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1.  The impact of FANCD2 deficiency on formaldehyde-induced toxicity in human lymphoblastoid cell lines 
Archives of toxicology  2012;87(1):189-196.
Formaldehyde (FA), a major industrial chemical and ubiquitous environmental pollutant, has recently been classified by the International Agency for Research on Cancer as a human leukemogen. The major mode of action of FA is thought to be the formation of DNA-protein crosslinks (DPCs). Repair of DPCs may be mediated by the Fanconi anemia pathway; however, data supporting the involvement of this pathway is limited, particularly in human hematopoietic cells. Therefore, we assessed the role of FANCD2, a critical component of the Fanconi anemia pathway, in FA-induced toxicity in human lymphoblast cell models of FANCD2-deficiency (PD20 cells) and FANCD2-sufficiency (PD20-D2 cells). After treatment of the cells with 0-150 μM FA for 24 hours, DPCs were increased in a dose-dependent manner in both cell lines, with greater increases in FANCD2-deficient PD20 cells. FA also induced cytotoxicity, micronuclei, chromosome aberrations, and apoptosis in a dose-dependent manner in both cell lines, with greater increases in cytotoxicity and apoptosis in PD20 cells. Increased levels of γ-ATR and γ-H2AX in both cell lines suggested the recognition of FA-induced DNA damage; however, the induction of BRCA2 was compromised in FANCD2-deficient PD20 cells, potentially reducing the capacity to repair DPCs. Together, these findings suggest that FANCD2 protein and the Fanconi anemia pathway are essential to protect human lymphoblastoid cells against FA toxicity. Future studies are needed to delineate the role of this pathway in mitigating FA-induced toxicity, particularly in hematopoietic stem cells, the target cells in leukemia.
doi:10.1007/s00204-012-0911-6
PMCID: PMC4312773  PMID: 22872141
DNA-protein crosslinks; micronuclei; chromosome aberrations; apoptosis; DNA damage and repair; Fanconi anemia
2.  A genome-wide association study of marginal zone lymphoma shows association to the HLA region 
Vijai, Joseph | Wang, Zhaoming | Berndt, Sonja I. | Skibola, Christine F. | Slager, Susan L. | de Sanjose, Silvia | Melbye, Mads | Glimelius, Bengt | Bracci, Paige M. | Conde, Lucia | Birmann, Brenda M. | Wang, Sophia S. | Brooks-Wilson, Angela R. | Lan, Qing | de Bakker, Paul I. W. | Vermeulen, Roel C. H. | Portlock, Carol | Ansell, Stephen M. | Link, Brian K. | Riby, Jacques | North, Kari E. | Gu, Jian | Hjalgrim, Henrik | Cozen, Wendy | Becker, Nikolaus | Teras, Lauren R. | Spinelli, John J. | Turner, Jenny | Zhang, Yawei | Purdue, Mark P. | Giles, Graham G. | Kelly, Rachel S. | Zeleniuch-Jacquotte, Anne | Ennas, Maria Grazia | Monnereau, Alain | Bertrand, Kimberly A. | Albanes, Demetrius | Lightfoot, Tracy | Yeager, Meredith | Chung, Charles C. | Burdett, Laurie | Hutchinson, Amy | Lawrence, Charles | Montalvan, Rebecca | Liang, Liming | Huang, Jinyan | Ma, Baoshan | Villano, Danylo J. | Maria, Ann | Corines, Marina | Thomas, Tinu | Novak, Anne J. | Dogan, Ahmet | Liebow, Mark | Thompson, Carrie A. | Witzig, Thomas E. | Habermann, Thomas M. | Weiner, George J. | Smith, Martyn T. | Holly, Elizabeth A. | Jackson, Rebecca D. | Tinker, Lesley F. | Ye, Yuanqing | Adami, Hans-Olov | Smedby, Karin E. | De Roos, Anneclaire J. | Hartge, Patricia | Morton, Lindsay M. | Severson, Richard K. | Benavente, Yolanda | Boffetta, Paolo | Brennan, Paul | Foretova, Lenka | Maynadie, Marc | McKay, James | Staines, Anthony | Diver, W. Ryan | Vajdic, Claire M. | Armstrong, Bruce K. | Kricker, Anne | Zheng, Tongzhang | Holford, Theodore R. | Severi, Gianluca | Vineis, Paolo | Ferri, Giovanni M. | Ricco, Rosalia | Miligi, Lucia | Clavel, Jacqueline | Giovannucci, Edward | Kraft, Peter | Virtamo, Jarmo | Smith, Alex | Kane, Eleanor | Roman, Eve | Chiu, Brian C. H. | Fraumeni, Joseph F. | Wu, Xifeng | Cerhan, James R. | Offit, Kenneth | Chanock, Stephen J. | Rothman, Nathaniel | Nieters, Alexandra
Nature Communications  2015;6:5751.
Marginal zone lymphoma (MZL) is the third most common subtype of B-cell non-Hodgkin lymphoma. Here we perform a two-stage GWAS of 1,281 MZL cases and 7,127 controls of European ancestry and identify two independent loci near BTNL2 (rs9461741, P=3.95 × 10−15) and HLA-B (rs2922994, P=2.43 × 10−9) in the HLA region significantly associated with MZL risk. This is the first evidence that genetic variation in the major histocompatibility complex influences MZL susceptibility.
Marginal zone lymphoma (MZL) is a common subtype of B-cell non-Hodgkin lymphoma. Here the authors carry out a two-stage genome-wide association study in over 8,000 Europeans and identify two new MZL risk loci at chromosome 6p, implicating the major histocompatibility complex in the disease for the first time.
doi:10.1038/ncomms6751
PMCID: PMC4287989  PMID: 25569183
3.  Formaldehyde Induces Micronuclei in Mouse Erythropoietic Cells and Suppresses the Expansion of Human Erythroid Progenitor Cells 
Toxicology letters  2013;224(2):233-239.
Although formaldehyde (FA) has been classified as a human leukemogen, the mechanisms of leukemogenesis remain elusive. Previously, using colony-forming assays in semi-solid media, we showed that FA exposure in vivo and in vitro was toxic to human hematopoietic stem/progenitor cells. In the present study, we have applied new liquid in vitro erythroid expansion systems to further investigate the toxic effects of FA (0–150 µM) on cultured mouse and human hematopoietic stem/progenitor cells. We determined micronucleus (MN) levels in polychromatic erythrocytes (PCEs) differentiated from mouse bone marrow. We measured cell growth, cell cycle distribution, and chromosomal instability, in erythroid progenitor cells (EPCs) expanded from human peripheral blood mononuclear cells. FA significantly induced MN in mouse PCEs and suppressed human EPC expansion in a dose-dependent manner, compared with untreated controls. In the expanded human EPCs, FA slightly increased the proportion of cells in G2/M at 100 µM and aneuploidy frequency in chromosomes 7 and 8 at 50 µM. Our findings provide further evidence of the toxicity of FA to hematopoietic stem/progenitor cells and support the biological plausibility of FA-induced leukemogenesis.
doi:10.1016/j.toxlet.2013.10.028
PMCID: PMC3891867  PMID: 24188930
formaldehyde; erythroid progenitor; micronuclei; aneuploidy
4.  Towards incorporating epigenetic mechanisms into carcinogen identification and evaluation 
Carcinogenesis  2013;34(9):1955-1967.
Remarkable progress in the field of epigenetics has turned academic, medical and public attention to the potential applications of these new advances in medicine and various fields of biomedical research. The result is a broader appreciation of epigenetic phenomena in the a etiology of common human diseases, most notably cancer. These advances also represent an exciting opportunity to incorporate epigenetics and epigenomics into carcinogen identification and safety assessment. Current epigenetic studies, including major international sequencing projects, are expected to generate information for establishing the ‘normal’ epigenome of tissues and cell types as well as the physiological variability of the epigenome against which carcinogen exposure can be assessed. Recently, epigenetic events have emerged as key mechanisms in cancer development, and while our search of the Monograph Volume 100 revealed that epigenetics have played a modest role in evaluating human carcinogens by the International Agency for Research on Cancer (IARC) Monographs so far, epigenetic data might play a pivotal role in the future. Here, we review (i) the current status of incorporation of epigenetics in carcinogen evaluation in the IARC Monographs Programme, (ii) potential modes of action for epigenetic carcinogens, (iii) current in vivo and in vitro technologies to detect epigenetic carcinogens, (iv) genomic regions and epigenetic modifications and their biological consequences and (v) critical technological and biological issues in assessment of epigenetic carcinogens. We also discuss the issues related to opportunities and challenges in the application of epigenetic testing in carcinogen identification and evaluation. Although the application of epigenetic assays in carcinogen evaluation is still in its infancy, important data are being generated and valuable scientific resources are being established that should catalyse future applications of epigenetic testing.
doi:10.1093/carcin/bgt212
PMCID: PMC3765050  PMID: 23749751
5.  Tobacco Smoke Exposure and the Risk of Childhood Acute Lymphoblastic and Myeloid Leukemias by Cytogenetic Subtype 
Background
Tobacco smoke contains carcinogens known to damage somatic and germ cells. We investigated the effect tobacco smoke on the risk of childhood acute lymphoblastic leukemia (ALL) and myeloid leukemia (AML), especially subtypes of pre-natal origin like ALL with translocation t(12;21) or high-hyperdiploidy (51–67 chromosomes).
Methods
We collected information on exposures to tobacco smoking before conception, during pregnancy, and after birth in 767 ALL cases, 135 AML cases, and 1,139 controls (1996–2008). Among cases, chromosome translocations, deletions, or aneuploidy were identified by conventional karyotype and fluorescence in-situ hybridization.
Results
Multivariable regression analyses for ALL and AML overall showed no definite evidence of associations with self-reported (yes/no) parental prenatal active smoking and child's passive smoking. However, children with history of paternal prenatal smoking combined with postnatal passive smoking had a 1.5-fold increased risk of ALL (95% CI: 1.01–2.23), compared to those without smoking history (ORs for pre- or postnatal smoking only were close to one). This joint effect was seen for B-cell precursor ALL with t(12;21) (OR=2.08; 95% CI: 1.04–4.16), but not high hyperdiploid B-cell ALL. Similarly, child's passive smoking was associated with an elevated risk of AML with chromosome structural changes (OR=2.76; 95% CI: 1.01–7.58), but not aneuploidy.
Conclusions
our data suggest that exposure to tobacco smoking before were associated with increased risks of childhood ALL and AML; and risks varied by timing of exposure (before and/or after birth) and cytogenetic subtype, based on imprecise estimates.
Impact
Parents should limit exposures to tobacco smoke before and after the child's birth.
doi:10.1158/1055-9965.EPI-13-0350
PMCID: PMC3769478  PMID: 23853208
Childhood leukemia; tobacco smoking; cytogenetics
7.  Characterization of Changes in Gene Expression and Biochemical Pathways at Low Levels of Benzene Exposure 
PLoS ONE  2014;9(5):e91828.
Benzene, a ubiquitous environmental pollutant, causes acute myeloid leukemia (AML). Recently, through transcriptome profiling of peripheral blood mononuclear cells (PBMC), we reported dose-dependent effects of benzene exposure on gene expression and biochemical pathways in 83 workers exposed across four airborne concentration ranges (from <1 ppm to >10 ppm) compared with 42 subjects with non-workplace ambient exposure levels. Here, we further characterize these dose-dependent effects with continuous benzene exposure in all 125 study subjects. We estimated air benzene exposure levels in the 42 environmentally-exposed subjects from their unmetabolized urinary benzene levels. We used a novel non-parametric, data-adaptive model selection method to estimate the change with dose in the expression of each gene. We describe non-parametric approaches to model pathway responses and used these to estimate the dose responses of the AML pathway and 4 other pathways of interest. The response patterns of majority of genes as captured by mean estimates of the first and second principal components of the dose-response for the five pathways and the profiles of 6 AML pathway response-representative genes (identified by clustering) exhibited similar apparent supra-linear responses. Responses at or below 0.1 ppm benzene were observed for altered expression of AML pathway genes and CYP2E1. Together, these data show that benzene alters disease-relevant pathways and genes in a dose-dependent manner, with effects apparent at doses as low as 100 ppb in air. Studies with extensive exposure assessment of subjects exposed in the low-dose range between 10 ppb and 1 ppm are needed to confirm these findings.
doi:10.1371/journal.pone.0091828
PMCID: PMC4006721  PMID: 24786086
8.  Alterations in serum immunoglobulin levels in workers occupationally exposed to trichloroethylene 
Carcinogenesis  2012;34(4):799-802.
Trichloroethylene (TCE) has been associated with a variety of immunotoxic effects and may be associated with an increased risk of non-Hodgkin lymphoma (NHL). Altered serum immunoglobulin (Ig) levels have been reported in NHL patients and in animals exposed to TCE. Recently, we reported that occupational exposure to TCE is associated with immunosuppressive effects and immune dysfunction, including suppression of B-cell counts and activation, even at relatively low levels. We hypothesized that TCE exposure would also affect Ig levels in humans. We measured serum levels of IgG, IgM and IgE, by enzyme-linked immunosorbent assay, in TCE-exposed workers (n = 80) and unexposed controls (n = 45), matched by age and gender, in a cross-sectional, molecular epidemiology study of occupational exposure to TCE in Guangdong, China. Exposed workers had about a 17.5% decline in serum levels of IgG compared with unexposed controls (P = 0.0002). Similarly, serum levels of IgM were reduced by about 38% in workers exposed to TCE compared with unexposed controls (P < 0.0001). Serum levels of both IgG and IgM were significantly decreased in workers exposed to TCE levels below 12 p.p.m., the median exposure level. Adjustment for B-cell counts had minimal impact on our findings. IgE levels were not significantly different between exposed and control subjects. These results provide further evidence that TCE is immunotoxic at relatively low exposure levels and provide additional biologic plausibility for the reported association of TCE with NHL.
doi:10.1093/carcin/bgs403
PMCID: PMC3616671  PMID: 23276795
9.  Occupational exposure to formaldehyde and alterations in lymphocyte subsets 
Background
Formaldehyde is used in many occupational settings, most notably in manufacturing, health care, and embalming. Formaldehyde has been classified as a human carcinogen, but its mechanism of action remains uncertain.
Methods
We carried out a cross-sectional study of 43 formaldehyde exposed-workers and 51 unexposed age and sex-matched controls in Guangdong, China to study formaldehyde’s early biologic effects. To follow-up our previous report that the total lymphocyte count was decreased in formaldehyde-exposed workers compared to controls, we evaluated each major lymphocyte subset (i.e., CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and B cells) and T cell lymphocyte subset (CD4+ naïve and memory T cells, CD8+ naïve and memory T cells, and regulatory T cells). Linear regression of each subset was used to test for differences between exposed workers and controls, adjusting for potential confounders.
Results
Total NK cell and T cell counts were about 24% (p=0.037) and 16% (p=0.0042) lower, respectively, among exposed workers. Among certain T cell subsets, decreased counts among exposed workers were observed for CD8+ T cells (p=0.026), CD8+ effector memory T cells (p=0.018), and regulatory T cells (CD4+FoxP3+: p=0.04; CD25+FoxP3+: p=0.008).
Conclusions
Formaldehyde exposed-workers experienced decreased counts of NK cells, regulatory T cells, and CD8+ effector memory T cells; however, due to the small sample size these findings need to be confirmed in larger studies.
doi:10.1002/ajim.22088
PMCID: PMC3493854  PMID: 22767408
formaldehyde; NK cell; B cell; T cell; T cell subset
10.  Genome-wide Association Study Identifies Multiple Risk Loci for Chronic Lymphocytic Leukemia 
Berndt, Sonja I. | Skibola, Christine F. | Joseph, Vijai | Camp, Nicola J. | Nieters, Alexandra | Wang, Zhaoming | Cozen, Wendy | Monnereau, Alain | Wang, Sophia S. | Kelly, Rachel S. | Lan, Qing | Teras, Lauren R. | Chatterjee, Nilanjan | Chung, Charles C. | Yeager, Meredith | Brooks-Wilson, Angela R. | Hartge, Patricia | Purdue, Mark P. | Birmann, Brenda M. | Armstrong, Bruce K. | Cocco, Pierluigi | Zhang, Yawei | Severi, Gianluca | Zeleniuch-Jacquotte, Anne | Lawrence, Charles | Burdette, Laurie | Yuenger, Jeffrey | Hutchinson, Amy | Jacobs, Kevin B. | Call, Timothy G. | Shanafelt, Tait D. | Novak, Anne J. | Kay, Neil E. | Liebow, Mark | Wang, Alice H. | Smedby, Karin E | Adami, Hans-Olov | Melbye, Mads | Glimelius, Bengt | Chang, Ellen T. | Glenn, Martha | Curtin, Karen | Cannon-Albright, Lisa A. | Jones, Brandt | Diver, W. Ryan | Link, Brian K. | Weiner, George J. | Conde, Lucia | Bracci, Paige M. | Riby, Jacques | Holly, Elizabeth A. | Smith, Martyn T. | Jackson, Rebecca D. | Tinker, Lesley F. | Benavente, Yolanda | Becker, Nikolaus | Boffetta, Paolo | Brennan, Paul | Foretova, Lenka | Maynadie, Marc | McKay, James | Staines, Anthony | Rabe, Kari G. | Achenbach, Sara J. | Vachon, Celine M. | Goldin, Lynn R | Strom, Sara S. | Lanasa, Mark C. | Spector, Logan G. | Leis, Jose F. | Cunningham, Julie M. | Weinberg, J. Brice | Morrison, Vicki A. | Caporaso, Neil E. | Norman, Aaron D. | Linet, Martha S. | De Roos, Anneclaire J. | Morton, Lindsay M. | Severson, Richard K. | Riboli, Elio | Vineis, Paolo | Kaaks, Rudolph | Trichopoulos, Dimitrios | Masala, Giovanna | Weiderpass, Elisabete | Chirlaque, María-Dolores | Vermeulen, Roel C H | Travis, Ruth C. | Giles, Graham G. | Albanes, Demetrius | Virtamo, Jarmo | Weinstein, Stephanie | Clavel, Jacqueline | Zheng, Tongzhang | Holford, Theodore R | Offit, Kenneth | Zelenetz, Andrew | Klein, Robert J. | Spinelli, John J. | Bertrand, Kimberly A. | Laden, Francine | Giovannucci, Edward | Kraft, Peter | Kricker, Anne | Turner, Jenny | Vajdic, Claire M. | Ennas, Maria Grazia | Ferri, Giovanni M. | Miligi, Lucia | Liang, Liming | Sampson, Joshua | Crouch, Simon | Park, Ju-hyun | North, Kari E. | Cox, Angela | Snowden, John A. | Wright, Josh | Carracedo, Angel | Lopez-Otin, Carlos | Bea, Silvia | Salaverria, Itziar | Martin, David | Campo, Elias | Fraumeni, Joseph F. | de Sanjose, Silvia | Hjalgrim, Henrik | Cerhan, James R. | Chanock, Stephen J. | Rothman, Nathaniel | Slager, Susan L.
Nature genetics  2013;45(8):868-876.
doi:10.1038/ng.2652
PMCID: PMC3729927  PMID: 23770605
11.  Modulation of Ras signaling alters the toxicity of hydroquinone, a benzene metabolite and component of cigarette smoke 
BMC Cancer  2014;14:6.
Background
Benzene is an established human leukemogen, with a ubiquitous environmental presence leading to significant population exposure. In a genome-wide functional screen in the yeast Saccharomyces cerevisiae, inactivation of IRA2, a yeast ortholog of the human tumor suppressor gene NF1 (Neurofibromin), enhanced sensitivity to hydroquinone, an important benzene metabolite. Increased Ras signaling is implicated as a causal factor in the increased pre-disposition to leukemia of individuals with mutations in NF1.
Methods
Growth inhibition of yeast by hydroquinone was assessed in mutant strains exhibiting varying levels of Ras activity. Subsequently, effects of hydroquinone on both genotoxicity (measured by micronucleus formation) and proliferation of WT and Nf1 null murine hematopoietic precursors were assessed.
Results
Here we show that the Ras status of both yeast and mammalian cells modulates hydroquinone toxicity, indicating potential synergy between Ras signaling and benzene toxicity. Specifically, enhanced Ras signaling increases both hydroquinone-mediated growth inhibition in yeast and genotoxicity in mammalian hematopoetic precursors as measured by an in vitro erythroid micronucleus assay. Hydroquinone also increases proliferation of CFU-GM progenitor cells in mice with Nf1 null bone marrow relative to WT, the same cell type associated with benzene-associated leukemia.
Conclusions
Together our findings show that hydroquinone toxicity is modulated by Ras signaling. Individuals with abnormal Ras signaling could be more vulnerable to developing myeloid diseases after exposure to benzene. We note that hydroquinone is used cosmetically as a skin-bleaching agent, including by individuals with cafe-au-lait spots (which may be present in individuals with neurofibromatosis who have a mutation in NF1), which could be unadvisable given our findings.
doi:10.1186/1471-2407-14-6
PMCID: PMC3898384  PMID: 24386979
Hydroquinone; in vitro micronucleus assay; IRA2; NF1; Ras; Yeast
12.  Acute lymphoblastic leukaemia in children – is there a role for MTHFR? 
British journal of haematology  2010;149(5):10.1111/j.1365-2141.2010.08101.x.
doi:10.1111/j.1365-2141.2010.08101.x
PMCID: PMC3876734  PMID: 20148884
childhood leukaemia; epidemiology; folic acid
13.  Individual Differences in Arsenic Metabolism and Lung Cancer in a Case-Control Study in Cordoba, Argentina 
Toxicology and applied pharmacology  2010;247(2):10.1016/j.taap.2010.06.006.
In humans, ingested inorganic arsenic is metabolized to monomethylarsenic (MMA) then to dimethylarsenic (DMA), although in most people this process is not complete. Previous studies have identified associations between the proportion of urinary MMA (%MMA) and increased risks of several arsenic-related diseases, although none of these reported on lung cancer. In this study, urinary arsenic metabolites were assessed in 45 lung cancer cases and 75 controls from arsenic-exposed areas in Cordoba, Argentina. Folate has also been linked to arsenic-disease susceptibility, thus an exploratory assessment of associations between single nucleotide polymorphisms in folate metabolizing genes, arsenic methylation, and lung cancer was also conducted. In analyses limited to subjects with metabolite concentrations above detection limits, the mean %MMA was higher in cases than in controls (17.5% versus 14.3%, p = 0.01). The lung cancer odds ratios for subjects with %MMA in the upper tertile compared to those in the lowest tertile was 3.09 (95% CI, 1.08–8.81). Although the study size was too small for a definitive conclusion, there was an indication that lung cancer risks might be highest in those with a high %MMA who also carried cystathionine β-synthase (CBS) rs234709 and rs4920037 variant alleles. This study is the first to report an association between individual differences in arsenic metabolism and lung cancer, a leading cause of arsenic-related mortality. These results add to the increasing body of evidence that variation in arsenic metabolism plays an important role in arsenic-disease susceptibility.
doi:10.1016/j.taap.2010.06.006
PMCID: PMC3849353  PMID: 20600216
arsenic; lung cancer; drinking water; metabolism
14.  Connexin40 regulates platelet function 
Nature Communications  2013;4:2564.
The presence of multiple connexins was recently demonstrated in platelets, with notable expression of Cx37. Studies with Cx37-deficient mice and connexin inhibitors established roles for hemichannels and gap junctions in platelet function. It was uncertain, however, whether Cx37 functions alone or in collaboration with other family members through heteromeric interactions in regulation of platelet function. Here we report the presence and functions of an additional platelet connexin, Cx40. Inhibition of Cx40 in human platelets or its deletion in mice reduces platelet aggregation, fibrinogen binding, granule secretion and clot retraction. The effects of the Cx37 inhibitor 37,43Gap27 on Cx40−/− mouse platelets and of the Cx40 inhibitor 40Gap27 on Cx37−/− mouse platelets revealed that each connexin is able to function independently. Inhibition or deletion of Cx40 reduces haemostatic responses in mice, indicating the physiological importance of this protein in platelets. We conclude that multiple connexins are involved in regulating platelet function, thereby contributing to haemostasis and thrombosis.
Hemichannels and gap junctions containing the connexin Cx37 are required for platelet functions such as aggregation and granule secretion through poorly defined mechanisms. Vaiyapuri et al. show that Cx40 is also required and can act independently of Cx37 in mouse platelets.
doi:10.1038/ncomms3564
PMCID: PMC3806366  PMID: 24096827
15.  Arsenic immunotoxicity: a review 
Environmental Health  2013;12:73.
Exposure to arsenic (As) is a global public health problem because of its association with various cancers and numerous other pathological effects, and millions of people worldwide are exposed to As on a regular basis. Increasing lines of evidence indicate that As may adversely affect the immune system, but its specific effects on immune function are poorly understood. Therefore, we conducted a literature search of non-cancer immune-related effects associated with As exposure and summarized the known immunotoxicological effects of As in humans, animals and in vitro models. Overall, the data show that chronic exposure to As has the potential to impair vital immune responses which could lead to increased risk of infections and chronic diseases, including various cancers. Although animal and in vitro models provide some insight into potential mechanisms of the As-related immunotoxicity observed in human populations, further investigation, particularly in humans, is needed to better understand the relationship between As exposure and the development of disease.
doi:10.1186/1476-069X-12-73
PMCID: PMC3848751  PMID: 24004508
Arsenic; Immune system; Immunotoxicity; Immunocompromised; Immunosuppression
16.  Elevated urinary levels of kidney injury molecule-1 among Chinese factory workers exposed to trichloroethylene 
Carcinogenesis  2012;33(8):1538-1541.
Epidemiological studies suggest that trichloroethylene (TCE) exposure may be associated with renal cancer. The biological mechanisms involved are not exactly known although nephrotoxicity is believed to play a role. Studies on TCE nephrotoxicity among humans, however, have been largely inconsistent. We studied kidney toxicity in Chinese factory workers exposed to TCE using novel sensitive nephrotoxicity markers. Eighty healthy workers exposed to TCE and 45 comparable unexposed controls were included in the present analyses. Personal TCE exposure measurements were taken over a 2-week period before urine collection. Ninety-six percent of workers were exposed to TCE below the current US Occupational Safety and Health Administration permissible exposure limit (100 ppm 8h TWA), with a mean (SD) of 22.2 (35.9) ppm. Kidney injury molecule-1 (KIM-1) and Pi-glutathione S transferase (GST) alpha were elevated among the exposed subjects as compared with the unexposed controls with a strong exposure-response association between individual estimates of TCE exposure and KIM-1 (P < 0.0001). This is the first report to use a set of sensitive nephrotoxicity markers to study the possible effects of TCE on the kidneys. The findings suggest that at relatively low occupational exposure levels a toxic effect on the kidneys can be observed. This finding supports the biological plausibility of linking TCE exposure and renal cancer.
Abbreviations:GSTglutathione-S-transferaseKIM-1kidney injury molecule-1NAGN-acetyl-beta-(d)-glucosaminidaseOVMorganic vapour monitoringTCEtrichloroethyleneVEGFvascular endothelial growth factor.
doi:10.1093/carcin/bgs191
PMCID: PMC3499056  PMID: 22665366
17.  Single molecule quantitation and sequencing of rare translocations using microfluidic nested digital PCR 
Nucleic Acids Research  2013;41(16):e159.
Cancers are heterogeneous and genetically unstable. New methods are needed that provide the sensitivity and specificity to query single cells at the genetic loci that drive cancer progression, thereby enabling researchers to study the progression of individual tumors. Here, we report the development and application of a bead-based hemi-nested microfluidic droplet digital PCR (dPCR) technology to achieve ‘quantitative’ measurement and single-molecule sequencing of somatically acquired carcinogenic translocations at extremely low levels (<10−6) in healthy subjects. We use this technique in our healthy study population to determine the overall concentration of the t(14;18) translocation, which is strongly associated with follicular lymphoma. The nested dPCR approach improves the detection limit to 1 × 10−7 or lower while maintaining the analysis efficiency and specificity. Further, the bead-based dPCR enabled us to isolate and quantify the relative amounts of the various clonal forms of t(14;18) translocation in these subjects, and the single-molecule sensitivity and resolution of dPCR led to the discovery of new clonal forms of t(14;18) that were otherwise masked by the conventional quantitative PCR measurements. In this manner, we created a quantitative map for this carcinogenic mutation in this healthy population and identified the positions on chromosomes 14 and 18 where the vast majority of these t(14;18) events occur.
doi:10.1093/nar/gkt613
PMCID: PMC3763562  PMID: 23873959
18.  Leukemia-related chromosomal loss detected in hematopoietic progenitor cells of benzene-exposed workers 
Benzene exposure causes acute myeloid leukemia, and hematotoxicity, shown as suppression of mature blood and myeloid progenitor cell numbers. As the leukemia-related aneuploidies monosomy 7 and trisomy 8 previously had been detected in the mature peripheral blood cells of exposed workers, we hypothesized that benzene could cause leukemia through the induction of these aneuploidies in hematopoietic stem and progenitor cells. We measured loss and gain of chromosomes 7 and 8 by fluorescence in situ hybridization in interphase colony-forming unit-granulocyte-macrophage (CFU-GM) cells cultured from otherwise healthy benzene-exposed (n=28) and unexposed (n=14) workers. CFU-GM monosomy 7 and 8 levels (but not trisomy) were significantly increased in subjects exposed to benzene overall, compared to levels in the control subjects (p=0.0055 and p=0.0034, respectively). Levels of monosomy 7 and 8 were significantly increased in subjects exposed to <10 ppm (20%, p=0.0419 and 28%, p=0.0056, respectively) and ≥10 ppm (48%, p=0.0045 and 32%, p=0.0354) benzene, compared with controls, and significant exposure-response trends were detected (ptrend=0.0033 and 0.0057). These data show that monosomies 7 and 8 are produced in a dose-dependent fashion in the blood progenitor cells of workers exposed to benzene and may be mechanistically relevant biomarkers of early effect for benzene and other leukemogens.
doi:10.1038/leu.2012.143
PMCID: PMC3472034  PMID: 22643707
Benzene; leukemia; monosomy; hematopoietic progenitor
19.  Multi-locus HLA class I and II allele and haplotype associations with follicular lymphoma 
Tissue Antigens  2012;79(4):279-286.
Follicular lymphoma (FL) is an indolent, sometimes fatal disease characterized by recurrence at progressively shorter intervals and is frequently refractive to therapy. Genome-wide association studies have identified SNPs in the human leukocyte antigen (HLA) region on chromosome 6p21.32–33 that are statistically significantly associated with FL risk. Low to medium resolution typing of single or multiple HLA genes has provided an incomplete picture of the total genetic risk imparted by this highly variable region. To gain further insight into the role of HLA alleles in lymphomagenesis and to investigate the independence of validated SNPs and HLA alleles with FL risk, high-resolution HLA typing was conducted using next-generation sequencing in 222 non-Hispanic white FL cases and 220 matched controls from a larger San Francisco Bay Area population-based case-control study of lymphoma. A novel protective association was found between the DPB1*03:01 allele and FL risk (OR=0.39, 95% CI 0.21–0.68). Extended haplotypes DRB1*01:01-DQA1*01:01-DQB1*05:01 (OR=2.01, 95% CI 1.22–3.38) and DRB1*15-DQA1*01-DQB1*06 (OR=0.55, 95% CI 0.36–0.82) also influenced FL risk. Moreover, DRB1*15-DQA1*01-DQB1*06 was highly correlated with an established FL risk locus, rs2647012. These results provide further insight into the critical roles of HLA alleles and SNPs in FL pathogenesis that involve multi-locus effects across the HLA region.
doi:10.1111/j.1399-0039.2012.01845.x
PMCID: PMC3293942  PMID: 22296171
follicular lymphoma; HLA; genetic risk factors; next-generation sequencing
20.  Performance in Omics Analyses of Blood Samples in Long-Term Storage: Opportunities for the Exploitation of Existing Biobanks in Environmental Health Research 
Environmental Health Perspectives  2013;121(4):480-487.
Background: The suitability for omic analysis of biosamples collected in previous decades and currently stored in biobanks is unknown.
Objectives: We evaluated the influence of handling and storage conditions of blood-derived biosamples on transcriptomic, epigenomic (CpG methylation), plasma metabolomic [UPLC-ToFMS (ultra performance liquid chromatography–time-of-flight mass spectrometry)], and wide-target proteomic profiles.
Methods: We collected fresh blood samples without RNA preservative in heparin, EDTA, or citrate and held them at room temperature for ≤ 24 hr before fractionating them into buffy coat, erythrocytes, and plasma and freezing the fractions at –80oC or in liquid nitrogen. We developed methodology for isolating RNA from the buffy coats and conducted omic analyses. Finally, we analyzed analogous samples from the EPIC-Italy and Northern Sweden Health and Disease Study biobanks.
Results: Microarray-quality RNA could be isolated from buffy coats (including most biobank samples) that had been frozen within 8 hr of blood collection by thawing the samples in RNA preservative. Different anticoagulants influenced the metabolomic, proteomic, and to a lesser extent transcriptomic profiles. Transcriptomic profiles were most affected by the delay (as little as 2 hr) before blood fractionation, whereas storage temperature had minimal impact. Effects on metabolomic and proteomic profiles were noted in samples processed ≥ 8 hr after collection, but no effects were due to storage temperature. None of the variables examined significantly influenced the epigenomic profiles. No systematic influence of time-in-storage was observed in samples stored over a period of 13–17 years.
Conclusions: Most samples currently stored in biobanks are amenable to meaningful omics analysis, provided that they satisfy collection and storage criteria defined in this study.
doi:10.1289/ehp.1205657
PMCID: PMC3620742  PMID: 23384616
biomarkers; epigenomics; metabolomics; metabonomics; molecular epidemiology; proteomics; transcriptomics
21.  Genome-Wide Functional and Stress Response Profiling Reveals Toxic Mechanism and Genes Required for Tolerance to Benzo[a]pyrene in S. cerevisiae 
Frontiers in Genetics  2013;3:316.
Benzo[a]pyrene (BaP) is a ubiquitous, potent, and complete carcinogen resulting from incomplete organic combustion. BaP can form DNA adducts but other mechanisms may play a role in toxicity. We used a functional toxicology approach in S. cerevisiae to assess the genetic requirements for cellular resistance to BaP. In addition, we examined translational activities of key genes involved in various stress response pathways. We identified multiple genes and processes involved in modulating BaP toxicity in yeast which support DNA damage as a primary mechanism of toxicity, but also identify other potential toxicity pathways. Gene ontology enrichment analysis indicated that DNA damage and repair as well as redox homeostasis and oxidative stress are key processes in cellular response to BaP suggesting a similar mode of action of BaP in yeast and mammals. Interestingly, toxicant export is also implicated as a potential novel modulator of cellular susceptibility. In particular, we identified several transporters with human orthologs (solute carrier family 22) which may play a role in mammalian systems.
doi:10.3389/fgene.2012.00316
PMCID: PMC3567348  PMID: 23403841
benzo[a]pyrene; toxicity; yeast; stress; resistance; sensitivity; S-9; ontology
22.  Current understanding of the mechanism of benzene-induced leukemia in humans: implications for risk assessment 
Carcinogenesis  2011;33(2):240-252.
Benzene causes acute myeloid leukemia and probably other hematological malignancies. As benzene also causes hematotoxicity even in workers exposed to levels below the US permissible occupational exposure limit of 1 part per million, further assessment of the health risks associated with its exposure, particularly at low levels, is needed. Here, we describe the probable mechanism by which benzene induces leukemia involving the targeting of critical genes and pathways through the induction of genetic, chromosomal or epigenetic abnormalities and genomic instability, in a hematopoietic stem cell (HSC); stromal cell dysregulation; apoptosis of HSCs and stromal cells and altered proliferation and differentiation of HSCs. These effects modulated by benzene-induced oxidative stress, aryl hydrocarbon receptor dysregulation and reduced immunosurveillance, lead to the generation of leukemic stem cells and subsequent clonal evolution to leukemia. A mode of action (MOA) approach to the risk assessment of benzene was recently proposed. This approach is limited, however, by the challenges of defining a simple stochastic MOA of benzene-induced leukemogenesis and of identifying relevant and quantifiable parameters associated with potential key events. An alternative risk assessment approach is the application of toxicogenomics and systems biology in human populations, animals and in vitro models of the HSC stem cell niche, exposed to a range of levels of benzene. These approaches will inform our understanding of the mechanisms of benzene toxicity and identify additional biomarkers of exposure, early effect and susceptibility useful for risk assessment.
doi:10.1093/carcin/bgr297
PMCID: PMC3271273  PMID: 22166497
23.  The role of inflammation in age-related disease 
Aging (Albany NY)  2013;5(1):84-93.
The National Institutes of Health (NIH) Geroscience Interest Group (GSIG) sponsored workshop, The Role of Inflammation in Age-Related Disease, was held September 6th-7th, 2012 in Bethesda, MD. It is now recognized that a mild pro-inflammatory state is correlated with the major degenerative diseases of the elderly. The focus of the workshop was to better understand the origins and consequences of this low level chronic inflammation in order to design appropriate interventional studies aimed at improving healthspan. Four sessions explored the intrinsic, environmental exposures and immune pathways by which chronic inflammation are generated, sustained, and lead to age-associated diseases. At the conclusion of the workshop recommendations to accelerate progress toward understanding the mechanistic bases of chronic disease were identified.
PMCID: PMC3616233  PMID: 23474627
24.  Gap Junctions and Connexin Hemichannels Underpin Haemostasis and Thrombosis 
Circulation  2012;125(20):2479-2491.
Background
Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have now examined the role of connexins in platelets, blood cells that circulate in isolation, but upon tissue injury adhere to each other and the vessel wall to prevent blood loss and facilitate wound repair.
Methods and Results
We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses prior to platelet-platelet contact, and reduced laser induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion and clot retraction indicating an important role for Cx37 hemichannels and gap junctions in platelet thrombus function.
Conclusions
Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of haemostasis and thrombosis and represent potential therapeutic targets.
doi:10.1161/CIRCULATIONAHA.112.101246
PMCID: PMC3378664  PMID: 22528526
Connexin37; Gap junction; Haemostasis; Platelets; Thrombosis
25.  For re-submission to Mutation Research, 7/30/07 Depletion of WRN Enhances DNA Damage in HeLa Cells Exposed to the Benzene Metabolite, Hydroquinone 
Mutation research  2007;649(1-2):54-61.
Werner syndrome is a progeroid disorder caused by mutations of the WRN gene. The encoded WRN protein belongs to the family of RecQ helicases that plays a role in the maintenance of genomic stability. Single nucleotide polymorphisms in WRN have been associated with an increased risk for some cancers and were recently linked to benzene hematotoxicity. To further address the role of WRN in benzene toxicity, we employed RNA interference (RNAi) to silence endogenous WRN in HeLa cells and examined the susceptibility of these WRN-depleted cells to the toxic effects of the benzene metabolite hydroquinone. HeLa cells were used as the experimental model because RNAi is highly effective in this system producing almost complete depletion of the target protein. Depletion of WRN led to a decrease in cell proliferation and an enhanced susceptibility to hydroquinone cytotoxicity as revealed by an increase in necrosis. WRN-depleted HeLa cells treated with hydroquinone also displayed an increase in the amount of DNA double strand breaks as determined by the Comet assay, and an elevated DNA damage response as indicated by the 7-fold induction of γH2AX and acetyl-p53 (Lys373 and Lys382) over control levels. Together, these results show that WRN plays an important role in the protection of HeLa cells against the toxicity of the benzene metabolite hydroquinone, specifically in mounting a normal DNA damage response following the induction of DNA double-strand breaks. Further studies in bone marrow-derived stem or progenitor cells are required to confirm our findings in HeLa cells and expand our ability to extrapolate the results to benzene toxicity in humans.
doi:10.1016/j.mrgentox.2007.07.011
PMCID: PMC3461953  PMID: 17875398
RNA interference; siRNA; γH2AX; p53; leukemia; lymphoma; DNA repair; strand breaks

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