Previously, we reported an analysis of NHANES 2003/04, in which we found associations between raised urinary BPA concentrations and coronary heart disease, diabetes and raised liver enzymes in adults. There was no evidence of association with other reported health outcomes, suggesting specificity of the findings. Here we sought to determine if these associations were present again in the NHANES 2005/06 sample, and to calculate pooled estimates of the effect sizes. These analyses use the only large-scale (and high-quality) population-representative datasets available. Urinary BPA concentrations were lower in 2005/06 than in the earlier NHANES wave, with a geometric mean value of 1.79 compared with the earlier mean of 2.49. This represents a fall of around 30%. After adjusting for potential confounders, we found that higher BPA concentrations were again associated with heart disease diagnoses. Associations of BPA concentration with diabetes and liver enzymes were not statistically significant in the 2005/06 data, although pooled estimates remained significant.
Cardiovascular disease results from a complex interaction of genetic, lifestyle and environmental factors. Whilst several personal risk factors for developing heart disease have been identified, including smoking, diabetes and dyslipidemia, the contribution of environmental contaminants has received comparatively less attention. To date, the most widely reported associations between cardiovascular disease and pollutants have centred on exposure to air particulates, heavy metals, notably arsenic and lead, and persistent organic pollutants.
It is pertinent to consider what is known of these previously reported associations, to place the current findings of associations with BPA into context. Studies of over 100 million people across the USA and Europe have shown that for every 10 mg/m3
increase in aerial fine particulate matter (PM10), there is a 0.3%–0.7% increase in cardiovascular mortality 
. These effects can appear and disappear quickly; transient increases in very fine particulate matter are associated with an increased incidence of acute myocardial infarction within a few hours, highlighting the high sensitivity of cardiovascular tissues to the effects of environmental contaminants 
. Such associations are plausible through several biological mechanisms, including alterations to vasomotor tone 
, thrombogenic effects 
and the induction of systemic inflammation 
. In the case of arsenic, altered vascular tone appears to be of more importance to the elevation in cardiovascular risk than vascular inflammation or dislipidemia 
. Animal studies have implicated dysregulation of nitric oxide production and reactivity in arsenic's adverse effects on cardiovascular risk 
, potentially via oxidation of protein thiol groups, including endothelial nitric oxide synthase (eNOS) 
. A similar mechanism has been suggested for lead, which has been associated with hypertension in both animal and human epidemiology studies 
. Both polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p
-dioxin (TCDD) can induce oxidative stress-mediated alterations to normal vascular endothelial cell function, leading to pro-inflammatory changes 
. TCDDs can in addition cause an increase in atherogenic serum lipid levels in animals and humans and a decrease in low density lipoprotein receptors in the liver 
. Such a combination of damage to endothelial cells with elevation in serum lipid levels could be expected to increase cardiovascular risk.
The relevance of these mechanisms to the reported association between BPA and cardiovascular disease in the current study remains subjective. For example, inflammation is a dynamic response of vascularised tissues to injury, whilst excessive or prolonged inflammation is associated with various diseases in addition to cardiovascular disease 
. Following oral ingestion, BPA is not considered a persistent compound, but neither is it immediately cleared from the body, and there appears to be virtually continuous human exposure 
. It is lipophilic (the log of the octanol-water partition co-efficient (Kow
) for BPA is between 2.2 and 3.82 (NTP, 2008)) and it is plausible that partitioning to lipid rich tissues could occur with frequent exposure. This possibility is supported by recent findings that the population-based half life for BPA is significantly longer than the previous estimates of around 6 hours 
. The metabolism of BPA is reported to induce oxidative stress in rat hepatocytes following long term (30 day) oral intake 
and oxidative cellular damage has been reported in a number of other experimental contexts 
. Slow release of BPA or metabolites from tissue sites could theoretically lead to oxidative endothelial cell damage.
Urinary BPA concentrations have previously been shown to be positively associated with oxidative stress markers. Hong et al 
found a significant dose-responsive relationship between BPA and the oxidative stress markers malondialdehyde (MDA) and 8-deoxyguanosine (8-OHG) in a study of 960 adults, although the relationship was not present in multiple regression models adjusted for confounders including age and sex. Positive associations were also reported between urinary BPA, MDA and 8-OHD, and with C-reactive protein, an inflammatory marker, in a cross-sectional study of 134 post-menopausal women, but not in men or pre-menopausal women from the same study 
. These gender differences suggest that occupancy and activation of the estrogen receptor may have been a contributing factor in the induction of oxidative stress.
BPA is believed to exert its biological effects largely through loose occupancy of the estrogen receptor, and there are reports of both estrogen agonist 
and androgen antagonist 
activity. BPA also binds strongly to the estrogen-related receptor-gamma (ERR-gamma), the function of which is unknown 
. BPA exposure of cell suspensions results in lipid accumulation in adipocytes and hepatoma cell lines 
. A variety of other effects of BPA have been noted, including disrupted pancreatic Beta-cell function producing insulin resistance in mice exposed to oral BPA doses well below the current lowest observed adverse effect level considered by the US-EPA 
. Two days of low-dose BPA injections also produced insulin resistance in mice 
. Associations have been described previously between environmental toxins, body weight, and diabetes 
leading to suggestions that exposure to certain environmental pollutants may initiate or exacerbate the development of obesity 
and associated health problems 
. Diabetes and dyslipidemia are recognized as personal risk factors for developing heart disease. However, associations with diabetes seen in the 2003/04 NHANES survey was not evident in the current study, possibly because of the reduction in statistical power due to the lower levels of BPA in 2005/06, resulting in wide confidence intervals around estimates.
It is possible that an as yet unidentified mechanism could be involved in the effects of BPA on cardiovascular function. The peroxisome proliferation activated receptor gamma, PPARγ plays a central role in the control of energy balance and lipid homeostasis 
and PPARγ agonists such as rosiglitazone are used to treat insulin sensitivity. There have been reports that in addition to PPARγ-mediated genome effects on genes important for maintaining vascular tone (including eNOS), PPARγ agonists may activate or inhibit ion channel activity in vessel walls directly. In studies of isolated vascular smooth muscle, rosiglitazone was able to attenuate inward calcium currents and enhance calcium-activated potassium currents 
. Certain BPA derivatives, including bisphenol A diglycidyl ether (BADGE) are peroxisome proliferation activated receptor gamma (PPARγ) antagonists and BADGE can block the adipogenic action of the receptor 
. Although BPA has been reported to increase adipocyte differentiation, it does not appear to affect PPARγ through the same mechanism as BADGE. Additional studies are clearly called for to examine in detail the relationship between BPA and its derivates and PPARγ signaling.
A major limitation of our original analysis on NHANES 2003/04 
was that no replication data were available to help exclude the possibility of a chance finding. The 2005/06 analyses presented here are based on an entirely new population sample, providing an opportunity for independent replication of the earlier findings. BPA concentrations in 2005/06 were substantially lower than in 2003/04, resulting in a reduced power to detect real associations. Despite this, we found near or significant associations with responses to the three questions about heart disease in NHANES 2005/06, and highly significant p-values for the pooled estimates of association. We can now conclude that chance is an implausible explanation for the BPA association with heart disease. Replication of the BPA associations with diabetes and liver enzymes are less certain, with estimates for 2005/06 having wide confidence intervals and not reaching statistical significance. Because of the lower BPA concentration levels in 2005/06 a reduction in the strengths of association is to be expected. The BPA measures in NHANES are based on single spot specimens, so misclassification from this single snapshot of body burden will have resulted in a smaller (diluted) estimate of the strength of association between BPA and the conditions of interest: the true associations are likely to be much stronger. In addition, the pooled estimates of association suggest that the new data is consistent with the original findings: larger samples will be needed to provide accurate estimates of BPA associations with diabetes or liver enzymes to exclude the possibility of real associations being present.
The cross sectional nature of the associations reported need to be treated with caution, as it is theoretically possible, for example, that those with cardiovascular disease change their diets in such a way as to increase BPA exposure. In addition, it is possible that BPA exposure is associated with differential survival in cardiovascular disease. Longitudinal data demonstrating that high BPA concentrations predict later onsets of heart disease or diabetes would strengthen the evidence for BPA playing a causal role. More work is also needed to understand the mechanisms of effect underlying the BPA exposure heart disease association, including in vitro and in vivo studies.
Urinary BPA concentrations in the NHANES adult population representative sample of the United States in 2005/06 were substantially lower than in 2003/04. Despite this, we have replicated earlier findings that higher urinary concentrations of Bisphenol A are associated with an increased prevalence of coronary heart disease.
Associations between urinary BPA concentration and diabetes or liver enzyme increases were not statistically significant in 2005/06, but confidence intervals were wide and associations remained in pooled data. Detailed studies are needed to clarify the mechanisms explaining the statistical association between BPA and adult morbidity.