2014
There have been many efforts to characterize the toxicology of environmental chemicals, ranging from traditional approaches such as long-term toxicity assessments in animal models and epidemiological studies in human populations to modern systems biology approaches to map gene and protein expression changes to high-throughput screening efforts to identify cytotoxic agents. However, nearly all of these approaches have been largely correlative, have not provided sufficient granularity on toxicological mechanisms, and have likely still missed subtler or insidious pathological effects that may arise from long-term chemical exposures. This is because animal, cell, or human phenotypes as well as genetic, epigenetic, protein, or metabolite changes that arise from chemical exposure are all indirect outcomes that arise from direct chemical interactions with specific molecular targets. Deconvoluting these indirect effects to predict the direct molecular targets has been very challenging, if not impossible. We believe that understanding the direct chemical-protein interactions will inform our understanding of downstream molecular, metabolic, and pathophysiological effects that may arise from chemical exposure, providing a more direct approach towards identifying environmental drivers of human disease. In our most recent and innovative efforts, we have developed and applied a next-generation chemoproteomic approach termed reactivity-based proteomic profiling (RBPP), which uses reactivity-based chemical probes to map functional sites in the proteome, to comprehensively map proteome-wide interactions of environmental chemicals in complex biological systems, towards understanding their toxicological action.
2013
The Co-Leaders for Core C are now Professors Christopher Vulpe and Daniel Nomura. The overall aims of Core C have not changed. However, we are now performing proteomic and metabolomic experiments and data analysis in the lab of Professor Daniel Nomura. The Nomura lab has dedicated mass-spectrometry equipment, methodologies, and bioinformatics platforms to support the proteomic and metabolomic needs of the UC Berkeley Superfund Research Program.
We have added an additional Aim to Core C: Provide facilities and methodologies for chemical proteomic and metabolomic technologies for identifying protein targets and mechanism of action of Superfund chemicals. This aim will utilize state-of-the-art chemical approaches that combine bioorthogonal probe development with proteomics and metabolomics for affinity enriching, identifying, and characterizing direct protein interactions of Superfund chemicals in complex living systems (e.g. mammalian cells, mice). This aim will provide in-depth characterization of the mechanisms of toxicity associated with Superfund chemicals by identifying direct chemical-protein interactions and the metabolic alterations that these interactions cause in living cells and animals.
We have made many important discoveries during the past year of funding. In one project, we have used advanced untargeted mass-spectrometry-based metabolomic platforms to identify small-molecule chemical biomarkers of lymphoma. We profiled serum samples from healthy individuals and lymphoma patients. Through this analysis, we found that 2-arachidonoylglycerol (2-AG) levels are elevated by ~2-fold in lymphoma patient serum-samples compared to healthy samples. 2-AG is an endogenous metabolite and agonist for the cannabinoid receptor type 1 (CB1). CB1 signaling, most popularly known through marijuana and CB1 agonist tetrahydrocannabinol, affects a wide-range of (patho)physiology including memory, pain, mood, food intake and metabolism, and cellular proliferation. In collaboration with Christine Skibola, a former PI of Core C and now a professor at University of Alabama at Birmingham, we have found that 2-AG is a driver of lymphoma cell proliferation through stimulating the CB1 receptor. Thus, we were able to not only find a biomarker of lymphoma in human serum, but also a potential metabolic driver of disease.
In another project, we have used advanced targeted and untargeted mass-spectrometry-based metabolomic platforms to map altered metabolism in arsenic-exposed macrophages. We treated mouse bone-marrow-derived primary macrophages with different forms of arsenic and identified several metabolic pathways that were dysregulated. These pathways included heightened eicosanoid (prostaglandin E2 and D2, PGE2/PGD2) and lysophosphatidic acid signaling pathways. Both eicosanoids and lysophosphatidic acid (LPA) are highly potent signaling lipids that act as pro-inflammatory or chemoattractant agents through binding their respective G-protein coupled receptors. Both LPA and PGE2/PGD2 are also drivers of cancer progression acting as agents that can stimulate proliferation, motility, and invasiveness of cancer cells. We are now investigating whether arsenic induces a pro-inflammatory response in vivo in mice through autocrine stimulation of macrophage-derived prostaglandins and LPA. Since chronic inflammation is a necessary driver of cancer progression, we hypothesize that arsenic may stimulate chronic inflammatory responses as well as stimulate cancer cell motility and invasiveness through generation of LPA and PGE2/PGD2. We anticipate that these studies will provide key insights into the mechanisms behind arsenic exposure and development of cancer.
In a more recently started project area, we have been developing innovative chemoproteomic and metabolomic platforms for assessing the direct biological targets of environmental chemicals and their mode of action in vivo in complex living systems. In one strategy, we have been utilizing a chemical proteomic platform termed activity-based protein profiling (ABPP), which uses active-site directed chemical probes to direct assess the functional state of large numbers of enzymes in complex biological systems, to directly identify functional targets of widely used organophosphorus (OP) pesticides. We found that these pesticides directly inhibit over 20 serine hydrolases in vivo leading to widespread disruptions in lipid metabolism. Through identifying direct biological targets of OP pesticides, we showed heretofore unrecognized modes of toxicity that may be associated with these agents. Overall, our study underscored the utility of utilizing multidimensional profiling approaches to obtain a more complete understanding of toxicities associated with environmental chemicals. We recently published this study in ACS Chemical Biology.
We have also developed a broader chemoproteomic strategy that incorporates the power of bioorthogonal chemistry with proteomics and environmental toxicology to inform toxicological mechanisms of OP flame retardants. We developed a chemical probe mimic of the OP flame retardant triphenyl phosphate (TPP) that incorporates a bioorthogonal alkyne handle (TPP-alkyne (TPPyne)). This probe was administered to mice to allow the chemical to interact with macromolecular targets in vivo. Then, we removed tissues from the mouse and appended an analytical handle (biotin-azide) onto the TPPyne using “click chemistry” to generate a triazole conjugate with biotin. Then the TPPyne-bound targets were enriched by biotin and bound proteins were identified by proteomics. The specificity of the TPPyne bound protein targets were confirmed by competition with the parent compound TPP. We found that TPP in vivo inhibits several carboxylesterase enzymes in mouse liver. Using metabolomics, we found that TPP inhibition of carboxylesterases led to wide-spread metabolic dysregulation, including hypertriglyceridemia in liver and serum. Thus, using chemoproteomic and metabolomic strategies, we found that TPP elicits metabolic toxicities through a unique mechanism of inhibiting carboxylesterases in vivo. We have submitted this manuscript to Nature Chemical Biology.
Thus, Core C has made substantial progress in the past year by: 1) identifying novel biomarkers and metabolic drivers of lymphoma; 2) identifying potential metabolic mechanism of action of arsenic in promoting inflammation and cancer; and 3) developing and deploying chemoproteomic and metabolomic strategies for broadly identifying functional biological targets of environmental chemicals and characterizing their mode of action.
Significance
We were able to not only find a biomarker of lymphoma in human serum, but also a potential metabolic driver of disease, which could lead to better risk diagnosis and treatment. Our work has provided key insights into the mechanisms behind arsenic exposure and development of cancer and a more complete understanding of toxicities associated with environmental chemicals.
Future Plans
We will continue to support research methods and analysis for Project research and will use state-of-the-art chemical approaches that combine bioorthogonal probe development with proteomics and metabolomics to provide facilities and methodologies for chemical proteomic and metabolomic technologies for identifying protein targets and mechanism of action of Superfund chemicals.
2012
Currently under review.
2011
Currently under review.
2010
The Toxicogenomics Laboratory Core provides a centralized source of specialized facilities and equipment, services, well-tested collection and storage protocols, and expert technical support using the latest “-omics” technologies and analytical instruments for project investigators. These services greatly enhance the ability of project investigators to achieve their overall goals.
Overall goal
The goal of this laboratory core is to provide the infrastructure and core expertise for Projects 1-4 to achieve their goals. The overall goal of the Program is to apply functional genomics, proteomics, transcriptomics, and nanotechnology to better detect arsenic, mercury, benzene, polycyclic aromatic hydrocarbons, trichloroethylene, and other Superfund priority chemicals in the environment; evaluate their effects on human health, especially the health of susceptible populations such as children; and remediate their presence and reduce their toxicity. Projects 1 through 4 use functional genomics, proteomics, and transcriptomics in their studies. Further, Projects 1 and 3 are epidemiological studies that require sophisticated sample processing so that these technologies can be applied. The success of Projects 1-4 largely depends on the effective handling and management of biological samples, as well as access to and expertise in the latest “-omic” technologies. Thus, detailed collection and storage protocols have been designed and core facilities provided for the cytogenetic, genotyping, gene expression and proteomic analyses proposed in Projects 1-4.
Important accomplishments so far
The core has processed, maintained and stored biological samples and cell lines; provided facilities and methodologies for cytogenetic analysis (study of chromosome structure); provided facilities for gene expression profiling using Affymetrix, Illumina, and custom array technologies; provided facilities for proteomic analyses using various mass spectrometric technologies; and provided facilities and methodologies for the analysis of genetic polymorphisms by Taqman-based and bead array technologies using the ABI 7900 Sequence Detection System and Illumina Bead Station platforms.
Accomplishments for the last year
We conducted protein expression profiling in arsenic target cell lines to help us identify candidate biomarkers of response to arsenic, thereby helping to elucidate the mechanisms of arsenic toxicity. The most significantly altered peptides were chosen for protein identification via mass spectrometry. To follow up, we performed analyses to confirm the identified expression changes of 3 proteins, HSPA5, MCM6 and GNB1, which are involved in protein folding, DNA replication and signal transduction, respectively. For this, we treated human HK-2 kidney cells and HOK-16B keratinocytes to both inorganic arsenic and MMA, a toxic, methylated form of arsenic found as a common metabolite in the human body.
What we plan to do next
Future experiments will involve 2D DIGE analysis on cells exposed for multiple time points and multiple biologically relevant concentrations of arsenic to determine the specificity of response to treatment, followed by measurement of expression changes in urine specimens from children exposed to high and low levels of arsenic.
2009
Christine Skibola and Chris Vulpe, co-leaders of the Core C laboratory facilities, have continued to provide significant support in cytogenetic, cell culture, genomic and proteomic analyses to meet the goals of Projects 1-3. Specifically, in our work with Project 1, Core C continues to process childhood leukemia bone marrow samples and peripheral blood samples and perform cytogenetic analyses by fluorescent in situ hybridization on a subset of cases as part of the Northern California Childhood Leukemia Study.
In previous work with Project 3 investigators, we found that genetic variation in the cystathionine-b-synthase (CBS) gene were associated with significant increases in the toxic arsenic metabolite, MMA, found in the urine in a case-control study population exposed to either high or low arsenic levels from Argentina. These SNPs also were associated with lower urinary levels of an arsenic metabolite, DMA, considered to be less toxic than MMA. To follow up on these findings, we are currently performing further genotyping of CBS polymorphisms to help us determine what actual CBS gene variants are responsible for the differences in the percent of arsenic metabolites found in the urine. These studies will help us to further understand the genetic basis for susceptibility to arsenic-induced disease.
In additional work related to Project 3, our laboratory conducted proteomic analyses on biological specimens from persons exposed to arsenic where we found decreased β-defensin-1 (HBD1) peptides in urine from individuals in Nevada and Chile exposed to arsenic in drinking water (Hegedus et al., 2008). Subsequent in vitro analysis revealed suppressed HBD1 mRNA following arsenic treatment, providing evidence that HBD1 may be a biomarker of response to arsenic. To further explore these findings, we recently investigated effects of arsenic in human cells derived from the skin, kidney and bladder, which are targets of arsenic toxicity. First, we performed RT-PCR analysis to measure HBD1 expression, and found highest and lowest expression in kidney and bladder cells, respectively. Our data revealed significant decreases in HBD1 levels following treatment with biologically relevant doses of arsenic. We also found that the kidney cells were more sensitive to arsenic than other cell types. Taken together, these data suggest that decreased urinary HBD1 measured in our epidemiological studies is most likely a direct result of decreased production of HBD1 in the kidneys, rather than the bladder, resulting from arsenic exposure. We are currently investigating the mechanism of HBD1 suppression and measuring HBD1 protein levels in As-treated cell lines. HBD1 is an antimicrobial peptide produced in the skin and mucosal epithelia of multiple tissues, and acts in both innate and adaptive immune responses. Decreased HBD1 expression has been reported in various cancers, suggesting that HBD1 may be a tumor suppressor gene. Therefore, it is plausible that suppression of HBD1 may play a role in arsenic-related carcinogenesis.
2008
Christine Skibola and Chris Vulpe, co-leaders of the Toxicogenomics laboratory facilities, have continued to provide significant support in cytogenetic, cell culture, proteomic and genetic analyses to meet the goals of multiple projects. Specifically, in their work with Drs. Buffler and Smith, they processed 45 childhood leukemia bone marrow samples and 40 peripheral blood samples, and performed cytogenetic analyses by fluorescent in situ hybridization on an additional 25 cases.
As part of their work with project investigators Alan Smith and Craig Steinmaus, they studied the role of genetics in arsenic metabolism by examining the percent of ingested arsenic excreted in the urine as monomethylarsonic/monomethylarsonous acid (%MMA) and as dimethylarsinic/dimethylarsinous acid (%DMA). They investigated polymorphisms in methylenetetrahydrofolate reductase, cystathionine-β-synthase (CBS), methionine synthase, thymidylate synthase, dihydrofolate reductase and serine hydroxymethyltransferase 1, genes that encode enzymes involved in folate metabolism. Two additional polymorphisms in glutathione-S-transferase-1 (GST01) also were chosen due to the modest influence of GST01 SNPs on urinary %MMA in previous reports. In a case-control study population exposed to either high or low arsenic levels from Cordoba Province, Argentina, they found statistically significant increases in %MMA associated with variant genotypes for CBS rs234709 and rs4920037 polymorphisms. Taking the mean level of %MMA, these gene variants accounted for a 26% increase in MMA levels in this population. These gene variants also accounted for a modest negative association with %DMA. MMA is considered to be the ultimate toxic arsenic metabolite (manuscript submitted). The researchers also went on to show potential gene-environment interaction with CBS SNPs, %MMA and risk of lung cancer (manuscript in preparation). These findings are the first to suggest that CBS SNPs may influence arsenic metabolism in humans and susceptibility to arsenic-induced disease.
The discovery of gene variants that influence arsenic metabolism may help to elucidate the mechanisms of arsenic-induced disease which are currently unknown. The Core plans to further study the influence of additional CBS gene variants on the potential toxic effects of MMA3 in humans and the role of other folate-metabolizing genes in arsenic toxicity.
2007
The Toxicogenomics Laboratory Core facilities have continued to provide significant support in cytogenetic, gene expression, cell culture and proteomic analyses to meet the goals of many Berkeley SBRP Projects. Specifically, in our work with The Biomarkers of Chemical Exposure and Leukemia Risk project, the lab continues to process childhood leukemia bone marrow samples (n=60) and peripheral blood samples (n=72), and the researchers have performed cytogenetic analyses by fluorescent in situ hybridization on another 50 subjects. The Core is also actively working on methods to isolate all forms of RNA, DNA and protein simultaneously from the samples.
As a continuation of the Toxicogenomics Labwork with The Arsenic Biomarker Epidemiology project investigators and the novel finding of reduced human beta defensin 1 (HBD-1) protein levels in urine from men highly exposed to arsenic, the investigators went on to determine the relevance of this finding in a replication study of an arsenic exposed population from Chile. Proteomic analyses of urine from this second study confirmed the decrease of the HBD-1 peptides in men and not women suggesting that HBD-1 may be a biomarker of response in men exposed to high levels of arsenic. In separate in vitro experiments, gene expression analysis of 293T and HeLa cell lines treated with organic and inorganic arsenic demonstrated reduced HBD1 mRNA confirming that the observed decrease in HBD-1 resulted from arsenic exposure. HBD-1 is an antimicrobial peptide constitutively expressed in multiple tissues including epithelial cells of the respiratory and urogenital systems. Recent studies support its role as a tumor suppressor gene for urological cancers suggesting that decreased HBD-1 levels may play a role in the development of cancers associated with As exposure.
The discovery of these biomarkers may be significant because they can be used to aid in the identification, diagnosis, and treatment of affected individuals and people who may be at risk but do not yet exhibit symptoms.
2006
The Toxicogenomics laboratory Core facilities have provided significant support in cytogenetic analyses, genotyping, gene expression and proteomic analyses to begin to meet the goals of the Biomarkers of Chemical Exposure and Leukemia Risk, the Functional Profiling of Susceptibility Genes, and the Arsenic Biomarker Epidemiology projects. Proteomic analyses of biological samples from the Biomarkers of Chemical Exposure and Leukemia Risk project and the Arsenic Biomarker Epidemiology project have led to the identification of two potentially important biomarkers: a human beta defensin 1 peptide that is down regulated in arsenic exposed men, and pro-thymosin-a, a protein that was differentially expressed in acute lymphoblastic leukemia (ALL) versus acute myelogenous leukemia (AML) childhood leukemias.
The Core researchers have maintained ALL cell lines REH [t(12:21)], TK6 and 697 [t(1:19)] for processing for the Biomarkers of Chemical Exposure and Leukemia Risk project and have compared the expression of specific proteins to childhood leukemia samples. Core researchers found that pro-thymosin-a (a small acidic nuclear protein that is thought to be involved in cell cycle progression, proliferation, and cell differentiation) was differentially expressed in ALL versus AML cell lines and patient samples.
In the Cores work with the Arsenic Biomarker Epidemiology project, the researchers detected three proteins significantly decreased in men with high exposure to arsenic when compared to men with low exposure. One of these proteins has tentatively been identified as a human beta defensin 1 peptide.
The discovery of these biomarkers may be significant because they can be used to aid in the identification, diagnosis, and treatment of affected individuals and people who may be at risk but do not yet exhibit symptoms.