Project 2 Update Archive
Our goal is to help understand why toxic chemicals can affect some people more severely than others. We think that differences in our genes could influence the response to these chemicals. We are working to discover which genes are important for certain chemicals that are of concern at Superfund sites. In particular, we are focusing on benzene and trichloroethylene which are both industrial chemicals, and arsenic, a toxic metal also present at many sites. We are using model organisms to identify the genes that are important in people. We have recently found that a gene involved in a rare form of cancer in people, neurofibromatosis, is also involved in people’s response to benzene. Our work suggests that people with variations in this gene, called NF1, could be more likely to develop blood related toxicity after exposure to benzene. Similarly, we have recently found that genes involved in DNA repair are very important in the toxicity of trichloroethylene to the kidney. Together this work will help predict which people are likely to develop disease when exposed to superfund chemicals.
TCE is an environmental contaminant and human carcinogen that remains an environmental health hazard decades after its introduction. Studies have identified the metabolite dichlorovinyl cysteine (DCVC) as the penultimate mediator of TCE renal toxicity and ultimately, renal cancer. We demonstrated that DCVC causes DNA damage that results in substrates for translesion synthesis and recombination. These findings are the first to provide mechanistic and genetic evidence supporting DCVC genotoxicity as a likely contributor to TCE-induced renal cancer. Additioanlly, our newly developed semi-solid medium based screening method in human haploid cells allows efficiently and simultaneously screening and generating mutant colonies from cells resistant to exposures of chemicals of interest.
We have demonstrated that exposure to trichloroethylene (TCE) metabolite dichlorovinyl cysteine (DCVC) can cause DNA damage that does distort the helix, but results in a decrease in a cell’s ability to correct the damage. Since the last update we have set up an automated approach to screen DNA samples after exposure to an increased number of toxicants.
Human haploid cell models are advantageous as an induced gene mutation can result in a clear phenotype due to the absence of a second gene copy. We recently developed a more efficient semi-solid medium based screening platform that employs a human haploid cell mutant library (KBM7-Mu) to identify genes that modulate sensitivity to chemical exposures. Compared to the liquid medium-based approach, our method allows for simultaneously screening and generating mutant colonies from cells resistant to the chemical of interest. This shortens the entire screening process and decreases the rate of false positives.
Both of these developments in DNA analysis have dramatically shortened the entire screening process and decreased the rate of false positives, increasing the effectiveness of our research.
We will continue DNA studies and analyses using the yeast model and expanding the application of our findings when possible. We continue to use our human haploid cell system to screen for genes that are involved in toxicity of other environmental chemicals.
Currently under review.
Currently under review.
People clearly differ in their susceptibility to the toxic effects of Superfund chemicals and a genetic (familial) component is strongly suspected.
The aim of Project 2 is to identify genes that confer susceptibility to Superfund chemicals, through the use of a simple yeast model system that allows rapid identification of these genes. Further studies are then done to confirm the genes in both human cells in a Petri dish and eventually in human populations. The investigators are studying Superfund-related chemicals including benzene, arsenic, trichloroethylene (TCE), and flame retardants. An increased understanding of the genetic variability in response to these toxicants will enable more accurate risk assessments for sites contaminated with these compounds.
Important discoveries so far
To date, the investigators have been able to examine thousands of genes in yeast cells for their importance in an individual’s vulnerability to toxic chemicals at Superfund sites, including various metals, arsenic compounds, and benzene and its metabolites. They have discovered several key genes (including NF1, SRXN1, PRDX1) associated with the response to oxidative stress following exposures to benzene and its metabolites. Benzene is an established cause of leukemia and other blood disorders, and oxidative DNA damage may be a potential mechanism to cause the disease.
They also found that yeast deletion mutants lacking the gene MTQ2 were highly resistant to arsenic exposure. The human equivalent of yeast MTQ2 is N-6 adenine-specific DNA methyltransferase 1 (N6AMT1). Enhanced expression of N6AMT1 in human bladder cells significantly increased their resistance to the toxicity of arsenic and its metabolites and N6AMT1 was shown to methylate arsenic to a non-toxic metabolite. The newly discovered protein N6AMT1 could play a role in susceptibility to arsenic toxicity and carcinogenicity.
Highlight for last year
The most important thing we discovered over the last year was that a recently discovered protein called N6AMT1 can metabolize arsenic compounds and lower toxicity. This is a novel finding. A paper on this is in press in Environmental Health Perspectives.
What we plan to do next
The investigators will extend the studies with the yeast method to several persistent bio-accumulative halogenated toxicants of emerging concern at Superfund sites. Further, they will apply a novel and complementary human cell screening approach to identify additional candidate human susceptibility genes. Together, these studies will provide a comprehensive high-throughput approach to identify important genes and cellular processes involved in susceptibility to Superfund chemicals.
People are exposed to a variety of toxic chemicals present in the environment on a daily basis. However, some people develop disease as a result and some not. An individual’s chances depend in part on subtle genetic differences, which make up each person’s unique genetic blueprint. The goal of Project 2 is to identify these key genes efficiently using a yeast model system then to confirm the association in both population and human cells in vitro studies.
To-date, we have been able to examine thousands of genes in yeast cells for their importance in an individual’s vulnerability to toxic chemicals at Superfund sites, including various metals, arsenic compounds, benzene and its metabolites. In the past year, we have discovered several key genes associated with the response to oxidative stress following exposures to benzene and its metabolites. Benzene is an established cause of leukemia and other blood disorders, and oxidative DNA damage may be a potential mechanism to cause the disease. We have currently focused on one of the genes identified in yeast cells, showing that absence of the gene (NF1) leads to a reduced ability to form colonies by hematopoietic cells in the presence of benzene metabolites, such as hydroquinone.
In addition to yeast cells, we continue to investigate roles of the human gene, WRN, in benzene-induced toxicity in human cells. We have shown that single-nucleotide polymorphisms (SNPs) in this gene are associated with susceptibility to benzene-induced hematotoxicity in exposed workers, and have demonstrated that the loss of WRN increases genomic instability, and its depletion enhances genotoxicity of hydroquinone.
Like benzene, arsenic is also an established human carcinogen. We have identified a group of genes (MYST1, N6AMT1, etc) that are essential and detrimental for yeast growth in inorganic arsenic (AsIII) and mono-methylated arsenic compound (MMA) exposures. We recently published a study showing that the alteration of histone modification regulated by MYST1 as a consequence of arsenic exposure plays an important epigenetic role in arsenic-induced toxicity. Additionally, we have identified a new putative methyltransferase, which may participate in the metabolic conversion of arsenicals into methylated products. Considering the important role of methylated metabolites in arsenic induced toxicity, we have highlighted the importance of our findings and proposed emerging roles of epigenetic mechanisms in arsenic carcinogenesis.
In 2009, our research team in Project 2 published numerous papers that greatly increased understanding of genetic determinants of human susceptibility in chemical exposure to benzene and arsenic. These findings have been reported in many national and international meetings. Dr. Xuefeng Ren was invited to present his arsenic studies in human cells at the 2009 Superfund annual meeting in New York, which won Dr. Ren the 2009 Postdoctoral Fellowship Award from the U.S. National Society of Toxicology (SOT). Along with Dr. Matthew North and Dr. Zhiying Ji, who are also vital members of the project team, all three were invited to present their novel research results at the 2009 SOT annual meeting in Baltimore. Drs. Ji, Ren and North also each won 2009 Young Investigator Awards from Northern California SOT.
Many people are exposed to a variety of toxic chemicals present in the environment on a daily basis. However, only some people develop disease (get sick) as a result. An individual’s chances depend in part on subtle differences in the genes which comprise the genetic blueprint for each person. Unfortunately, for most toxic chemicals, researchers don’t know where to look for these important variations because they don’t know which of the tens of thousands of genes are important for dealing with each toxic chemical. In this project, Drs. Christopher Vulpe and Luoping Zhang are figuring out which genes are the important ones to focus on by using baker’s yeast. By using yeast, they have been able to check thousands of genes for their importance in an individual’s vulnerability to Superfund chemicals. In the past year, the research group led by Dr. Chris Vulpe found several genes associated with the packaging of DNA in a cell (the chromatin) as key to sensitivity to arsenic. Dr. Luoping Zhang’s group further demonstrated the importance of one of these genes in human cells. Arsenic is an established cause of bladder cancer in humans and these findings suggest that alterations in the way that DNA is packaged into chromatin may affect arsenic’s toxicity and carcinogenicity. In the next year, the group plans to look to see if these variations in these genes play a role in the likelihood that a person will develop bladder cancer or other diseases after being exposed to arsenic. Understanding the genetic determinants of chemical susceptibility in humans can help identify groups of people that could be at increased risk of developing disease following exposure to toxic agents, and to design prevention and intervention strategies with the aim of preserving public health.
The focus for this year has been the selection of candidate biomarkers in yeast for susceptibility to sodium arsenite (AsIII) and monomethyl arsonous acid III (MMAIII) toxicity. MMAIII is a toxic metabolite of arsenic. The adverse effects of AsIII exposure in people, as well as increased sensitivity relative to other species, are thought to be due mainly to MMAIII. By screening a collection of yeast deletion mutants, Berkeley researchers Christopher Vulpe and Luoping Zhang have identified several susceptibility genes in yeast, that is, genes that are essential for yeast growth in AsIII, MMAIII or both. Vulpe and Zhang have also confirmed the sensitivity to arsenicals of selected yeast strains that carry deletions in the genes of interest.
Some of the identified genes are associated with chromatin remodeling, DNA repair, cell cycle regulation and oxidative stress response. For the selected yeast genes, there is a high degree of conservation with human genes, not only in sequence but also in function. Moreover, some of these genes encode for components in the same molecular complex that are conserved in humans and, interestingly, some of these have been associated with human cancer in the literature. The research team is preparing a manuscript for publication of these results and has already presented them at the Annual SBRP meeting in North Carolina.
Vulpe and Zhang hypothesized that these homologous human genes can play a role in sensitivity to arsenic in man. To test this, they are currently performing experiments in human cell lines in order to determine the relevance of these findings in humans.
Both researchers have concluded the yeast screens for cadmium, lead and zinc and are starting to analyze the data with bioinformatics and systems biology tools that they previously established for their arsenic data analysis, to compare the responses of these three metals at a cellular level. In addition, the research group has identified some candidate susceptibility genes in the same manner as for arsenic although the analysis for these metals is not yet complete. The project leaders are currently conducting the screens for the remaining two metals, chromium and nickel and have begun working on benzene metabolites, including hydroquinone.
Using siRNA, the researchers demonstrated that the Werner syndrome (WRN) gene was involved in protecting HeLa cells against hydroquinone-induced damage. Further, SNPs in WRN have been associated with significant decreases in total white blood cell counts among benzene-exposed workers. To further investigate WRN‘s role in benzene hematotoxicity, they employed shRNA to establish two stably knock down WRN in two hematopoietic cell lines, lymphoblastic (TK6) and promyeloblastic (HL60) cells, and achieved >90% knock down in both cell lines. The preliminary results show increased levels of apoptosis and S/G2 phase arrest in the WRN-depleted cells compared to wild-type controls when exposed to hydroquinone, suggesting increased sensitivity to hydroquinone. Furthermore, in WRN-depleted cells research has shown increased levels of the protein BLM, a DNA repair protein implicated in Bloom syndrome (BLM), which suggests that it may be compensating for the loss of WRN. Vulpe and Zhang are currently exploring WRN‘s relationship and interactions with other DNA repair genes in the modulation of benzene related cytotoxicity and genotoxicity.
The Functional Profiling of Susceptibility genes project takes advantage of the similarities between the yeast S. cerevisiae and humans to identify genes that are sensitive to the chemicals on the Superfund list. The benefits of this new approach are that an expansive selection of candidate genes can be tested and there is no need to find an exposed population and carry out an epidemiological study, which can be costly and is narrow in focus.
From the 20 toxicants the project researchers planned to test, the researchers decided to start with the Metals/Metalloids group and have been working with sodium arsenite (NaAsO2), zinc chloride (ZnCl2), lead chloride (PbCl2) and cadmium chloride (CdCl2) to determine which toxicants inhibit growth of yeast the most. In this analysis the researchers found that cadmium was the most potent, followed by arsenic, lead and zinc. Project researchers were able to identify several genes particularly sensitive to arsenic. One of the most sensitive strains in this first dataset was Rad10”. The researchers also found that the gene WRN is important in protecting against benzene-induced toxicity, playing a key role in mounting a normal DNA damage response following DNA double-strand breaks.
These findings should enable future work in examining associations between common variants in these genes and adverse outcomes. In addition, this project will likely provide important insights in the cellular processes leading to toxicity for priority Superfund chemicals.