Danielle E. Buttke

Exposures to endocrine-disrupting chemicals and age of menarche in adolescent girls in NHANES (2003-2008).

Background: The observed age of menarche has fallen, which may have important adverse social and health consequences. Increased exposure to endocrine-disrupting compounds (EDCs) has been associated with adverse reproductive outcomes. Objective: Our objective was to assess the relationship between EDC exposure and the age of menarche in adolescent girls. Methods: We used data from female participants 12–16 years of age who had completed the reproductive health questionnaire and laboratory examination for the Centers for Disease Control and Prevention’s National Health and Nutrition Examination Survey (NHANES) for years 2003–2008 (2005–2008 for analyses of phthalates and parabens). Exposures were assessed based on creatinine-corrected natural log urine concentrations of selected environmental chemicals and metabolites found in at least 75% of samples in our study sample. We used Cox proportional hazards analysis in SAS 9.2 survey procedures to estimate associations after accounting for censored data among participants who had not reached menarche. We evaluated body mass index (BMI; kilograms per meter squared), family income-to-poverty ratio, race/ethnicity, mother’s smoking status during pregnancy, and birth weight as potential confounders. Results: The weighted mean age of menarche was 12.0 years of age. Among 440 girls with both reproductive health and laboratory data, after accounting for BMI and race/ethnicity, we found that 2,5-dichlorophenol (2,5-DCP) and summed environmental phenols (2,5-DCP and 2,4-DCP) were inversely associated with age of menarche [hazard ratios of 1.10; 95% confidence interval (CI): 1.01, 1.19 and 1.09; 95% CI: 1.01, 1.19, respectively]. Other exposures (total parabens, bisphenol A, triclosan, benzophenone-3, total phthalates, and 2,4-DCP) were not significantly associated with age of menarche. Conclusions: Our findings suggest an association between 2,5-DCP, a potential EDC, and earlier age of menarche in the general U.S. population.

Segregation of micron-scale membrane sub-domains in live murine sperm.

Lipid rafts, membrane sub‐domains enriched in sterols and sphingolipids, are controversial because demonstrations of rafts have often utilized fixed cells. We showed in living sperm that the ganglioside GM1 localized to a micron‐scale membrane sub‐domain in the plasma membrane overlying the acrosome. We investigated four models proposed for membrane sub‐domain maintenance. GM1 segregation was maintained in live sperm incubated under non‐capacitating conditions, and after sterol efflux, a membrane alteration necessary for capacitation. The complete lack of GM1 diffusion to the post‐acrosomal plasma membrane (PAPM) in live cells argued against the transient confinement zone model. However, within seconds after cessation of sperm motility, GM1 dramatically redistributed several microns from the acrosomal sub‐domain to the post‐acrosomal, non‐raft sub‐domain. This redistribution was not accompanied by movement of sterols, and was induced by the pentameric cholera toxin subunit B (CTB). These data argued against a lipid–lipid interaction model for sub‐domain maintenance. Although impossible to rule out a lipid shell model definitively, mice lacking caveolin‐1 maintained segregation of both sterols and GM1, arguing against a role for lipid shells surrounding caveolin‐1 in sub‐domain maintenance. Scanning electron microscopy of sperm freeze‐dried without fixation identified cytoskeletal structures at the sub‐domain boundary. Although drugs used to disrupt actin and intermediate filaments had no effect on the segregation of GM1, we found that disulfide‐bonded proteins played a significant role in sub‐domain segregation. Together, these data provide an example of membrane sub‐domains extreme in terms of size and stability of lipid segregation, and implicate a protein‐based membrane compartmentation mechanism.

BIOCHEMICAL CHARACTERIZATION OF MEMBRANE FRACTIONS IN MURINE SPERM: IDENTIFICATION OF THREE DISTINCT SUB-TYPES OF MEMBRANE RAFTS

Despite enormous interest in membrane raft micro‐domains, no studies in any cell type have defined the relative compositions of the raft fractions on the basis of their major components—sterols, phospholipids, and proteins—or additional raft‐associating lipids such as the ganglioside, GM1. Our previous localization data in live sperm showed that the plasma membrane overlying the acrosome represents a stabilized platform enriched in GM1 and sterols. These findings, along with the physiological requirement for sterol efflux for sperm to function, prompted us to characterize sperm membrane fractions biochemically. After confirming limitations of commonly used detergent‐based approaches, we utilized a non‐detergent‐based method, separating membrane fractions that were reproducibly distinct based on sterol, GM1, phospholipid, and protein compositions (both mass amounts and molar ratios). Based on fraction buoyancy and biochemical composition, we identified at least three highly reproducible sub‐types of membrane raft. Electron microscopy revealed that raft fractions were free of visible contaminants and were separated by buoyancy rather than morphology. Quantitative proteomic comparisons and fluorescence localization of lipids suggested that different organelles contributed differentially to individual raft sub‐types, but that multiple membrane micro‐domain sub‐types could exist within individual domains. This has important implications for scaffolding functions broadly associated with rafts. Most importantly, we show that the common practice of characterizing membrane domains as either “raft” or “non‐raft” oversimplifies the actual biochemical complexity of cellular membranes. J. Cell. Physiol. 218: 537–548, 2009.

Mechanisms underlying the micron-scale segregation of sterols and GM1 in live mammalian sperm

We demonstrate for the first time that a stable, micron‐scale segregation of focal enrichments of sterols exists at physiological temperature in the plasma membrane of live murine and human sperm. These enrichments of sterols represent microheterogeneities within this membrane domain overlying the acrosome. Previously, we showed that cholera toxin subunit B (CTB), which binds the glycosphingolipid, GM1, localizes to this same domain in live sperm. Interestingly, the GM1 undergoes an unexplained redistribution upon cell death. We now demonstrate that GM1 is also enriched in the acrosome, an exocytotic vesicle. Transfer of lipids between this and the plasma membrane occurs at cell death, increasing GM1 in the plasma membrane without apparent release of acrosomal contents. This finding provides corroborative support for an emerging model of regulated exocytosis in which membrane communications might occur without triggering the “acrosome reaction.” Comparison of the dynamics of CTB‐bound endogenous GM1 and exogenous BODIPY–GM1 in live murine sperm demonstrate that the sub‐acrosomal ring (SAR) functions as a specialized diffusion barrier segregating specific lipids within the sperm head plasma membrane. Our data show significant differences between endogenous lipids and exogenous lipid probes in terms of lateral diffusion. Based on these studies, we propose a hierarchical model to explain the segregation of this sterol‐ and GM1‐enriched domain in live sperm, which is positioned to regulate sperm fertilization competence and mediate interactions with the oocyte. Moreover, our data suggest potential origins of subtypes of membrane raft microdomains enriched in sterols and/or GM1 that can be separated biochemically. J. Cell. Physiol. 218: 522–536, 2009.

Visualization of GM1 with cholera toxin B in live epididymal versus ejaculated bull, mouse, and human spermatozoa

Abstract The organization of membrane subdomains in mammalian sperm has recently generated controversy, with several reports describing widely differing localization patterns for the ganglioside GM1. Using the pentameric B subunit of cholera toxin (CTB), we found GM1 to be restricted to the plasma membrane overlying the acrosome in the heads of live murine sperm. Interestingly, CTB had minimal binding to live bovine and human sperm. To investigate whether this difference in GM1 localization was because of species differences or differences between collection from the epididymis (mouse) or an ejaculate (bull, human), we examined epididymal bovine and human sperm. We found that GM1 localized to the plasma membrane overlying the acrosome in sperm from these species. To determine whether some component of seminal plasma was interfering with the ability of CTB to access GM1, we incubated epididymal mouse sperm with fluid from murine seminal vesicles and epididymal bull sperm with bovine seminal plasma. This treatment largely abolished the ability of the CTB to bind to GM1, producing a fluorescence pattern similar to that reported for the human. The most abundant seminal plasma protein, PDC-109, was not responsible for this loss. As demonstration that the seminal plasma was not removing GM1, sperm exposed to seminal plasma were fixed before CTB addition, and again displayed fluorescence over the acrosome. These observations reconcile inconsistencies reported for the localization of GM1 in sperm of different species, and provide evidence for the segregation of GM1 to a stable subdomain in the plasma membrane overlying the acrosome.

Lipid Modulation of Calcium Flux through CaV2.3 Regulates Acrosome Exocytosis and Fertilization

Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca(2+) influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3s pore-forming α1E subunit showed altered Ca(2+) responses, reduced AE, and a strong subfertility phenotype. Surprisingly, AE depended on spatiotemporal information encoded by flux through CaV2.3, not merely the presence/amplitude of Ca(2+) waves. Using studies in both sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1s lipid and sugar components and CaV2.3s α1E and α2δ subunits. Our results provide a mechanistic understanding of membrane lipid regulation of Ca(2+) flux and therefore Ca(2+)-dependent cellular and developmental processes such as exocytosis and fertilization.

Associations between serum levels of Polybrominated Diphenyl Ether (PBDE) flame retardants and environmental and behavioral factors in pregnant women

Polybrominated diphenyl ethers (PBDE) are flame retardants that were previously used in upholstery, fabrics, and household appliances. PBDEs have been linked to adverse health outcomes, including neurotoxicity, thyroid hormone dysregulation, endocrine disruption, and poor semen quality. Because PBDEs pass into placental circulation, maternal exposures can approximate fetal exposures. Our objectives were to determine whether diet and specific human behaviors were significantly associated with PBDE exposures in a cohort of pregnant women. Women between the 34th and 38th week of pregnancy were given a questionnaire about behavioral, environmental, and dietary factors and asked to provide blood samples. Serum PBDE levels were measured using GS-MS and lipid adjusted. An adjusted ordinary least squares regression model was run to identify potential associations between behaviors and serum PBDE levels. Serum concentrations of BDEs 47, 99, 100, and 153 were found above the limit of detection in at least 50% of study participants and used in our models. Associations with serum PBDEs were observed with self-reported hand-to-mouth behaviors, including biting nails and licking fingers. Serum BDE levels of 47, 99, 153, and total PBDEs were also significantly higher in those individuals owning a large-screen TV compared with those who did not. Serum PBDE levels were comparable to levels reported in the general population. Hand-to-mouth behaviors may influence serum PBDE concentrations in adults. Household electronics such as large-screen TVs appear to serve as a significant source of PBDEs in pregnant women. Together, hand-to-mouth behaviors and TV ownership may serve as a route of exposure to PBDEs in adults.

Mental Health Needs Assessment After the Gulf Coast Oil Spill—Alabama and Mississippi, 2010

INTRODUCTION Previous oil spills and disasters from other human-made events have shown that mental health effects to the affected population are widespread and can be significant. HYPOTHESIS/PROBLEM There has been concern regarding the likelihood that existing public health surveillance was not capturing the mental health effects to the population affected by the Gulf Coast oil spill. The objectives of this study were to assess the mental health needs of coastal communities in the states of Alabama and Mississippi following the Deepwater Horizon oil spill. METHODS A cluster sampling methodology was used to assess the mental health status of coastal residents in three counties in Alabama four months following the 2010 Deepwater Horizon oil spill, and in the Gulf Coast counties in Mississippi 5.5 months after the oil spill. RESULTS A total of 469 residents of the selected areas were interviewed. Between 15.4 and 24.5% of the respondents reported depressive symptoms, with 21.4-31.5% reporting symptoms consistent with an anxiety disorder, and 16.3-22.8% reporting ≥14 mentally unhealthy days within the past 30 days. Overall, there were more negative quality of life indicators and negative social context outcomes than in the states Behavioral Risk Factor Surveillance System (BRFSS) survey. Between 32.1% and 35.7% of all households reported decreased income since the oil spill, and 35.5-38.2% of all households reported having been exposed to oil. CONCLUSION The proportion of respondents reporting negative mental health parameters in the affected Alabama and Mississippi coastal communities is higher than the proportion reported in the 2008 and 2009 BRFSS state reports, suggesting that the public health response to the Deepwater Horizon oil spill should focus on mental health services in these communities.

Community Assessment for Public Health Emergency Response (CASPER) one year following the Gulf Coast oil spill: Alabama and Mississippi, 2011.

BACKGROUND On April 20, 2010, the Deepwater Horizon drilling unit exploded off the coast of Louisiana, resulting in 11 deaths and the largest marine petroleum release in history. Previous oil spill disasters have been associated with negative mental health outcomes in affected communities. In response to requests from Mississippi and Alabama, potential mental health issues resulting from this event were identified by implementing a novel use of a Community Assessment for Public Health Emergency Response (CASPER) in the months immediately following the Gulf Coast oil spill. PURPOSE This assessment was repeated one year later to determine long-term mental health needs and changes. METHODS A two-stage sampling method was used to select households, and a questionnaire including the Centers for Disease Control and Preventions Behavioral Risk Factor Surveillance System (BRFSS) questions was administered. Weighted cluster analysis was conducted, and BRFSS questions were compared to the most recent BRFSS reports and the 2010 results. RESULTS In 2011, 8.8%-15.1% of individuals reported depressive symptoms compared to 15.4%-24.5% of individuals in 2010, with 13.2%-20.3% reporting symptoms consistent with an anxiety disorder compared to 21.4%-31.5% of individuals in 2010. Respondents reporting decreased income following the oil spill were more likely to report mental health symptoms compared to respondents reporting no change in income. CONCLUSIONS Overall, mental health symptoms were higher in the three assessment areas compared to BRFSS reports, but lower than 2010 surveys. These results suggest that mental health services are still needed, particularly in households experiencing decreased income since the oil spill.

Toxicology, environmental health, and the "One Health" concept.

The One Health concept promotes collaboration among veterinarians, physicians, scientists, and other professions to promote human, animal, and ecosystem health. One Health illustrates the interconnectedness and interdependence of human, animal, and ecosystem health. This concept has traditionally focused on zoonoses that are infectious diseases, not on chemical- or poison-related illnesses in animals and their relationship to the detection and prevention of human illness. The purpose of this article is to describe key experiences of scientists in the Health Studies Branch within the National Center for Environmental of Health of the Centers for Disease Control and Prevention in which the study of animal illness facilitated a public health investigation into an outbreak of chemicalassociated human disease. The experiences highlight how utilizing the One Health approach may improve chemical-associated outbreak investigations and facilitate appropriate intervention strategies. An appropriate One Health approach in toxicology and environmental health in outbreak settings should include consideration of the common environments and food sources shared by humans and animals and consideration of the potential for contaminated animal products as food sources in human exposures.