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1.  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.
PMCID: PMC3616671  PMID: 23276795
2.  Occupational exposure to formaldehyde and alterations in lymphocyte subsets 
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.
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.
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).
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.
PMCID: PMC3493854  PMID: 22767408
formaldehyde; NK cell; B cell; T cell; T cell subset
3.  Modulation of Ras signaling alters the toxicity of hydroquinone, a benzene metabolite and component of cigarette smoke 
BMC Cancer  2014;14:6.
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.
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.
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.
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.
PMCID: PMC3898384  PMID: 24386979
Hydroquinone; in vitro micronucleus assay; IRA2; NF1; Ras; Yeast
4.  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.
PMCID: PMC3876734  PMID: 20148884
childhood leukaemia; epidemiology; folic acid
5.  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.
PMCID: PMC3849353  PMID: 20600216
arsenic; lung cancer; drinking water; metabolism
6.  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.
PMCID: PMC3806366  PMID: 24096827
7.  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.
PMCID: PMC3848751  PMID: 24004508
Arsenic; Immune system; Immunotoxicity; Immunocompromised; Immunosuppression
8.  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.
PMCID: PMC3499056  PMID: 22665366
9.  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.
PMCID: PMC3763562  PMID: 23873959
10.  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.
PMCID: PMC3472034  PMID: 22643707
Benzene; leukemia; monosomy; hematopoietic progenitor
11.  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.
PMCID: PMC3293942  PMID: 22296171
follicular lymphoma; HLA; genetic risk factors; next-generation sequencing
12.  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.
PMCID: PMC3620742  PMID: 23384616
biomarkers; epigenomics; metabolomics; metabonomics; molecular epidemiology; proteomics; transcriptomics
13.  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.
PMCID: PMC3567348  PMID: 23403841
benzo[a]pyrene; toxicity; yeast; stress; resistance; sensitivity; S-9; ontology
14.  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.
PMCID: PMC3271273  PMID: 22166497
15.  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
16.  Gap Junctions and Connexin Hemichannels Underpin Haemostasis and Thrombosis 
Circulation  2012;125(20):2479-2491.
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.
Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of haemostasis and thrombosis and represent potential therapeutic targets.
PMCID: PMC3378664  PMID: 22528526
Connexin37; Gap junction; Haemostasis; Platelets; Thrombosis
17.  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.
PMCID: PMC3461953  PMID: 17875398
RNA interference; siRNA; γH2AX; p53; leukemia; lymphoma; DNA repair; strand breaks
18.  Benzene, the Exposome and Future Investigations of Leukemia Etiology 
Chemico-biological interactions  2011;192(1-2):155-159.
Benzene exposure is associated with acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and probably lymphoma and childhood leukemia. Biological plausibility for a causal role of benzene in these diseases comes from its toxicity to hematopoietic stem cells (HSC) or progenitor cells, from which all leukemias and related disorders arise. The effect of this toxicity is manifest as lowered blood counts (hematotoxicity), even in individuals occupationally exposed to low levels of benzene. Benzene can induce AML/MDS via several well-characterized pathways associated with these diseases. Through its metabolites, benzene induces multiple alterations that likely contribute to the leukemogenic process, and appears to operate via multiple modes of action. To improve mechanistic understanding and for risk assessment purposes, it may be possible to measure several of the key events in these modes of action in an in vitro model of the bone marrow stem cell niche. Even though benzene is leukemogenic at relatively low occupational levels of exposure, it seems unlikely that it is a major cause of leukemia in the general population exposed to benzene in the ppb range. Other established non-genetic causes of AML, e.g. smoking, ionizing radiation and cancer chemotherapy, also only explain about 20% of AML incidence, leaving ~80% unexplained. The question arises as to how to find the causes of the majority of de novo AMLs that remain unexplained. We propose that we should attempt to characterize the ‘exposome’ of human leukemia by using unbiased laboratory-based methods to find the unknown ‘environmental’ factors that contribute to leukemia etiology.
PMCID: PMC3461963  PMID: 21333640
Benzene; leukemia; myeloid; AML; mode of action; mechanism; blood; biomarker; metabolism; hydroquinone; stem cell niche
19.  Multiplexed, ligation-dependent probe amplification for rapid and inexpensive HLA-DQB1 allelotyping 
Tissue antigens  2011;78(4):275-280.
Many effective options exist to accurately type DNA for HLA alleles. However, most of the existing methods are excessively costly in terms of overall monetary costs, DNA requirements, and proprietary software. We present a novel assay able to resolve heterozygous HLA-DQB1 allelotypes at two digits, with even greater specificity for the HLA-DQB1*06 allele family, by using the multiplexed ligation-dependent probe amplification (MLPA) technology. This assay provides more specific allele data than genome-wide analysis and is more affordable than sequencing, making it a useful intermediate for researchers seeking to accurately allelotype human DNA samples.
PMCID: PMC3431962  PMID: 21762399
DQB1; genotyping; MHC class II; MLPA
20.  Heat Shock Protein 90 Inhibitors Reduce Trafficking of ATP-gated P2X1 Receptors and Human Platelet Responsiveness* 
The Journal of Biological Chemistry  2012;287(39):32747-32754.
Background: Heat shock protein 90 (HSP90) has previously been co-purified with P2X1 receptors for ATP.
Results: P2X1 receptor trafficking and responses (currents and calcium rises) were reduced by HSP90 inhibitors.
Conclusion: P2X1 receptors are constitutively regulated by HSP90.
Significance: In addition to a role in cancer treatment, HSP90 inhibitors may provide protection from thrombosis.
We have used selective inhibitors to determine whether the molecular chaperone heat shock protein 90 (HSP90) has an effect on both recombinant and native human P2X1 receptors. P2X1 receptor currents in HEK293 cells were reduced by ∼70–85% by the selective HSP90 inhibitor geldanamycin (2 μm, 20 min). This was associated with a speeding in the time course of desensitization as well as a reduction in cell surface expression. Imaging in real time of photoactivatable GFP-tagged P2X receptors showed that they are highly mobile. Geldanamycin almost abolished this movement for P2X1 receptors but had no effect on P2X2 receptor trafficking. P2X1/2 receptor chimeras showed that the intracellular N and C termini were involved in geldanamycin sensitivity. Geldanamycin also inhibited native P2X1 receptor-mediated responses. Platelet P2X1 receptors play an important role in hemostasis, contribute to amplification of signaling to a range of stimuli including collagen, and are novel targets for antithrombotic therapies. Platelet P2X1 receptor-, but not P2Y1 receptor-, mediated increases in intracellular calcium were reduced by 40–45% following HSP90 inhibition with geldanamycin or radicicol. Collagen stimulation leads to ATP release from platelets, and calcium increases to low doses of collagen were also reduced by ∼40% by the HSP90 inhibitors consistent with an effect on P2X1 receptors. These studies suggest that HSP90 inhibitors may be as effective as selective antagonists in regulating platelet P2X1 receptors, and their potential effects on hemostasis should be considered in clinical studies.
PMCID: PMC3463321  PMID: 22851178
Collagen; Electrophysiology; HSP90; Platelets; Purinergic Receptor; Trafficking; Calcium; Patch Clamp
21.  Correction: Effect of Chemical Mutagens and Carcinogens on Gene Expression Profiles in Human TK6 Cells 
PLoS ONE  2012;7(7):10.1371/annotation/cee00b5b-4def-4684-b02c-fdcc5eaf51e6.
PMCID: PMC3398057
22.  Effect of Chemical Mutagens and Carcinogens on Gene Expression Profiles in Human TK6 Cells 
PLoS ONE  2012;7(6):e39205.
Characterization of toxicogenomic signatures of carcinogen exposure holds significant promise for mechanistic and predictive toxicology. In vitro transcriptomic studies allow the comparison of the response to chemicals with diverse mode of actions under controlled experimental conditions. We conducted an in vitro study in TK6 cells to characterize gene expression signatures of exposure to 15 genotoxic carcinogens frequently used in European industries. We also examined the dose-responsive changes in gene expression, and perturbation of biochemical pathways in response to these carcinogens. TK6 cells were exposed at 3 dose levels for 24 h with and without S9 human metabolic mix. Since S9 had an impact on gene expression (885 genes), we analyzed the gene expression data from cells cultures incubated with S9 and without S9 independently. The ribosome pathway was affected by all chemical-dose combinations. However in general, no similar gene expression was observed among carcinogens. Further, pathways, i.e. cell cycle, DNA repair mechanisms, RNA degradation, that were common within sets of chemical-dose combination were suggested by clustergram. Linear trends in dose–response of gene expression were observed for Trichloroethylene, Benz[a]anthracene, Epichlorohydrin, Benzene, and Hydroquinone. The significantly altered genes were involved in the regulation of (anti-) apoptosis, maintenance of cell survival, tumor necrosis factor-related pathways and immune response, in agreement with several other studies. Similarly in S9+ cultures, Benz[a]pyrene, Styrene and Trichloroethylene each modified over 1000 genes at high concentrations. Our findings expand our understanding of the transcriptomic response to genotoxic carcinogens, revealing the alteration of diverse sets of genes and pathways involved in cellular homeostasis and cell cycle control.
PMCID: PMC3377624  PMID: 22723965
23.  Association of HLA-DQB1 alleles with risk of follicular lymphoma 
Leukemia & lymphoma  2010;52(1):53-58.
In a recent genome-wide association study of follicular lymphoma (FL), we identified novel risk alleles on chromosome 6p21.33 that appeared to be part of an extended haplotype including HLA-DRB1*0101, DQA1*0101, and DQB1*0501. To follow up on these findings, we obtained 2–4 digit HLA-DQB1 allelotypes on a subset of 265 FL cases and 757 controls using a novel assay that applies multiplexed ligation-dependent probe amplification (MLPA). We confirmed a positive association between FL and the HLA-DQB1*05 allele group (OR=1.70, 95% CI 1.28–2.27; adjusted p-value=0.013) and also identified an allele group inversely associated with FL risk, HLA-DQB1*06 (OR=0.51, 95% CI 0.38–0.69; adjusted p-value=4.46×10−5). Although these findings require verification, the role of HLA class II proteins in B-cell survival and proliferation make this a biologically plausible association.
PMCID: PMC3331678  PMID: 21133715
follicular lymphoma; MHC; HLA; DQ; NHL
24.  Chromosome-wide aneuploidy study (CWAS) in workers exposed to an established leukemogen, benzene 
Carcinogenesis  2011;32(4):605-612.
Evidence suggests that de novo, therapy-related and benzene-induced acute myeloid leukemias (AML) occur via similar cytogenetic and genetic pathways, several of which involve aneuploidy, the loss or gain of chromosomes. Aneuploidy of specific chromosomes has been detected in benzene-related leukemia patients as well as in healthy benzene-exposed workers, suggesting that aneuploidy precedes and may be a potential mechanism underlying benzene-induced leukemia. Here, we analyzed the peripheral blood lymphocytes of 47 exposed workers and 27 unexposed controls using a novel OctoChrome fluorescence in situ hybridization (FISH) technique that simultaneously detects aneuploidy in all 24 chromosomes. Through this chromosome-wide aneuploidy study (CWAS) approach, we found heterogeneity in the monosomy and trisomy rates of the 22 autosomes when plotted against continuous benzene exposure. In addition, statistically significant, chromosome-specific increases in the rates of monosomy [5, 6, 7, 10, 16 and 19] and trisomy [5, 6, 7, 8, 10, 14, 16, 21 and 22] were found to be dose dependently associated with benzene exposure. Furthermore, significantly higher rates of monosomy and trisomy were observed in a priori defined ‘susceptible’ chromosome sets compared with all other chromosomes. Together, these findings confirm that benzene exposure is associated with specific chromosomal aneuploidies in hematopoietic cells, which suggests that such aneuploidies may play roles in benzene-induced leukemogenesis.
PMCID: PMC3066415  PMID: 21216845
25.  FRET-Based Detection of M1 Muscarinic Acetylcholine Receptor Activation by Orthosteric and Allosteric Agonists 
PLoS ONE  2012;7(1):e29946.
Background and Objective
Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M1 mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET).
Variants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M1 mAChR, respectively. The optimized FRET receptor construct (M1-cam5) was expressed stably in HEK293 cells.
The variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with Gαq/11 proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M1 FRET (FEYFP/FECFP) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine.
The M1 FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for pharmacological and structural investigations of M1 mAChR activation.
PMCID: PMC3260180  PMID: 22272263

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