Formaldehyde-induced hematopoietic stem and progenitor cell toxicity in mouse lung and nose.

Formaldehyde (FA), an economically important and ubiquitous chemical, has been classified as a human carcinogen and myeloid leukemogen. However, the underlying mechanisms of leukemogenesis remain unclear. Unlike many classical leukemogens that damage hematopoietic stem/progenitor cells (HSC/HPC) directly in the bone marrow, FA-as the smallest, most reactive aldehyde-is thought to be incapable of reaching the bone marrow through inhalation exposure. A recent breakthrough study discovered that mouse lung contains functional HSC/HPC that can produce blood cells and travel bi-directionally between the lung and bone marrow, while another early study reported the presence of HSC/HPC in rat nose. Based on these findings, we hypothesized that FA inhalation could induce toxicity in HSC/HPC present in mouse lung and/or nose rather than in the bone marrow. To test this hypothesis, we adapted a commercially available protocol for culturing burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte, macrophage (CFU-GM) colonies from bone marrow and spleen to also enable culture of these colonies from mouse lung and nose, a novel application of this assay. We reported that in vivo exposure to FA at 3 mg/m3 or ex vivo exposure up to 400 µM FA decreased the formation of both colony types from mouse lung and nose as well as from bone marrow and spleen. These findings, to the best of our knowledge, are the first empirically to show that FA exposure can damage mouse pulmonary and olfactory HSC/HPC and provide potential biological plausibility for the induction of leukemia at the sites of entry rather than the bone marrow.

Formaldehyde induces toxicity in mouse bone marrow and hematopoietic stem/progenitor cells and enhances benzene-induced adverse effects.

Formaldehyde (FA) is a human leukemogen and is hematotoxic in human and mouse. The biological plausibility of FA-induced leukemia is controversial because few studies have reported FA-induced bone marrow (BM) toxicity, and none have reported BM stem/progenitor cell toxicity. We sought to comprehensively examine FA hematoxicity in vivo in mouse peripheral blood, BM, spleen and myeloid progenitors. We included the leukemogen and BM toxicant, benzene (BZ), as a positive control, separately and together with FA as co-exposure occurs frequently. We exposed BALB/c mice to 3 mg/m3 FA in air for 2 weeks, mimicking occupational exposure, then measured complete blood counts, nucleated BM cell count, and myeloid progenitor colony formation. We also investigated potential mechanisms of FA toxicity, including reactive oxygen species (ROS) generation, apoptosis, and hematopoietic growth factor and receptor levels. FA exposure significantly reduced nucleated BM cells and BM-derived colony-forming unit-granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E); down-regulated GM-CSFRα and EPOR expression; increased ROS in nucleated BM, spleen and CFU-GM cells; and increased apoptosis in nucleated spleen and CFU-GM cells. FA and BZ each similarly altered BM mature cells and stem/progenitor counts, BM and CFU-GM ROS, and apoptosis in spleen and CFU-GM but had differential effects on other end points. Co-exposure was more potent for several end points. Thus, FA is toxic to the mouse hematopoietic system, including BM stem/progenitor cells, and it enhances BZ-induced toxic effects. Our findings suggest that FA may induce BM toxicity by affecting myeloid progenitor growth and survival through oxidative damage and reduced expression levels of GM-CSFRα and EPOR.

Chromosome-wide aneuploidy study of cultured circulating myeloid progenitor cells from workers occupationally exposed to formaldehyde.

Formaldehyde (FA) is an economically important industrial chemical to which millions of people worldwide are exposed environmentally and occupationally. Recently, the International Agency for Cancer Research concluded that there is sufficient evidence that FA causes leukemia, particularly myeloid leukemia. To evaluate the biological plausibility of this association, we employed a chromosome-wide aneuploidy study approach, which allows the evaluation of aneuploidy and structural chromosome aberrations (SCAs) of all 24 chromosomes simultaneously, to analyze cultured myeloid progenitor cells from 29 workers exposed to relatively high levels of FA and 23 unexposed controls. We found statistically significant increases in the frequencies of monosomy, trisomy, tetrasomy and SCAs of multiple chromosomes in exposed workers compared with controls, with particularly notable effects for monosomy 1 [P = 6.02E-06, incidence rate ratio (IRR) = 2.31], monosomy 5 (P = 9.01E-06; IRR = 2.24), monosomy 7 (P = 1.57E-05; IRR = 2.17), trisomy 5 (P = 1.98E-05; IRR = 3.40) and SCAs of chromosome 5 (P = 0.024; IRR = 4.15). The detection of increased levels of monosomy 7 and SCAs of chromosome 5 is particularly relevant as they are frequently observed in acute myeloid leukemia. Our findings provide further evidence that leukemia-related cytogenetic changes can occur in the circulating myeloid progenitor cells of healthy workers exposed to FA, which may be a potential mechanism underlying FA-induced leukemogenesis.

Alterations in serum immunoglobulin levels in workers occupationally exposed to trichloroethylene.

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.

The impact of FANCD2 deficiency on formaldehyde-induced toxicity in human lymphoblastoid cell lines.

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 cross-links (DPCs). Repair of DPCs may be mediated by the Fanconi anemia pathway; however, data supporting the involvement of this pathway are 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 h, 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.

Current understanding of the mechanism of benzene-induced leukemia in humans: implications for risk assessment.

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.

Epigenome-wide association studies of occupational exposure to benzene and formaldehyde.

Sufficient evidence supports a relationship between certain myeloid neoplasms and exposure to benzene or formaldehyde. DNA methylation could underlie benzene- and formaldehyde-induced health outcomes, but data in exposed human populations are limited. We conducted two cross-sectional epigenome-wide association studies (EWAS), one in workers exposed to benzene and another in workers exposed to formaldehyde. Using HumanMethylation450 BeadChips, we investigated differences in blood cell DNA methylation among 50 benzene-exposed subjects and 48 controls, and among 31 formaldehyde-exposed subjects and 40 controls. We performed CpG-level and regional-level analyses. In the benzene EWAS, we found genome-wide significant alterations, i.e., FWER-controlled P-values <0.05, in the mean and variance of methylation at 22 and 318 CpG sites, respectively, and in mean methylation of a large genomic region. Pathway analysis of genes corresponding to benzene-associated differential methylation sites revealed an impact on the AMPK signalling pathway. In formaldehyde-exposed subjects compared to controls, 9 CpGs in the DUSP22 gene promoter had genome-wide significant decreased methylation variability and a large region of the HOXA5 promoter with 44 CpGs was hypomethylated. Our findings suggest that DNA methylation may contribute to the pathogenesis of diseases related to benzene and formaldehyde exposure. Aberrant expression and methylation of HOXA5 previously has been shown to be clinically significant in myeloid leukaemias. The tumour suppressor gene DUSP22 is a potential biomarker of exposure to formaldehyde, and irregularities have been associated with multiple exposures and diseases.

Consensus on the Key Characteristics of Immunotoxic Agents as a Basis for Hazard Identification.

BACKGROUND: Key characteristics (KCs), properties of agents or exposures that confer potential hazard, have been developed for carcinogens and other toxicant classes. KCs have been used in the systematic assessment of hazards and to identify assay and data gaps that limit screening and risk assessment. Many of the mechanisms through which pharmaceuticals and occupational or environmental agents modulate immune function are well recognized. Thus KCs could be identified for immunoactive substances and applied to improve hazard assessment of immunodulatory agents. OBJECTIVES: The goal was to generate a consensus-based synthesis of scientific evidence describing the KCs of agents known to cause immunotoxicity and potential applications, such as assays to measure the KCs. METHODS: A committee of 18 experts with diverse specialties identified 10 KCs of immunotoxic agents, namely, 1) covalently binds to proteins to form novel antigens, 2) affects antigen processing and presentation, 3) alters immune cell signaling, 4) alters immune cell proliferation, 5) modifies cellular differentiation, 6) alters immune cell-cell communication, 7) alters effector function of specific cell types, 8) alters immune cell trafficking, 9) alters cell death processes, and 10) breaks down immune tolerance. The group considered how these KCs could influence immune processes and contribute to hypersensitivity, inappropriate enhancement, immunosuppression, or autoimmunity. DISCUSSION: KCs can be used to improve efforts to identify agents that cause immunotoxicity via one or more mechanisms, to develop better testing and biomarker approaches to evaluate immunotoxicity, and to enable a more comprehensive and mechanistic understanding of adverse effects of exposures on the immune system. https://doi.org/10.1289/EHP10800.

Chromosome-wide aneuploidy study (CWAS) in workers exposed to an established leukemogen, benzene.

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.

Reproductive and developmental toxicity of formaldehyde: a systematic review.

Formaldehyde, the recently classified carcinogen and ubiquitous environmental contaminant, has long been suspected of causing adverse reproductive and developmental effects, but previous reviews were inconclusive, due in part, to limitations in the design of many of the human population studies. In the current review, we systematically evaluated evidence of an association between formaldehyde exposure and adverse reproductive and developmental effects, in human populations and in vivo animal studies, in the peer-reviewed literature. The mostly retrospective human studies provided evidence of an association of maternal exposure with adverse reproductive and developmental effects. Further assessment of this association by meta-analysis revealed an increased risk of spontaneous abortion (1.76, 95% CI 1.20-2.59, p=0.002) and of all adverse pregnancy outcomes combined (1.54, 95% CI 1.27-1.88, p<0.001), in formaldehyde-exposed women, although differential recall, selection bias, or confounding cannot be ruled out. Evaluation of the animal studies including all routes of exposure, doses and dosing regimens studied, suggested positive associations between formaldehyde exposure and reproductive toxicity, mostly in males. Potential mechanisms underlying formaldehyde-induced reproductive and developmental toxicities, including chromosome and DNA damage (genotoxicity), oxidative stress, altered level and/or function of enzymes, hormones and proteins, apoptosis, toxicogenomic and epigenomic effects (such as DNA methylation), were identified. To clarify these associations, well-designed molecular epidemiologic studies, that include quantitative exposure assessment and diminish confounding factors, should examine both reproductive and developmental outcomes associated with exposure in males and females. Together with mechanistic and animal studies, this will allow us to better understand the systemic effect of formaldehyde exposure.

Using the Key Characteristics of Carcinogens to Develop Research on Chemical Mixtures and Cancer.

BACKGROUND: People are exposed to numerous chemicals throughout their lifetimes. Many of these chemicals display one or more of the key characteristics of carcinogens or interact with processes described in the hallmarks of cancer. Therefore, evaluating the effects of chemical mixtures on cancer development is an important pursuit. Challenges involved in designing research studies to evaluate the joint action of chemicals on cancer risk include the time taken to perform the experiments because of the long latency and choosing an appropriate experimental design. OBJECTIVES: The objectives of this work are to present the case for developing a research program on mixtures of environmental chemicals and cancer risk and describe recommended approaches. METHODS: A working group comprising the coauthors focused attention on the design of mixtures studies to inform cancer risk assessment as part of a larger effort to refine the key characteristics of carcinogens and explore their application. Working group members reviewed the key characteristics of carcinogens, hallmarks of cancer, and mixtures research for other disease end points. The group discussed options for developing tractable projects to evaluate the joint effects of environmental chemicals on cancer development. RESULTS AND DISCUSSION: Three approaches for developing a research program to evaluate the effects of mixtures on cancer development were proposed: a chemical screening approach, a transgenic model-based approach, and a disease-centered approach. Advantages and disadvantages of each are discussed. https://doi.org/10.1289/EHP8525.

Applying genome-wide CRISPR to identify known and novel genes and pathways that modulate formaldehyde toxicity.

Formaldehyde (FA), a ubiquitous environmental pollutant, is classified as a Group I human carcinogen by the International Agency for Research on Cancer. Previously, we reported that FA induced hematotoxicity and chromosomal aneuploidy in exposed workers and toxicity in bone marrow and hematopoietic stem cells of experimental animals. Using functional toxicogenomic profiling in yeast, we identified genes and cellular processes modulating eukaryotic FA cytotoxicity. Although we validated some of these findings in yeast, many specific genes, pathways and mechanisms of action of FA in human cells are not known. In the current study, we applied genome-wide, loss-of-function CRISPR screening to identify modulators of FA toxicity in the human hematopoietic K562 cell line. We assessed the cellular genetic determinants of susceptibility and resistance to FA at 40, 100 and 150 µM (IC10, IC20 and IC60, respectively) at two time points, day 8 and day 20. We identified multiple candidate genes that increase sensitivity (e.g. ADH5, ESD and FANC family) or resistance (e.g. FASN and KDM6A) to FA when disrupted. Pathway analysis revealed a major role for the FA metabolism and Fanconi anemia pathway in FA tolerance, consistent with findings from previous studies. Additional network analyses revealed potential new roles for one-carbon metabolism, fatty acid synthesis and mTOR signaling in modulating FA toxicity. Validation of these novel findings will further enhance our understanding of FA toxicity in human cells. Our findings support the utility of CRISPR-based functional genomics screening of environmental chemicals.

Key Characteristics of Cardiovascular Toxicants.

BACKGROUND: The concept of chemical agents having properties that confer potential hazard called key characteristics (KCs) was first developed to identify carcinogenic hazards. Identification of KCs of cardiovascular (CV) toxicants could facilitate the systematic assessment of CV hazards and understanding of assay and data gaps associated with current approaches. OBJECTIVES: We sought to develop a consensus-based synthesis of scientific evidence on the KCs of chemical and nonchemical agents known to cause CV toxicity along with methods to measure them. METHODS: An expert working group was convened to discuss mechanisms associated with CV toxicity. RESULTS: The group identified 12 KCs of CV toxicants, defined as exogenous agents that adversely interfere with function of the CV system. The KCs were organized into those primarily affecting cardiac tissue (numbers 1-4 below), the vascular system (5-7), or both (8-12), as follows: 1) impairs regulation of cardiac excitability, 2) impairs cardiac contractility and relaxation, 3) induces cardiomyocyte injury and death, 4) induces proliferation of valve stroma, 5) impacts endothelial and vascular function, 6) alters hemostasis, 7) causes dyslipidemia, 8) impairs mitochondrial function, 9) modifies autonomic nervous system activity, 10) induces oxidative stress, 11) causes inflammation, and 12) alters hormone signaling. DISCUSSION: These 12 KCs can be used to help identify pharmaceuticals and environmental pollutants as CV toxicants, as well as to better understand the mechanistic underpinnings of their toxicity. For example, evidence exists that fine particulate matter [PM ≤2.5µm in aerodynamic diameter (PM2.5)] air pollution, arsenic, anthracycline drugs, and other exogenous chemicals possess one or more of the described KCs. In conclusion, the KCs could be used to identify potential CV toxicants and to define a set of test methods to evaluate CV toxicity in a more comprehensive and standardized manner than current approaches. https://doi.org/10.1289/EHP9321.

Toxicogenomic profiling of chemically exposed humans in risk assessment.

Gene-environment interactions contribute to complex disease development. The environmental contribution, in particular low-level and prevalent environmental exposures, may constitute much of the risk and contribute substantially to disease. Systematic risk evaluation of the majority of human chemical exposures, has not been conducted and is a goal of regulatory agencies in the U.S. and worldwide. With the recent recognition that toxicological approaches more predictive of effects in humans are required for risk assessment, in vitro human cell line data as well as animal data are being used to identify toxicity mechanisms that can be translated into biomarkers relevant to human exposure studies. In this review, we discuss how data from toxicogenomic studies of exposed human populations can inform risk assessment, by generating biomarkers of exposure, early effect, and/or susceptibility, elucidating mechanisms of action underlying exposure-related disease, and detecting response at low doses. Good experimental design incorporating precise, individual exposure measurements, phenotypic anchors (pre-disease or traditional toxicological markers), and a range of relevant exposure levels, is necessary. Further, toxicogenomic studies need to be designed with sufficient power to detect true effects of the exposure. As more studies are performed and incorporated into databases such as the Comparative Toxicogenomics Database (CTD) and Chemical Effects in Biological Systems (CEBS), data can be mined for classification of newly tested chemicals (hazard identification), and, for investigating the dose-response, and inter-relationship among genes, environment and disease in a systems biology approach (risk characterization).

Changes in the peripheral blood transcriptome associated with occupational benzene exposure identified by cross-comparison on two microarray platforms.

Benzene is an established cause of leukemia, and possibly lymphoma, in humans, but the underlying molecular pathways remain largely undetermined. We used two microarray platforms to identify global gene expression changes associated with well-characterized occupational benzene exposure in the peripheral blood mononuclear cells (PBMC) of a population of shoe-factory workers. Differential expression of 2692 genes (Affymetrix) and 1828 genes (Illumina) was found and the concordance was 50% (based on an average fold-change > or =1.3 from the two platforms), with similar expression ratios among the concordant genes. Four genes (CXCL16, ZNF331, JUN and PF4), which we previously identified by microarray and confirmed by real-time PCR, were among the top 100 genes identified by both platforms in the current study. Gene ontology analysis showed overrepresentation of genes involved in apoptosis among the concordant genes while pathway analysis identified pathways related to lipid metabolism. The two-platform approach allows for robust changes in the PBMC transcriptome of benzene-exposed individuals to be identified.

The Key Characteristics of Carcinogens: Relationship to the Hallmarks of Cancer, Relevant Biomarkers, and Assays to Measure Them.

The key characteristics (KC) of human carcinogens provide a uniform approach to evaluating mechanistic evidence in cancer hazard identification. Refinements to the approach were requested by organizations and individuals applying the KCs. We assembled an expert committee with knowledge of carcinogenesis and experience in applying the KCs in cancer hazard identification. We leveraged this expertise and examined the literature to more clearly describe each KC, identify current and emerging assays and in vivo biomarkers that can be used to measure them, and make recommendations for future assay development. We found that the KCs are clearly distinct from the Hallmarks of Cancer, that interrelationships among the KCs can be leveraged to strengthen the KC approach (and an understanding of environmental carcinogenesis), and that the KC approach is applicable to the systematic evaluation of a broad range of potential cancer hazards in vivo and in vitro We identified gaps in coverage of the KCs by current assays. Future efforts should expand the breadth, specificity, and sensitivity of validated assays and biomarkers that can measure the 10 KCs. Refinement of the KC approach will enhance and accelerate carcinogen identification, a first step in cancer prevention.See all articles in this CEBP Focus section, "Environmental Carcinogenesis: Pathways to Prevention."

The benzene metabolite, hydroquinone and etoposide both induce endoreduplication in human lymphoblastoid TK6 cells.

Both occupational exposure to the leukemogen benzene and in vitro exposure to its metabolite hydroquinone (HQ) lead to the induction of numerical and structural chromosome changes. Several studies have shown that HQ can form DNA adducts, disrupt microtubule assembly and inhibit DNA topoisomerase II (topo II) activity. As these are potential mechanisms underlying endoreduplication (END), a phenomenon that involves DNA amplification without corresponding cell division, we hypothesized that HQ could cause END. We measured END in the human lymphoblastoid cell line, TK6, treated with HQ (0-20 microM) and etoposide (0-0.2 microM) for 48 h. Etoposide was used as a positive control as it is a topo II poison and established human leukemogen that has previously been shown to induce END in Chinese hamster ovary cells. Both HQ and etoposide significantly induced END in a dose-dependent manner (P(trend) < 0.0001 and P(trend) = 0.0003, respectively). Since END may underlie the acquisition of high chromosome numbers by tumour cells, it may play a role in inducing genomic instability and subsequent carcinogenesis from HQ and etoposide. In order to further explore the cytogenetic effects of HQ and etoposide, we also examined specific structural changes. HQ did not induce translocations of chromosome 11 [t(11;?)] but significantly induced translocations of chromosome 21 [t(21;?)] and structural chromosome aberrations (SCA) (P(trend) = 0.0415 and P(trend) < 0.0001, respectively). Etoposide potently induced all these structural changes (P(trend) < 0.0001). The lack of an effect of HQ on t(11;?) and the reduced ability of HQ to induce t(21;?) and SCA, compared with etoposide, further suggests that HQ acts primarily as a topo II catalytic inhibitor rather than as a topo II poison in intact human cells.

In utero exposure to endocrine disrupting chemicals, micro-RNA profiles, and fetal growth: a pilot study protocol.

Background: The developing fetus is particularly vulnerable to the effects of endocrine disrupting chemicals (EDCs). Molecular fingerprints of EDCs can be identified via microRNA (miRNA) expression profiles and may be etiologically implicated in the developmental origin of disease (DOHaD). Methods/design: This pilot study includes pregnant women at high risk (smoking at conception), and low risk (non-smoking at conception) for SGA birth (birthweight<10th percentile for gestational age). We have randomly selected 12 mothers (3 high-risk SGA birth, 3 low-risk SGA birth, 3 high-risk non-SGA birth, 3 low-risk non-SGA birth), with EDC measurements from gestational week 17. All offspring are female. We aim to test the stability of our samples (maternal serum, cord blood, placenta tissue), observe the differential expression of miRNA profiles over time (gestational weeks 17, 25, 33, 37, birth), and study the consistency between maternal EDC measures and miRNA expression profiles across our repeated measures. Expected impact of the study for Public Health: Results from this pilot study will inform the development of a larger cohort wide analysis, and will impact the current state of knowledge in the fields of public health, epigenetics, and the DOHaD.

Proposed Key Characteristics of Female Reproductive Toxicants as an Approach for Organizing and Evaluating Mechanistic Data in Hazard Assessment.

BACKGROUND: Identification of female reproductive toxicants is currently based largely on integrated epidemiological and in vivo toxicology data and, to a lesser degree, on mechanistic data. A uniform approach to systematically search, organize, integrate, and evaluate mechanistic evidence of female reproductive toxicity from various data types is lacking. OBJECTIVE: We sought to apply a key characteristics approach similar to that pioneered for carcinogen hazard identification to female reproductive toxicant hazard identification. METHODS: A working group of international experts was convened to discuss mechanisms associated with chemical-induced female reproductive toxicity and identified 10 key characteristics of chemicals that cause female reproductive toxicity: 1) alters hormone receptor signaling; alters reproductive hormone production, secretion, or metabolism; 2) chemical or metabolite is genotoxic; 3) induces epigenetic alterations; 4) causes mitochondrial dysfunction; 5) induces oxidative stress; 6) alters immune function; 7) alters cell signal transduction; 8) alters direct cell­cell interactions; 9) alters survival, proliferation, cell death, or metabolic pathways; and 10) alters microtubules and associated structures. As proof of principle, cyclophosphamide and diethylstilbestrol (DES), for which both human and animal studies have demonstrated female reproductive toxicity, display at least 5 and 3 key characteristics, respectively. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), for which the epidemiological evidence is mixed, exhibits 5 key characteristics. DISCUSSION: Future efforts should focus on evaluating the proposed key characteristics against additional known and suspected female reproductive toxicants. Chemicals that exhibit one or more of the key characteristics could be prioritized for additional evaluation and testing. A key characteristics approach has the potential to integrate with pathway-based toxicity testing to improve prediction of female reproductive toxicity in chemicals and potentially prevent some toxicants from entering common use. https://doi.org/10.1289/EHP4971.

Chromosome translocations in workers exposed to benzene.

As benzene has been linked with elevated risk of both acute myeloid leukemia and lymphoma, we explored the effect of benzene exposure on levels of t(8;21), t(15;17), and t(14;18) translocations. Circulating lymphocytes of normal individuals also often contain t(14;18). Quantitative polymerase chain reaction analysis showed that 37 workers with benzene exposure had a decreased level of t(14;18) in their blood with only 16.2% having 10 or more copies of the t(14;18) BCL-2/IgH fusion gene/microg DNA, as opposed to 55% of 20 controls (P = .0063 by Fisher's exact test). This decline may be related to the immunotoxicity to specific subtypes of circulating B-lymphocytes, but the data do not support the use of t(14;18) as a biomarker of increased lymphoma risk in benzene-exposed populations. None of 88 individuals (31 controls and 57 exposed) exhibited detectable t(8;21) transcripts, and while t(15;17) transcripts were detected in two individuals, the result is inconclusive as one was exposed and the other was unexposed.