Stephanie J. Frisbee

The C8 Health Project: Design, Methods, and Participants

Background The C8 Health Project was created, authorized, and funded as part of the settlement agreement reached in the case of Jack W. Leach, et al. v. E.I. du Pont de Nemours & Company (no. 01-C-608 W.Va., Wood County Circuit Court, filed 10 April 2002). The settlement stemmed from the perfluorooctanoic acid (PFOA, or C8) contamination of drinking water in six water districts in two states near the DuPont Washington Works facility near Parkersburg, West Virginia. Objectives This study reports on the methods and results from the C8 Health Project, a population study created to gather data that would allow class members to know their own PFOA levels and permit subsequent epidemiologic investigations. Methods Final study participation was 69,030, enrolled over a 13-month period in 2005–2006. Extensive data were collected, including demographic data, medical diagnoses (both self-report and medical records review), clinical laboratory testing, and determination of serum concentrations of 10 perfluorocarbons (PFCs). Here we describe the processes used to collect, validate, and store these health data. We also describe survey participants and their serum PFC levels. Results The population geometric mean for serum PFOA was 32.91 ng/mL, 500% higher than previously reported for a representative American population. Serum concentrations for perfluorohexane sulfonate and perfluorononanoic acid were elevated 39% and 73% respectively, whereas perfluorooctanesulfonate was present at levels similar to those in the U.S. population. Conclusions This largest known population study of community PFC exposure permits new evaluations of associations between PFOA, in particular, and a range of health parameters. These will contribute to understanding of the biology of PFC exposure. The C8 Health Project also represents an unprecedented effort to gather basic data on an exposed population; its achievements and limitations can inform future legal settlements for populations exposed to environmental contaminants.

Association of Perfluorooctanoic Acid and Perfluorooctane Sulfonate With Serum Lipids Among Adults Living Near a Chemical Plant

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are compounds that do not occur in nature but have been widely used since World War II and persist indefinitely in the environment. They are present in the serum of Americans with median levels of 4 ng/mL and 21 ng/mL, respectively. PFOA has been positively associated with cholesterol in several studies of workers. A cross-sectional study of lipids and PFOA and PFOS was conducted among 46,294 community residents aged 18 years or above, who drank water contaminated with PFOA from a chemical plant in West Virginia. The mean levels of serum PFOA and PFOS in 2005-2006 were 80 ng/mL (median, 27 ng/mL) and 22 ng/mL (median, 20 ng/mL), respectively. All lipid outcomes except high density lipoprotein cholesterol showed significant increasing trends by increasing decile of either compound; high density lipoprotein cholesterol showed no association. The predicted increase in cholesterol from lowest to highest decile for either compound was 11-12 mg/dL. The odds ratios for high cholesterol (>/=240 mg/dL), by increasing quartile of PFOA, were 1.00, 1.21 (95% confidence interval (CI): 1.12, 1.31), 1.33 (95% CI: 1.23, 1.43), and 1.40 (95% CI: 1.29, 1.51) and were similar for PFOS quartiles. Because these data are cross-sectional, causal inference is limited. Nonetheless, the associations between these compounds and lipids raise concerns, given their common presence in the general population.

Perfluorooctanoic acid, perfluorooctanesulfonate, and serum lipids in children and adolescents: results from the C8 Health Project

BACKGROUND Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS) are man-made compounds with widespread presence in human sera. In previous occupational and adult studies, PFOA and PFOS were positively associated with serum lipid levels. OBJECTIVE To interrogate associations between PFOA and PFOS and serum lipids in children and adolescents. DESIGN Cross-sectional community-based study. SETTING Mid-Ohio River Valley. PARTICIPANTS A total of 12 476 children and adolescents included in the C8 Health Project, which resulted from the pretrial settlement of a class action lawsuit pursuant to PFOA contamination of the drinking water supply. MAIN OUTCOME MEASURES Serum lipids (total, high-density lipoprotein [HDL-C], and low-density lipoprotein [LDL-C] cholesterol and fasting triglycerides). RESULTS Mean (SD) serum PFOA and PFOS concentrations were 69.2 (111.9) ng/mL and 22.7 (12.6) ng/mL, respectively. In linear regression after adjustment for covariables, PFOA was significantly associated with increased total cholesterol and LDL-C, and PFOS was significantly associated with increased total cholesterol, HDL-C, and LDL-C. Using general linear model analysis of covariance, between the first and fifth quintiles of PFOA there was a 4.6-mg/dL and a 3.8-mg/dL increase in the adjusted mean levels of total cholesterol and LDL-C levels, respectively, and an 8.5-mg/dL and a 5.8-mg/dL increase in the adjusted mean levels of total cholesterol and LDL-C, respectively, between the first and fifth quintiles of PFOS. Increases were 10 mg/dL for some age- and sex-group strata. Observed effects were nonlinear, with larger increases in total cholesterol and LDL-C levels occurring at the lowest range, particularly of PFOA. CONCLUSION Although the epidemiologic and cross-sectional natures of this study limit causal inferences, the consistently observed associations between increasing PFOA and PFOS and elevated total cholesterol and LDL-C levels warrant further study.

Implications of Early Menopause in Women Exposed to Perfluorocarbons

CONTEXT Perfluorocarbons (PFC) are man-made chemicals used in numerous household products. They have a long half-life in humans and complex animal toxicity, and accumulating evidence points toward associations with multiple human health endpoints. OBJECTIVE Our objective was to investigate whether PFC are associated with endocrine disruption in women. DESIGN Cross-sectional analyses were made between quintiles of serum PFC, serum estradiol, and menopause onset. SETTING The C8 Health Project, with cohort of 69,030 adults and children, was conducted due to PFC contamination of drinking water from six water districts in two states. PARTICIPANTS Participants included 25,957 women aged 18-65 yr. MAIN OUTCOME MEASURES Serum estradiol levels and onset of menopause were assessed. The survey was the result of a class action suit, and survey designers (an independent corporation) had no a priori hypotheses. All hypotheses have been formulated by other investigators after data collection. RESULTS After excluding women who reported hysterectomy and adjusting for age within the group, smoking, alcohol consumption, body mass index, and exercise, the odds of having experienced menopause were significantly higher in the highest quintile relative to the lowest quintile of perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) in the perimenopausal [PFOS odds = 1.4, confidence interval (CI) = 1.1-1.8; PFOA odds =1.4, CI = 1.1-1.8] and menopausal age groups (PFOS odds = 2.1, CI=1.6-2.8; PFOA odds = 1.7, CI = 1.3-2.3). After appropriate exclusions and adjustment for covariates, there was a significant inverse association between PFOS and estradiol in perimenopausal (β = -3.65; P < 0.0001) and menopausal age groups (β = -0.83; P = 0.007) but not between PFOA and estradiol. CONCLUSIONS These data suggest that PFC are associated with endocrine disruption in women and that further research on mechanisms is warranted.

Serum perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) concentrations and liver function biomarkers in a population with elevated PFOA exposure.

Background: Perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) persist in the environment and are found in relatively high concentrations in animal livers. Studies in humans have reported inconsistent associations between PFOA and liver enzymes. Objectives: We examined the cross-sectional association between serum PFOA and PFOS concentrations with markers of liver function in adults. Methods: The C8 Health Project collected data on 69,030 persons; of these, a total of 47,092 adults were included in the present analysis. Linear regression models were fitted for natural log (ln)-transformed values of alanine transaminase (ALT), γ-glutamyltransferase (GGT), and direct bilirubin on PFOA, PFOS, and potential confounders. Logistic regression models were fitted comparing deciles of PFOA or PFOS in relation to high biomarker levels. A multilevel analysis comparing the evidence for association of PFOA with liver function at the individual level within water districts to that at the population level between water districts was also performed. Results: ln-PFOA and ln-PFOS were associated with ln-ALT in linear regression models [PFOA: coefficient, 0.022; 95% confidence interval (CI): 0.018, 0.025; PFOS: coefficient, 0.020; 95% CI: 0.014, 0.026] and with raised ALT in logistic regression models [with a steady increase in the odds ratio (OR) estimates across deciles of PFOA and PFOS; PFOA: OR = 1.10; 95% CI: 1.07, 1.13; PFOS: OR = 1.13; 95% CI: 1.07, 1.18]. There was less consistent evidence of an association of PFOA and GGT or bilirubin. The relationship with bilirubin appears to rise at low levels of PFOA and to fall again at higher levels. Conclusions: These results show a positive association between PFOA and PFOS concentrations and serum ALT level, a marker of hepatocellular damage.

Depressive behavior and vascular dysfunction: a link between clinical depression and vascular disease?

As chronic stress and depression have become recognized as significant risk factors for peripheral vascular disease in patients with no prior history of vasculopathy, we interrogated this relationship utilizing an established mouse model of chronic stress/depressive symptoms from behavioral research. Male mice were exposed to 8 wk of unpredictable chronic mild stress (UCMS; e.g., wet bedding, predator sound/smell, random disruption of light/dark cycle), with indexes of depressive behavior (coat status, grooming, and mobility) becoming exacerbated vs. controls. In vascular rings, constrictor (phenylephrine) and endothelium-independent dilator (sodium nitroprusside) responses were not different between groups, although endothelium-dependent dilation (methacholine) was attenuated with UCMS. Nitric oxide synthase (NOS) inhibition was without effect in UCMS but nearly abolished reactivity in controls, while cyclooxygenase inhibition blunted dilation in both. Combined blockade abolished reactivity in controls, although a significant dilation remained in UCMS that was abolished by catalase. Arterial NO production was attenuated by UCMS, although H2O2 production was increased. UCMS mice demonstrated an increased, although variable, insulin resistance and inflammation. However, while UCMS-induced vascular impairments were consistent, the predictive power of aggregate plasma levels of insulin, TNF-alpha, IL-1beta, and C-reactive peptide were limited. However, when separated into tertiles with regard to vascular outcomes, insulin resistance and hypertension were predictive of the most severe vascular impairments. Taken together, these data suggest that aggregate insulin resistance, inflammation, and hypertension in UCMS mice are not robust predictors of vascular dysfunction, suggesting that unidentified mechanisms may be superior predictors of poor vascular outcomes in this model.

Distinct patterns of fat metabolism in skeletal muscle of normal-weight, overweight, and obese humans

The link between body weight, lipid metabolism, and health risks is poorly understood and difficult to study. Magnetic resonance spectroscopy (MRS) permits noninvasive investigation of lipid metabolism. We extended existing two-dimensional MRS techniques to permit quantification of intra- and extramyocellular lipid (IMCL and EMCL, respectively) compartments and their degree of unsaturation in human subjects and correlated these results with body mass index (BMI). Using muscle creatine for normalization, we observed a statistically significant (P < 0.01) increase in the IMCL-to-creatine ratio with BMI (n = 8 subjects per group): 5.9 +/- 1.7 at BMI < 25, 10.9 +/- 1.82 at 25 < BMI < 30, and 13.1 +/- 0.87 at BMI > 30. Similarly, the degree of IMCL unsaturation decreased significantly (P < 0.01) with BMI: 1.51 +/- 0.08 at BMI < 25, 1.30 +/- 0.11 at 25 < BMI < 30, and 0.90 +/- 0.14 at BMI > 30. We conclude that important aspects of lipid metabolism can be evaluated by two-dimensional MRS and propose that degree of unsaturation measured noninvasively may serve as a biomarker for lipid metabolic defects associated with obesity.

Impact of Chronic Anticholesterol Therapy on Development of Microvascular Rarefaction in the Metabolic Syndrome

Objective: The obese Zucker rat (OZR) model of the metabolic syndrome is partly characterized by moderate hypercholesterolemia, in addition to other contributing comorbidities. Previous results suggest that vascular dysfunction in OZR is associated with chronic reduction in vascular nitric‐oxide (NO) bioavailability and chronic inflammation, both frequently associated with hypercholesterolemia. As such, we evaluated the impact of chronic cholesterol‐reducing therapy on the development of impaired skeletal muscle arteriolar reactivity and microvessel density in OZR and its impact on chronic inflammation and NO bioavailability.

Protective effect of sex on chronic stress- and depressive behavior-induced vascular dysfunction in BALB/cJ mice

The presence of chronic, unresolvable stresses leads to negative health outcomes, including development of clinical depression/depressive disorders, with outcome severity being correlated with depressive symptom severity. One of the major outcomes associated with chronic stress and depression is the development of cardiovascular disease (CVD) and an elevated CVD risk profile. However, in epidemiological research, sex disparities are evident, with premenopausal women suffering from depressive symptoms more acutely than men, but also demonstrating a relative protection from the onset of CVD. Given this, we investigated the differential effect of sex on conduit artery and resistance arteriolar function in male and female mice following 8 wk of an unpredictable chronic mild stress (UCMS) protocol. In males, plasma cortisol and depressive symptom severity (e.g., coat status, anhedonia, delayed grooming) were elevated by UCMS. Endothelium-dependent dilation to methacholine/acetylcholine was impaired in conduit arteries and skeletal muscle arterioles, suggesting a severe loss of nitric oxide bioavailability and increased production of thromboxane A2 vs. prostaglandin I2 associated with elevated reactive oxygen species (ROS) and an increased level of systemic inflammation. Endothelium-independent dilation was intact. In females, depressive symptoms and plasma cortisol increases were more severe than in males, although alterations to vascular reactivity were blunted, including the effects of elevated ROS and inflammation on dilator responses. These results suggest that compared with males, female rats are more susceptible to chronic stress in terms of the severity of depressive behaviors, but that the subsequent development of vasculopathy is blunted owing to an improved ability to tolerate elevated ROS and systemic inflammatory stress.

Microvascular perfusion heterogeneity contributes to peripheral vascular disease in metabolic syndrome

A major challenge facing public health is the increased incidence and prevalence of the metabolic syndrome, a clinical condition characterized by excess adiposity, impaired glycaemic control, dyslipidaemia and moderate hypertension. The greatest concern for this syndrome is the profound increase in risk for development of peripheral vascular disease (PVD) in afflicted persons. However, ongoing studies suggest that reductions in bulk blood flow to skeletal muscle may not be the primary contributor to the premature muscle fatigue that is a hallmark of PVD. Compelling evidence has been provided suggesting that an increasingly spatially heterogeneous and temporally stable distribution of blood flow at successive arteriolar bifurcations in metabolic syndrome creates an environment where a large number of the pre‐capillary arterioles have low perfusion, low haematocrit, and are increasingly confined to this state, with limited ability to adapt perfusion in response to a challenged environment. Single pharmacological interventions are unable to significantly restore function owing to a divergence in their spatial effectiveness, although combined therapeutic approaches to correct adrenergic dysfunction, elevated oxidant stress and increased thromboxane A2 improve perfusion‐based outcomes. Integrated, multi‐target therapeutic interventions designed to restore healthy network function and flexibility may provide for superior outcomes in subjects with metabolic syndrome‐associated PVD.