Researchers have developed a more accurate method of measuring bisphenol A (BPA) levels in humans and found that exposure to the endocrine-disrupting chemical is far higher than previously assumed.
The study, published in the journal The Lancet Diabetes & Endocrinology on Dec. 5, provides the first evidence that the measurements relied upon by regulatory agencies, including the U.S. Food and Drug Administration, are flawed, underestimating exposure levels by as much as 44 times.
“This study raises serious concerns about whether we’ve been careful enough about the safety of this chemical,” said Patricia Hunt, Washington State University professor and corresponding author on the paper.
“What it comes down to is that the conclusions federal agencies have come to about how to regulate BPA may have been based on inaccurate measurements.”
BPA can be found in a wide range of plastics, including food and drink containers, and animal studies have shown that it can interfere with the body’s hormones.
In particular, fetal exposure to BPA has been linked to problems with growth, metabolism, behavior, fertility and even greater cancer risk.
Despite this experimental evidence, the FDA has evaluated data from studies measuring BPA in human urine and determined that human exposure to the chemical is at very low, and therefore, safe levels.
This paper challenges that assumption and raises questions about other chemicals, including BPA replacements, that are also assessed using indirect methods.
Hunt’s colleague, Roy Gerona, assistant professor at University of California, San Francisco, developed a direct way of measuring BPA that more accurately accounts for BPA metabolites, the compounds that are created as the chemical passes through the human body.
Previously, most studies had to rely on an indirect process to measure BPA metabolites, using an enzyme solution made from a snail to transform the metabolites back into whole BPA, which could then be measured.
Gerona’s new method is able to directly measure the BPA metabolites themselves without using the enzyme solution.
In this study, a research team comprised of Gerona, Hunt and Fredrick vom Saal of University of Missouri compared the two methods, first with synthetic urine spiked with BPA and then with 39 human samples.
They found much higher levels of BPA using the direct method, as much as 44 times the mean reported by the National Health and Nutrition Examination Survey (NHANES).
The disparity between the two methods increased with more BPA exposure: the greater the exposure the more the previous method missed.
Gerona, the first author on the paper, said more replication is needed.
“I hope this study will bring attention to the methodology used to measure BPA, and that other experts and labs will take a closer look and assess independently what is happening,” he said.
They found much higher levels of BPA using the direct method, as much as 44 times the mean reported by the National Health and Nutrition Examination Survey (NHANES).
The research team is conducting further experiments into BPA measurement as well as other chemicals that may also have been measured in this manner, a category that includes environmental phenols such as parabens, benzophenone, triclosan found in some cosmetics and soaps, and phthalates found in many consumer products including toys, food packaging and personal care products.
“BPA is still being measured indirectly through NHANES, and it’s not the only endocrine-disrupting chemical being measured this way,” Gerona said.
“Our hypothesis now is that if this is true for BPA, it could be true for all the other chemicals that are measured indirectly.”
Funding: This study was supported by grants from the National Institutes of Health.
Due to concerns about its safety, the use of bisphenol A (BPA) has been restricted, and structural analogs, especially bisphenol S (BPS), have mainly replaced BPA in consumer products (ANSES 2013). As a result of its use to manufacture epoxy resins for inner coating of food cans and packaging containers, the dominant source of human exposure to BPS is considered to be contaminated food (Wu et al. 2018). The use of BPS as a color developer of thermal papers (Wu et al. 2018) may also contribute to human exposure through dermal and hand-to-mouth oral exposures. BPS has thus been detected in 89.4% of urine samples from a representative cohort of the U.S. population (n=868, Lehmler et al. 2018), with median urinary BPS concentrations of 0.37μg/L. Similar median levels of BPS exposure were reported in a study conducted in pregnant women from Netherlands (0.36μg/L; Philips et al. 2018). The increased frequency of BPS detection in urine samples collected between 2000 and 2014 (n=616) in U.S. adult volunteers reflects the reality of substituting BPA with BPS (Ye et al. 2015). The prevalence and level of human exposure may also be increased by potential accumulation of BPS in the environment resulting from its lower biodegradability, in comparison with BPA, in seawater (Danzl et al. 2009).
In vitro studies have demonstrated the endocrine activity of BPS, leading authors to raise the alert about its health hazard potential and the risk of such substitution (Rochester and Bolden 2015). BPS, like BPA, has been shown to display estrogenic activity via nuclear receptors, the potency of the effects depending on the in vitro assay systems used and their related endpoints. Hence, BPS showed less potent estrogen activities than BPA in a human ovarian adenocarcinoma cell line (Rosenmai et al. 2014) but was equipotent to BPA in human breast cancer MCF-7 cells (Kuruto-Niwa et al. 2005). The ability of BPS to act as an estrogen receptor (ER) agonist may result from its binding to ER α and β (Molina-Molina et al. 2013). The lower affinity of human ERα and ERβ for BPS than for BPA, reported by the former authors, is in agreement with its recently demonstrated lower potent estrogenic activity via human ERα and β in comparison with BPA (Kojima et al. 2019). Although these effects via nuclear receptors are observed at micromolar concentrations, some studies have demonstrated that at picomolar (pM) concentrations, BPS can activate nongenomic signaling pathways in pituitary cells, as does BPA (Viñas and Watson 2013). BPS has also been shown to decrease testosterone secretion ex vivo by mouse fetal testes at a lower concentration than BPA (Eladak et al. 2015), whereas, in vitro, the reduction of testosterone secretion by a human adrenocortical carcinoma cell line required higher BPS concentrations (Goldinger et al. 2015). Additionally, similar effects of BPA and BPS have been reported on lipid accumulation and the expression of adipogenic markers in human preadipocytes (Boucher et al. 2014, 2016), the potency of the adipogenic effects of BPS on a preadipocyte murine line being even greater than that of BPA (Ahmed and Atlas 2016). Although limited in number, in vivo studies have also evidenced similar effects of BPA and BPS treatments on mammary gland development that was accelerated in mice prenatally exposed to BPA or BPS (5mg/kg, Tucker et al. 2018), a higher incidence of mammary lesions being observed following prenatal exposure to BPS at 0.5mg/kg. The decrease of protein contents and testosterone concentrations of the rat testes after subchronic oral treatments with 50mg/kg of either BPA or BPS has been associated with a reduction in the height of the epithelial tissues of seminiferous tubules (Ullah et al. 2018). Adverse reproductive outcomes were also observed in female rats subcutaneously treated with either BPA or BPS (5 and 50mg/kg) during the neonatal period of life, including delayed onset of puberty, a disrupted pattern of estrous cyclicity, and detrimental effects on the ovarian development and function (Ahsan et al. 2018).
The harmful consequences of the chemical substitution of BPA with BPS for health may be further exacerbated if the toxicokinetic (TK) properties of BPS increase its bioavailability and enhance its persistence in the body, thereby resulting in higher plasma concentrations of BPS than of BPA for the same external exposure level. Indeed, the amounts of BPA that can reach the target tissues and exert effects are dependent on plasma concentrations, these latter being related to the dose by a key TK parameter, namely the blood (plasma) clearance in addition to the bioavailability. Bioavailability, which corresponds to the amount of substance reaching the systemic circulation unchanged, is determined by both the extent of gastrointestinal absorption and of gut and hepatic first-pass elimination when exposure occurs via the oral route. Due to the extensive first-pass glucuronidation of orally administered BPA (Völkel et al. 2002) and its high plasma clearance (Collet et al. 2015), the concentrations of unconjugated BPA in adult human plasma are predicted to be very low (in the pM range, Gauderat et al. 2017; Teeguarden et al. 2013), the predominant form of circulating BPA being BPA glucuronide (BPAG) (Völkel et al. 2002).
Due to their lack of estrogenicity (Matthews et al. 2001; Skledar et al. 2016), systemic exposure to bisphenol conjugated metabolites is not taken into account for risk assessment purposes. However, in vitro studies have suggested that BPAG may exert biological activities similar to (Boucher et al. 2015) or different from those of the parent compound (Viñas et al. 2013).
Although it cannot be excluded that a possible back conversion of BPAG to its unconjugated form might account for its effect on adipocyte differentiation (Gayrard et al. 2015), the effect of BPAG on some estrogenic signaling pathways in estrogen-responsive prolactinoma cells, as opposed to that of BPA, suggests that BPAG on its own may have the ability to interfere with the actions of estrogens (Viñas et al. 2013). The fact that bisphenol glucuronides cannot be considered as totally inactive raised the need to evaluate systemic exposures to both the parent and its glucuronidated metabolites.
Currently, the limited TK and metabolic data of BPS suggest that although BPS, like BPA, is predominantly metabolized by conjugation reactions (Le Fol et al. 2015; Skledar et al. 2016; Zhou et al. 2014), the TK behavior of BPS may differ from that of BPA. Indeed, Oh et al. (2018) showed that after oral dosing in humans, the fraction of plasma BPS that was unconjugated (28%) was much higher than the corresponding value for BPA (0.5%; Thayer et al. 2015).
Recently, these human biomonitoring data have been used to calibrate a physiologically based toxicokinetic model for BPS oral exposure (Karrer et al. 2018) and to predict human serum concentration profiles of unconjugated BPS. The modeled higher systemic exposure to BPS in comparison with BPA has highlighted the need for experimental data to further elucidate the TK behavior of BPS.
In that context, the objective of the present study was to develop a data-driven semiphysiologically based TK model from data obtained following intravenous administration of BPA, BPS, and BPS glucuronide (BPSG) and gavage administration of BPA and BPS in piglets, described as a relevant species for investigating oral TK in humans (Kararli 1995).
This approach, by enabling a comparison of key TK parameters of BPA and BPS, namely plasma and renal clearances, oral bioavailability, and glucuronidation, should provide new insights for assessing the hazards of BPA substitution.
BPA and BPS were not detected in any of the control samples obtained before the administrations, suggesting that little to no sample contamination had occurred during sample collection, processing, and assay.
Internal Exposure after IV and Oral Dosing
Figures 2A and 2B show the time course of individual plasma concentrations relative to the dose of BPA and BPAG (Figure 2A) and of BPS and BPSG (Figure 2B) after IV dosing. BPA, BPAG (Figure 2A), BPS, and BPSG plasma concentration–time plots (Figure 2B) were obtained after both a single IV administration of BPA at 21.9μmol/kg (Exp. 1 and 2, n=11) or BPS at 20μmol/kg (Exp. 3 and 4, n=12) and simultaneous IV administrations of BPA and BPS at 20μmol/kg (Exp. 5, n=4).
Table 2 gives the values estimated by noncompartmental analysis for TK parameters of BPA and BPAG vs. BPS and BPSG after the corresponding IV BPA and BPS administrations. After IV administration to piglets, plasma BPA and BPS concentrations decreased rapidly to reach values below the LOQ, beyond 12 h after administration. Mean BPS plasma clearance was about 3.5 times lower than BPA clearance (0.95±0.24 vs. 3.41±1.23L/kg×h, p<0.05).
The time–concentration curves of BPAG and BPSG plasma concentrations observed after IV BPA and BPS dosing showed a comparable decay, with maximal plasma BPAG and BPSG concentrations being obtained about 20 min after administration and decreasing slowly to values below the LOQ of the assay, beyond about 12–24 h and 8–12 h after the IV administrations, respectively.
The mean apparent clearance of BPSG was of the same order as that of BPAG (0.41±0.08 vs. 0.51±0.12L/kg×h). This value was close to the BPSG plasma clearance determined after direct IV BPSG dosing (0.56±0.12L/kg×h).
Toxicokinetic parameters of BPA and BPS (mean±SD) estimated by noncompartmental analysis after BPA and BPS IV dosing.
|Toxicokinetic parameter||BPA IV dosing (n=15)||BPS IV dosing (n=16)|
|Cmax/dose (μmol/L per μmol/kg BW)||0.48±0.43||1.80±0.85||2.12±1.82*||2.55±0.58|
|AUC0- tlast/dose (μmol×h/L per μmol/kg)||0.34±0.14||2.04±0.58||1.11±0.30*||2.59±0.77|
Note: The toxicokinetic parameters after IV dosing were estimated from datasets obtained after a single IV administration of BPA at 21.9μmol/kg (Exp. 1–2, n=11), a single IV administration of BPS at 20μmol/kg (Exp. 3–4, n=12), and simultaneous IV administrations of BPA and BPS at the same dosage (Exp. 5, n=4). AUC0- tlast/dose Dose scaled area under the plasma concentration–time curve from dosing time to the time of the last measurable plasma concentration;BPA, Bisphenol A; BPAG, Bisphenol A glucuronide; BPS, Bisphenol S; BPSG, Bisphenol S glucuronide.; BW, Body weight; Cl, Clearance; Cl_F, Apparent clearance; Cmax/dose, Dose scaled maximal plasma concentration; IV, Intravenous; NA, not applicable; Tmax, Time of Cmax.*Significantly different from mean values obtained with BPA (p<0.05, ANOVA test).
Figures 2C and 2D show the time course of individual plasma concentrations relative to the dose of BPA and BPAG (Fig 2C) and of BPS and BPSG (Fig 2D) after oral dosing. BPA, BPAG (Figure 2C), BPS, and BPSG plasma concentration–time plots (Figure 2D) were obtained after both a single oral administration of BPA at 438μmol/kg (Exp. 1, n=8) or BPS at 40μmol/kg (Exp. 4, n=6) and simultaneous oral administrations of BPA and BPS at 200μmol/kg (Exp. 5, n=4). Table 3 gives the values estimated by noncompartmental analysis for TK parameters of BPA and BPAG vs. BPS and BPSG after respective oral BPA and BPS administrations.
Toxicokinetic parameters of BPA and BPS (mean±SD) estimated by non-compartmental analysis after BPA and BPS oral dosing.
|Toxicokinetic parameter||BPA oral dosing (n=12)||BPS oral dosing (n=10)|
|Cmax/dose (μmol/L per μmol/kg BW)||0.0014±0.0018||0.58±0.34||0.51±0.29||2.94±4.78|
|AUClast/dose (μmol×h/L per μmol/kg BW)||0.0024±0.0027||1.75±0.73||0.63±0.29||2.87±0.99|
Note: The toxicokinetic parameters after oral dosing were estimated from data sets obtained after a single oral administration of BPA at 438μmol/kg (Exp. 1, n=8), a single oral administration of BPS at 40μmol/kg (Exp. 3–4, n=6), and simultaneous oral administrations of BPA and BPS at 200μmol/kg (Exp. 5, n=4). AUClast/dose, Dose scaled area under the plasma concentration–time curve from dosing time to the time of the last measurable plasma concentration; BPA, Bisphenol A; BPAG, Bisphenol A glucuronide; BPS, Bisphenol S; BPSG, Bisphenol S glucuronide; BW, Body weight; Cl_F, Apparent clearance; Cmax/dose, Dose scaled maximal plasma concentration; IV, Intravenous; Tmax, Time of Cmax.
After oral administrations, the BPA and BPS plasma concentrations increased to maximal values about 1 h and 30 min after dosing, respectively. The average maximal BPS plasma concentrations were, in relation to the dose administered, more than 300 times higher than the maximal BPA values ranging from 173 to 951 nmol/L (43−238 ng/ml) scaled for a nominal dose of 1μmol/kg BW for BPS and from 0.059 to 5.75 nmol/L (0.013−1.31 ng/ml) scaled for a nominal dose of 1μmol/kg BW for BPA. The plasma concentrations of BPS remained above the LOQ of the assay for 3 times longer than those of BPA.
The BPS systemic exposure after oral dosing was, on average, 262 times higher than that of BPA, the AUC0-tlast values relative to dose ranging from 323 to 1,220 nmol×h/L (81−305 ng×h/ml) scaled for a nominal dose of 1μmol/kg BW for BPS and from 0.23 to 9.21 nmol×h/L (0.053−2.10 ng×h/ml) scaled for a nominal dose of 1μmol/kg BW for BPA. The time course of BPAG and BPSG plasma concentrations observed after oral administrations of BPA and BPS, respectively, did not differ significantly.
Urinary Excretion of BPA and BPS
The cumulative urinary excretions of BPAG, BPS, and BPSG that were recovered at fixed intervals over 24 h (Exp. 2–5) are presented in Figure 3. By 24 h, the mean fractions (±SD) of the BPA dose recovered in urine as BPAG was approximately two times lower after oral BPA dosing (62.2±15.8%, range: 49.5–80.2%, n=4) than after IV BPA dosing (97.0±27.6%, 63.1–135.7%, n=7, Figure 3).
The mean fractional urinary excretion of BPS as BPSG was, respectively, 76.8±20.1% (36.3–107.9%, n=16) and 86.5±23.9% (65.5–126.8%, n=10) after IV and oral BPS dosing. About half of the BPA and BPS doses were eliminated 3 h after dosing. Two of the low BPSG recoveries (<70%) were attributed to loss of urine at one collection time corresponding to 6 h and 9 h post dosing.
Unconjugated BPA and BPS in urine represented, respectively, 0.044±0.028% and 0.027±0.0084% of the IV BPA and BPS doses. The mean fraction of the IV BPSG dose recovered in urine was 59.0±9.96% (40.1–69.8%), 24 h post dosing.
For the present paper, the NLME modeling enabled several historical information data sets to be used to generate a single set of parameters (with their SE) for BPA and BPS. The estimated primary parameters (noted vector thetas), namely VBPA (VBPS), VBPAG (VBPSG), and the 13 Kij of the model are reported with their SE and their coefficient of variation (Tables S3 and S4).
The mean volume of the central compartment of BPA (1.0L/kg) was the highest in comparison with BPS (0.30L/kg), BPSG (0.14L/kg), and BPAG (0.11L/kg). Tables S5 and S6 report the estimated variance matrix of η (ω²) and BSV for the structural parameters of BPA and BPS TK model for which the shrinkage for η was lower than 30% (K12, K23, K25, K30, K50, VBPA, VBPAG, VBPS and VBPSG).
The population predicted vs. observed plasma concentrations of BPA and BPAG and of BPS and BPSG (Figures S1 and S2) were evenly distributed around the line of identity of the diagnostic plots, suggesting that these data were appropriately described by the model. The goodness-of-fit plots for corresponding individual predicted values suggested the absence of any major bias in the random component of the model.
The population predicted vs. observed quantities of BPA, BPAG, BPS, and BPSG excreted in urine (Figures S3A1–S5A1 and Figures S3B1–S5B1) were distributed vertically, suggesting that the model did not adequately predict these population values, especially the very low quantities of BPA and BPS excreted in urine (Figures S3A1–S5A1). Rather than discard these urinary values, we kept them not only to ensure the transparency of our results but also because the corresponding individual predicted values that were obtained by including a random component in the structural model were reasonably well predicted by the model (Figures S3–S5). Figures S6–S9 show the results of the Visual Predictive Check of the model for BPA and BPS TK, respectively. For each figure except those related to the very small quantities of BPA (Figure S7A) and BPS (Figure S9A and B) excreted in the urine after BPA and BPS IV or oral BPS dosing, the observed quantiles (20%, 50%, and 80%) are reasonably well overlaid by the corresponding predictive check quantiles.
Tables 4 and and55 show the population secondary parameters for BPA/BPAG and BPS/BPSG, respectively, estimated from the primary parameters of the model. Figure 4 compares the fates of a same BPA and BPS dose given orally as predicted by the model.
The estimated fraction of the oral BPA dose absorbed by the enterocytes was 76.6%, whereas BPS oral absorption was near total. According to the model, about 34% of the BPA dose (44% of the absorbed dose) was glucuronidated in the gut wall, the near totality (99%) of the remaining fraction (40.3%, i.e., 53% of the absorbed dose) being subjected to an hepatic first-pass effect (39.9%).
In contrast, the model predicted that nearly 100% of the ingested BPS dose gained direct access to the liver, where the estimated hepatic first-pass effect was only 41%. The lack of a gut first-pass effect for BPS associated with a limited hepatic first-pass effect resulted in a much higher oral bioavailable fraction for BPS (57.4%) than for BPA (0.50%, Tables 4 and and5).5).
The renal clearance values of BPA (0.0020L/kg×h) and BPS (0.00035L/kg×h) represented 0.06 and 0.037% of their respective plasma clearance values estimated by noncompartmental analysis, whereas the renal clearance values of BPAG (0.277L/kg×h) and BPSG (0.243L/kg×h) were close to their respective apparent clearance values (Tables 4 and and55).
Population secondary parameters of BPA/BPAG in pigs as obtained with a 9-compartment model.
|BPA fraction absorbed by the gastro-intestinal tract||0.765||0.766||7.84|
|Fraction of absorbed BPA undergoing a first-pass effect (gut and liver)||0.988||0.989||0.61|
|Fraction of administered BPA reaching the liver by the portal blood flow||0.405||0.403||27.13|
|Fraction of administered BPA glucuronidated by the gastro-intestinal tract||0.360||0.340||24.39|
|BPA renal clearance (L/kg×h)||0.0020||0.0020||23.35|
|BPAG renal clearance (L/kg×h)||0.277||0.279||14.12|
|BPA Bioavailability (%)||0.005||0.005||31.73|
Note: The bootstrap procedure was used to estimate the mean, median, and precision (coefficient of variation, CV%) of parameter estimates. BPA: Bisphenol A; BPAG, Bisphenol A glucuronide.
Population secondary parameters of BPS/BPSG in pigs as obtained with a 9-compartment model.
|BPS fraction absorbed by the gastro-intestinal tract||0.986||0.989||0.956|
|Fraction of absorbed BPS undergoing a first-pass effect (liver)||0.435||0.408||17.412|
|Fraction of administered BPS reaching the liver by the portal blood flow||0.986||0.989||0.966|
|Fraction of administered BPS glucuronidated by the gastro-intestinal tract||0.00015||0.00014||66.823|
|BPS renal clearance (L/kg×h)||0.00035||0.00035||16.325|
|BPSG renal clearance (L/kg×h)||0.243||0.245||11.188|
|BPS Bioavailability (%)||0.557||0.574||12.935|
Note: The bootstrap procedure was used to estimate the mean, median, and precision (coefficient of variation, CV%) of parameter estimates. BPS, Bisphenol S; BPSG, Bisphenol S glucuronide.
Washington State University
Patricia Hunt – Washington State University
The image is credited to Washington State University.
Original Research: Open access
“BPA: have flawed analytical techniques compromised risk assessments?”. Roy Gerona, Frederick S vom Saal, Patricia A Hunt.
The Lancet Diabetes & Endocrinology doi:10.1016/S2213-8587(19)30381-X.