cancer risk, US EPA has not established a drinking water Maximum Contaminant Level (MCL) for DX and a wide range of drinking water targets have been established across the US and by Health Canada. The DX carcinogenic mechanism remains unknown; this information gap contributes to the varied approaches to its regulation. Our recent mice study indicated alterations in oxidative stress response accompanying DNA damage as an early change by high dose DX (5000 ppm) in drinking water. Herein, we report a follow-up study, in which we used glutathione (GSH)-deficient glutamate-cysteine ligase modifier subunit (Gclm)-null mice to investigate the role of redox homeostasis in DX-induced liver cytotoxicity and genotoxicity. Gclm-null and wild-type mice were exposed to DX for one week (1000 mg/kg/day by oral gavage) or three months (5000 ppm in drinking water). Subchronic exposure of high dose DX caused mild liver cytotoxicity. DX induced assorted molecular changes in the liver including: (i) a compensatory nuclear factor erythroid 2-related factor 2 (NRF2) anti-oxidative response at the early stage (one week), (ii) progressive CYP2E1 induction, (iii) development of oxidative stress, as evidenced by persistent NRF2 induction, oxidation of GSH pool, and accumulation of the lipid peroxidation by-product 4-hydroxynonenal, and (iv) elevations in oxidative DNA damage and DNA repair response. These DX-elicited changes were exaggerated in GSH-deficient mice. Collectively, the current study provides additional evidence linking redox dysregulation to DX liver genotoxicity, implying oxidative stress as a candidate mechanism of DX liver carcinogenicity.

AMPK activators for the prevention and treatment of neurodegenerative diseases.

INTRODUCTION: As the global population ages at an unprecedented rate, the burden of neurodegenerative diseases is expected to grow. Given the profound impact illness like dementia exert on individuals and society writ large, researchers, physicians, and scientific organizations have called for increased investigation into their treatment and prevention. Both metformin and aspirin have been associated with improved cognitive outcomes. These agents are related in their ability to stimulate AMP kinase (AMPK). Momordica charantia, another AMPK activator, is a component of traditional medicines and a novel agent for the treatment of cancer. It is also being evaluated as a nootropic agent. AREAS COVERED: This article is a comprehensive review which examines the role of AMPK activation in neuroprotection and the role that AMPK activators may have in the management of dementia and cognitive impairment. It evaluates the interaction of metformin, aspirin, and Momordica charantia, with AMPK, and reviews the literature characterizing these agents' impact on neurodegeneration. EXPERT OPINION: We suggest that AMPK activators should be considered for the treatment and prevention of neurodegenerative diseases. We identify multiple areas of future investigation which may have a profound impact on patients worldwide.

Molecular Mechanisms of Alcohol-Induced Colorectal Carcinogenesis.

The etiology of colorectal cancer (CRC) is complex. Approximately, 10% of individuals with CRC have predisposing germline mutations that lead to familial cancer syndromes, whereas most CRC patients have sporadic cancer resulting from a combination of environmental and genetic risk factors. It has become increasingly clear that chronic alcohol consumption is associated with the development of sporadic CRC; however, the exact mechanisms by which alcohol contributes to colorectal carcinogenesis are largely unknown. Several proposed mechanisms from studies in CRC models suggest that alcohol metabolites and/or enzymes associated with alcohol metabolism alter cellular redox balance, cause DNA damage, and epigenetic dysregulation. In addition, alcohol metabolites can cause a dysbiotic colorectal microbiome and intestinal permeability, resulting in bacterial translocation, inflammation, and immunosuppression. All of these effects can increase the risk of developing CRC. This review aims to outline some of the most significant and recent findings on the mechanisms of alcohol in colorectal carcinogenesis. We examine the effect of alcohol on the generation of reactive oxygen species, the development of genotoxic stress, modulation of one-carbon metabolism, disruption of the microbiome, and immunosuppression.

Impaired GSH biosynthesis disrupts eye development, lens morphogenesis and PAX6 function.

PURPOSE: The purpose of this study was to elucidate the role and molecular consequences of impaired glutathione (GSH) biosynthesis on eye development. METHODS: GSH biosynthesis was impaired in surface ectoderm-derived ocular tissues by crossing Gclcf/f mice with hemizygous Le-Cre transgenic mice to produce Gclcf/f/Le-CreTg/- (KO) mice. Control mice included Gclcf/fand Gclcwt/wt/Le-CreTg/- mice (CRE). Eyes from all mice (at various stages of eye development) were subjected to histological, immunohistochemical, Western blot, RT-qPCR, RNA-seq, and subsequent Gene Ontology, Ingenuity Pathway Analysis and TRANSFAC analyses. PAX6 transactivation activity was studied using a luciferase reporter assay in HEK293T cells depleted of GSH using buthionine sulfoximine (BSO). RESULTS: Deletion of Gclc diminished GSH levels, increased reactive oxygen species (ROS), and caused an overt microphthalmia phenotype characterized by malformation of the cornea, iris, lens, and retina that is distinct from and much more profound than the one observed in CRE mice. In addition, only the lenses of KO mice displayed reduced crystallin (α, ß), PITX3 and Foxe3 expression. RNA-seq analyses at postnatal day 1 revealed 1552 differentially expressed genes (DEGs) in the lenses of KO mice relative to those from Gclcf/f mice, with Crystallin and lens fiber cell identity genes being downregulated while lens epithelial cell identity and immune response genes were upregulated. Bioinformatic analysis of the DEGs implicated PAX6 as a key upstream regulator. PAX6 transactivation activity was impaired in BSO-treated HEK293T cells. CONCLUSIONS: These data suggest that impaired ocular GSH biosynthesis may disrupt eye development and PAX6 function.

Identification of Dose-Dependent DNA Damage and Repair Responses From Subchronic Exposure to 1,4-Dioxane in Mice Using a Systems Analysis Approach.

1,4-Dioxane (1,4-DX) is an environmental contaminant found in drinking water throughout the United States. Although it is a suspected liver carcinogen, there is no federal or state maximum contaminant level for 1,4-DX in drinking water. Very little is known about the mechanisms by which this chemical elicits liver carcinogenicity. In the present study, female BDF-1 mice were exposed to 1,4-DX (0, 50, 500, and 5,000mg/L) in their drinking water for 1 or 4 weeks, to explore the toxic effects. Histopathological studies and a multi-omics approach (transcriptomics and metabolomics) were performed to investigate potential mechanisms of toxicity. Immunohistochemical analysis of the liver revealed increased H2AXγ-positive hepatocytes (a marker of DNA double-strand breaks), and an expansion of precholangiocytes (reflecting both DNA damage and repair mechanisms) after exposure. Liver transcriptomics revealed 1,4-DX-induced perturbations in signaling pathways predicted to impact the oxidative stress response, detoxification, and DNA damage. Liver, kidney, feces, and urine metabolomic profiling revealed no effect of 1,4-DX exposure, and bile acid quantification in liver and feces similarly showed no effect of exposure. We speculate that the results may be reflective of DNA damage being counterbalanced by the repair response, with the net result being a null overall effect on the systemic biochemistry of the exposed mice. Our results show a novel approach for the investigation of environmental chemicals that do not elicit cell death but have activated the repair systems in response to 1,4-DX exposure.

Overview of PAX gene family: analysis of human tissue-specific variant expression and involvement in human disease.

Paired-box (PAX) genes encode a family of highly conserved transcription factors found in vertebrates and invertebrates. PAX proteins are defined by the presence of a paired domain that is evolutionarily conserved across phylogenies. Inclusion of a homeodomain and/or an octapeptide linker subdivides PAX proteins into four groups. Often termed "master regulators", PAX proteins orchestrate tissue and organ development throughout cell differentiation and lineage determination, and are essential for tissue structure and function through maintenance of cell identity. Mutations in PAX genes are associated with myriad human diseases (e.g., microphthalmia, anophthalmia, coloboma, hypothyroidism, acute lymphoblastic leukemia). Transcriptional regulation by PAX proteins is, in part, modulated by expression of alternatively spliced transcripts. Herein, we provide a genomics update on the nine human PAX family members and PAX homologs in 16 additional species. We also present a comprehensive summary of human tissue-specific PAX transcript variant expression and describe potential functional significance of PAX isoforms. While the functional roles of PAX proteins in developmental diseases and cancer are well characterized, much remains to be understood regarding the functional roles of PAX isoforms in human health. We anticipate the analysis of tissue-specific PAX transcript variant expression presented herein can serve as a starting point for such research endeavors.

Interplay between APC and ALDH1B1 in a newly developed mouse model of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer mortality worldwide. Mutations in the adenomatous polyposis coli (APC) gene are pivotal in colorectal tumorigenesis. Recently, we demonstrated that aldehyde dehydrogenase 1B1 (ALDH1B1) knockdown dramatically reduced colon tumor growth in a mouse xenograft model. The purpose of the present preliminary study is to examine the effect of loss of ALDH1B1 in CRC development in an inducible colon-specific Apc mouse model. METHODS: ApcW/FCdx2ERT2-Cre mice develop uni-allelic inactivation of Apc specifically in colon epithelial cells following tamoxifen treatment. Aldh1b1-/- KO mice were crossed with ApcW/FCdx2ERT2-Cre mice. Six-month-old male ApcW/FCdx2ERT2-Cre/Aldh1b1-/-, and ApcW/FCdx2ERT2-Cre/Aldh1b1+/+ mice were treated with tamoxifen (50 mg/kg, i.p.) for three consecutive days. ApcW/F/Aldh1b1-/- and ApcW/F/Aldh1b1+/+ mice were treated with corn oil (i.e., tamoxifen vehicle control) for three consecutive days. Eighteen days later, mice were sacrificed and their colons examined microscopically, macroscopically and histologically for the presence of adenoma. RESULTS: All ApcW/FCdx2ERT2-Cre/Aldh1b1+/+ and ApcW/FCdx2ERT2-Cre/Aldh1b1-/- mice treated with tamoxifen developed colorectal adenoma. The ApcW/FCdx2ERT2-Cre/Aldh1b1-/- mice showed a significant decrease in the total volume of all ileal and colonic adenomas, and decreased incidence of large colonic adenoma compared to ApcW/FCdx2ERT2-Cre/Aldh1b1+/+ mice. Immunohistochemical analysis of p53 and ß-catenin showed a trend toward decreased expression score in colonic adenomas of ApcW/FCdx2ERT2-Cre/Aldh1b1-/- mice. CONCLUSION: The present preliminary study suggests that deletion of ALDH1B1 may protect against the full development of colorectal cancer. Further mechanistic studies are required to elucidate how ALDH1B1 contributes for colorectal cancer.

Glutathione deficiency-elicited reprogramming of hepatic metabolism protects against alcohol-induced steatosis.

Depletion of glutathione (GSH) is considered a critical pathogenic event promoting alcohol-induced lipotoxicity. We recently show that systemic GSH deficiency in mice harboring a global disruption of the glutamate-cysteine ligase modifier subunit (Gclm) gene confers protection against alcohol-induced steatosis. While several molecular pathways have been linked to the observed hepatic protection, including nuclear factor erythroid 2-related factor 2 and AMP-activated protein kinase pathways, the precise mechanisms are yet to be defined. In this study, to gain insights into the molecular mechanisms underpinning the protective effects of loss of GCLM, global profiling of hepatic polar metabolites combined with liver microarray analysis was carried out. These inter-omics analyses revealed both low GSH- and alcohol-driven changes in multiple cellular pathways involving the metabolism of amino acids, fatty acid, glucose and nucleic acids. Notably, several metabolic changes were uniquely present in alcohol-treated Gclm-null mouse livers, including acetyl-CoA enrichment and diversion of acetyl-CoA flux from lipogenesis to alterative metabolic pathways, elevation in glutamate concentration, and induction of the glucuronate pathway and nucleotide biosynthesis. These metabolic features reflect low GSH-elicited cellular response to chronic alcohol exposure, which is beneficial for the maintenance of hepatic redox and metabolic homeostasis. The current study indicates that fine-tuning of hepatic GSH pool may evoke metabolic reprogramming to cope with alcohol-induced cellular stress.

1,4-Dioxane as an emerging water contaminant: State of the science and evaluation of research needs.

1,4-Dioxane has historically been used to stabilize chlorinated solvents and more recently has been found as a contaminant of numerous consumer and food products. Once discharged into the environment, its physical and chemical characteristics facilitate migration in groundwater, resulting in widespread contamination of drinking water supplies. Over one-fifth of U.S. public drinking water supplies contain detectable levels of 1,4-dioxane. Remediation efforts using common adsorption and membrane filtration techniques have been ineffective, highlighting the need for alternative removal approaches. While the data evaluating human exposure and health effects are limited, animal studies have shown chronic exposure to cause carcinogenic responses in the liver across multiple species and routes of exposure. Based on this experimental evidence, the U.S. Environmental Protection Agency has listed 1,4-dioxane as a high priority chemical and classified it as a probable human carcinogen. Despite these health concerns, there are no federal or state maximum contaminant levels for 1,4-dioxane. Effective public health policy for this emerging contaminant requires additional information about human health effects, chemical interactions, environmental fate, analytical detection, and treatment technologies. This review highlights the current state of knowledge, key uncertainties, and data needs for future research on 1,4-dioxane.

Hepatic metabolic adaptation in a murine model of glutathione deficiency.

Glutathione (GSH), the most abundant cellular non-protein thiol, plays a pivotal role in hepatic defense mechanisms against oxidative damage. Despite a strong association between disrupted GSH homeostasis and liver diseases of various etiologies, it was shown that GSH-deficient glutamate-cysteine ligase modifier subunit (Gclm)-null mice are protected against fatty liver development induced by a variety of dietary and environmental insults. The biochemical mechanisms underpinning this protective phenotype have not been clearly defined. The purpose of the current study was to characterize the intrinsic metabolic signature in the livers from GSH deficient Gclm-null mice. Global profiling of hepatic polar metabolites revealed a spectrum of changes in amino acids and metabolites derived from fatty acids, glucose and nucleic acids due to the loss of GCLM. Overall, the observed low GSH-driven metabolic changes represent metabolic adaptations, including elevations in glutamate, aspartate, acetyl-CoA and gluconate, which are beneficial for the maintenance of cellular redox and metabolic homeostasis.

The effect of anodal/cathodal biphasic electrical stimulation on insulin release.

We studied the effects of electrical stimulation on insulin release from rat insulinoma (INS-1) cells. The anodal/cathodal biphasic stimulation (ACBPS) electrical waveform resulted in a voltage- and stimulation duration-dependent increase in insulin release. ACBPS elicited insulin release both in the presence and absence of glucose. Basal and ACBPS-induced insulin secretion could be inhibited by mitochondrial poisons and calcium channel blockers, indicating that insulin release was dependent on adenosine triphosphate (ATP) and the influx of calcium. ACBPS parameters that released insulin caused no detectable plasma membrane damage or cytotoxicity, although temporary morphological changes could be observed immediately after ACBPS. ACBPS did not alter the plasma membrane transmembrane potential but did cause pronounced uptake of MitoTracker Red into the mitochondrial membrane, indicating an increased mitochondrial membrane potential. While the ATP:ADP ratio after ACBPS did not change, the guanosine triphosphate (GTP) levels increased and increased GTP levels have previously been associated with insulin release in INS-1 cells. These results provide evidence that ACBPS can have significant biological effects on cells. In the case of INS-1 cells, ACBPS promotes insulin release without causing cytotoxicity.

Evaluation of potential carcinogenicity of organic chemicals in synthetic turf crumb rubber.

Currently, there are >11,000 synthetic turf athletic fields in the United States and >13,000 in Europe. Concerns have been raised about exposure to carcinogenic chemicals resulting from contact with synthetic turf fields, particularly the infill material ("crumb rubber"), which is commonly fabricated from recycled tires. However, exposure data are scant, and the limited existing exposure studies have focused on a small subset of crumb rubber components. Our objective was to evaluate the carcinogenic potential of a broad range of chemical components of crumb rubber infill using computational toxicology and regulatory agency classifications from the United States Environmental Protection Agency (US EPA) and European Chemicals Agency (ECHA) to inform future exposure studies and risk analyses. Through a literature review, we identified 306 chemical constituents of crumb rubber infill from 20 publications. Utilizing ADMET Predictor™, a computational program to predict carcinogenicity and genotoxicity, 197 of the identified 306 chemicals met our a priori carcinogenicity criteria. Of these, 52 chemicals were also classified as known, presumed or suspected carcinogens by the US EPA and ECHA. Of the remaining 109 chemicals which were not predicted to be carcinogenic by our computational toxicology analysis, only 6 chemicals were classified as presumed or suspected human carcinogens by US EPA or ECHA. Importantly, the majority of crumb rubber constituents were not listed in the US EPA (n = 207) and ECHA (n = 262) databases, likely due to an absence of evaluation or insufficient information for a reliable carcinogenicity classification. By employing a cancer hazard scoring system to the chemicals which were predicted and classified by the computational analysis and government databases, several high priority carcinogens were identified, including benzene, benzidine, benzo(a)pyrene, trichloroethylene and vinyl chloride. Our findings demonstrate that computational toxicology assessment in conjunction with government classifications can be used to prioritize hazardous chemicals for future exposure monitoring studies for users of synthetic turf fields. This approach could be extended to other compounds or toxicity endpoints.