Nicolás Olea

Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: The case of Bisphenol A

Background In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. Objectives We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. Discussion Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., “good science”). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. Conclusions Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.

Urinary concentrations of phthalates and phenols in a population of Spanish pregnant women and children

BACKGROUND Phthalate and phenol exposure is prevalent among the general population and of potential concern for pregnant women and children because of their suspected susceptibility to endocrine effects. OBJECTIVES To evaluate the extent of exposure to several phthalates and phenols in a sample of Spanish pregnant women - according to their individual characteristics (age, social class, education, and body mass index) - and children who participated in the INMA - Infancia y Medio Ambiente (Environment and Childhood) project. METHODS One spot urine sample was taken during the third trimester of pregnancy from 120 pregnant women and from 30 4-year old children belonging to 5 Spanish birth cohorts, and analyzed for 11 phthalate metabolites and 9 phenols. RESULTS Three metabolites of di(2-ethylhexyl) phthalate, mono-2-ethyl-5-carboxypentyl phthalate, mono-2-ethyl-5-hydroxyhexyl phthalate, and mono-2-ethyl-5-oxohexyl phthalate; two metabolites of dibutyl phthalates, mono-isobutyl phthalate and mono-n-butyl phthalate; monoethyl phthalate (MEP), the main metabolite of diethyl phthalate; and two phenols, methyl paraben (M-PB) and 2,5-dichlorophenol were detected in the urine samples of all women. The highest urinary concentrations were for MEP and M-PB. Urinary concentrations of all phthalate metabolites and of 2,4-dichlorophenol, 2,5-dichlorophenol, and bisphenol A were lower in the pregnant women than in the children. Among women, a positive relationship with social class and education was shown for most of the phthalate metabolites and phenols. Almost all phthalate metabolites varied by region even after adjusting for social class and education. CONCLUSIONS Phthalate and phenol exposures are prevalent in a group of pregnant women and young children, two susceptible populations, and these exposures might be positively related to social class.

Human exposure to endocrine-disrupting chemicals and prenatal risk factors for cryptorchidism and hypospadias: a nested case-control study

Background Exposure to xenoestrogens during pregnancy may disturb the development and function of male sexual organs. Objective In this study we aimed to determine whether the combined effect of environmental estrogens measured as total effective xenoestrogen burden (TEXB) is a risk factor for male urogenital malformations. Methods In a case–control study, nested in a mother–child cohort (n = 702) established at Granada University Hospital, we compared 50 newborns with diagnosis of cryptorchidism and/or hypospadias with 114 boys without malformations matched by gestational age, date of birth, and parity. Controls did not differ from the total cohort in confounding variables. TEXB and levels of 16 organochlorine pesticides were measured in placenta tissues. Characteristics of parents, pregnancy, and birth were gathered by questionnaire. We used conditional and unconditional regression models to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Results TEXB from organohalogenated compounds was detectable in 72% and 54% of case and control placentas, respectively. Compared with controls, cases had an OR for detectable versus non-detectable TEXB of 2.82 (95% CI, 1.10–7.24). More pesticides were detected in cases than in controls (9.34 ± 3.19 vs. 6.97 ± 3.93). ORs for cases with detectable levels of pesticides, after adjusting for potential confounders in the conditional regression analysis, were o,p′-DDT (OR = 2.25; 95% CI, 1.03–4.89), p,p′-DDT (OR = 2.63; 95% CI, 1.21–5.72), lindane (OR = 3.38; 95% CI, 1.36–8.38), mirex (OR = 2.85; 95% CI, 1.22–6.66), and endosulfan alpha (OR = 2.19; 95% CI, 0.99–4.82). Engagement of mothers in agriculture (OR = 3.47; 95% CI, 1.33–9.03), fathers’ occupational exposure to xenoestrogens (OR = 2.98; 95% CI, 1.11–8.01), and history of previous stillbirths (OR = 4.20; 95% CI, 1.11–16.66) were also associated with risk of malformations. Conclusions We found an increased risk for male urogenital malformations related to the combined effect of environmental estrogens in placenta.

Breast cancer risk and the combined effect of environmental estrogens.

AbstractObjective: The present study aimed to determine whether the combined effects of environmental estrogens measured as the total effective xenoestrogen burden (TEXB-alpha) are a risk factor for breast cancer over and above the risk potentially linked to specific pesticides. Methods: We measured the levels of 16 organochlorine pesticides as well as TEXB in adipose tissue of 198 women at the time of breast cancer diagnosis. These were compared with findings in 260 age and hospital matched control women without breast cancer. Results: The median levels of p,p′-DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene), aldrin, endosulfan ether and lindane (the pesticides detected in >40% of the study population) were higher in cases than controls, although the differences did not reach statistical significance. After adjusting for potential confounders, the odds ratio (OR) for breast cancer in women with detectable levels of aldrin was 1.55 (95% confidence interval (CI) 1.00–2.40). Among the postmenopausal women, the OR for aldrin and lindane was 1.84 (95% CI 1.06–3.18) and 1.76 (95% CI 1.04–2.98), respectively. Among cases with body mass index (BMI) below the median (28.6 kg/m2), the OR was 3.42 (95% CI 1.22–9.58) for women in the highest quartile of TEXB-alpha versus those in the lowest. The subgroup of leaner postmenopausal women showed an increased risk (OR: 5.67; 95% CI 1.59–20.21) for those in the highest tertile versus those in the lowest. Conclusions: We found an increased risk for breast cancer in the leaner women, especially in the leaner postmenopausal subgroup, related to the TEXB-alpha. The pesticides aldrin and lindane are also individually associated with risk.

Exposure to pesticides and cryptorchidism: geographical evidence of a possible association

Synthetic hormone-disrupting chemicals may play a role in the increased frequency of cryptorchidism observed in some studies. We used a spatial ecological design to search for variations in orchidopexy rates in the province of Granada in Spain and to search for relationships between these differences and geographical variations in exposure to pesticides. Orchidopexy rates were estimated for the period from 1980 to 1991 in all municipalities and health care districts served by the University of Granada Hospital. A random sample of males of the same age (1-16 years) admitted for any reason during the same period was used to estimate inpatient control rates. Each municipality was assigned to one of four levels of pesticide use. We used Poisson homogeneity tests to detect significant differences in rates of orchidopexy between districts and between levels of pesticide use. Poisson and logistic regression models were also used to estimate the strength of association between orchidopexy and level of pesticide use. Orchidopexy rates tended to be higher in districts near the Mediterranean coast where intensive farming is widespread. The city of Granada, where the reference hospital is located, also had higher figures both for orchidopexy and inpatient control rates. Regression models showed that the strength of association between orchidopexy and level of pesticide use tended to increase with higher levels of use, with the exception of level 0 (mainly in the city of Granada). Our results are compatible with a hypothetical association between exposure to hormone-disruptive chemicals and the induction of cryptorchidism. Several methodological limitations in the design make it necessary to evaluate the results with caution. ImagesFigure 1.Figure 2.Figure 3.

In vitro study on the agonistic and antagonistic activities of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear receptors

Bisphenols are a group of chemicals structurally similar to bisphenol-A (BPA) in current use as the primary raw material in the production of polycarbonate and epoxy resins. Some bisphenols are intended to replace BPA in several industrial applications. This is the case of bisphenol-S (BPS), which has an excellent stability at high temperature and resistance to sunlight. Studies on the endocrine properties of BPS have focused on its interaction with human estrogen receptor alpha (hERα), but information on its interaction with other nuclear receptors is scarce. The aim of this study was to investigate interactions of BPS, BPF, BPA and its halogenated derivatives, tetrachlorobisphenol A (TCBPA), and tetrabromobisphenol A (TBBPA), with human estrogen receptors (hERα and hERβ), androgen receptor (hAR), and pregnane X receptor (hPXR), using a panel of in vitro bioassays based on competitive binding to nuclear receptors (NRs), reporter gene expression, and cell proliferation assessment. BPS, BPF, and BPA efficiently activated both ERs, while TCBPA behaved as weak hERα agonist. Unlike BPF and BPA, BPS was more active in the hERβ versus hERα assay. BPF and BPA were full hAR antagonists (BPA>BPF), whereas BPA and BPS were weak hAR agonists. Only BPA, TCBPA, and TBBPA, were hPXR agonists (TCBPA>TBBPA>BPA). These findings provide evidence that BPA congeners and derivatives disrupt multiple NRs and may therefore interfere with the endocrine system. Hence, further research is needed to evaluate the potential endocrine-disrupting activity of putative BPA substitutes.

Association of traffic-related air pollution with cognitive development in children

Background Air pollution from traffic has been associated with cardiorespiratory diseases in children and adults, but there is little information on its potential neurotoxic effects. This study aimed to investigate the association between exposure to nitrogen dioxide (NO2), as a marker of traffic-related air pollution, and cognitive development in children. Methods A population-based birth cohort from southern Spain was followed from the age of 4 years for 1 year. Complete data for analyses were gathered on 210 children living in urban and rural areas. NO2 exposure was predicted by means of land use regression models. A standardised version of the McCarthy Scales of Childrens Abilities (MSCA) was used to assess childrens motor and cognitive abilities. Multivariate analyses were performed to evaluate the relation between exposure to NO2 and MSCA outcomes, adjusting for potential confounders. Results A negative effect of NO2 was found across all MSCA subscales, despite low predicted NO2 exposure levels (5–36 μg/m3). Children exposed to higher NO2 (>24.75 μg/m3) showed a decrease of 4.19 points in the general cognitive score and decreases of 6.71, 7.37 and 8.61 points in quantitative, working memory and gross motor areas, respectively. However, except for gross motor function, associations were not statistically significant. Conclusion Although results were not statistically significant, the associations found between exposure to NO2 and cognitive functions suggest that traffic-related air pollution may have an adverse effect on neurodevelopment, especially early in life, even at low exposure levels.

Estrogenic effect of a series of bisphenol analogues on gene and protein expression in MCF-7 breast cancer cells.

Bisphenols constitute a family of compounds, which includes many substances that have as a common chemical structure two phenolic rings joined together through a bridging carbon. In the present study, we aimed to determine whether several events triggered by 17 beta-estradiol (E(2)) in MCF-7 breast cancer cells were also observed in response to various bisphenol-A (BPA) analogues. We studied the expression of estrogen controlled genes by measuring the induction of pS2 (mRNA and protein) and progesterone receptor (PgR) as well as the expression of a luciferase reporter gene transfected into MVLN cells. These data were compared to the cell proliferation potency and effectiveness as the latest expression of estrogen controlled functions. Bisphenols showed an agonistic effect in all our assays, suggesting that these compounds may act through all the response pathways triggered by the natural hormone. We found differences between the assays in the potency of bisphenols, defined as the minimum concentration required to produce a maximal effect. In the cell proliferation assay, all tested compounds needed a lower concentration than in the other assays to give maximal response. Our results suggest that the polarity and nature of the substituent in the central carbon determines the estrogenic potency. Presence of two propyl chains at the central carbon appears to confer the greatest potency in both gene and protein expression assays.

Human exposure to endocrine disrupters: Standardisation of a marker of estrogenic exposure in adipose tissue

In many epidemiological studies based on the direct measurement of exposure to organochlorines, the chemicals of concern are determined directly from adipose tissue samples. Although the measurement of all possible organochlorines, their metabolites, isomers and congeners may be desirable, it is expensive and time‐consuming and many chemicals with hormonal activity may not yet have been identified. Testing systems are therefore required to screen for estrogenicity and to identify appropriate biomarkers of human exposure. To address this issue, we developed and standardised a method to assess the total estrogenic xenobiotic burden in human adipose tissue. The method extracts and separates the more lipophilic xenoestrogens from ovarian estrogens, with a subsequent bioassay determination of the cumulative effect of the xenoestrogens. It was applied to 400 women, using 200 mg of adipose tissue: 65% of samples showed measurable estrogenicity in the fraction where most non‐polar xenoestrogens eluted, and 76% of fractions where ovarian estrogens eluted were positive for estrogenicity. Residues of 16 organochlorine pesticides were determined. No correlation was found between pesticide content and estrogenicity of the samples. The high percentage of positive samples suggests that the method is sensitive enough to be used as a biomarker of human exposure to estrogenic xenobiotics and can be applied in epidemiological studies.

Determination of Bisphenol A and its chlorinated derivatives in placental tissue samples by liquid chromatography-tandem mass spectrometry

The group of compounds commonly called endocrine disruptors covers a wide range of synthetic and natural substances able to alter the normal hormone function of wildlife and humans, consequently causing adverse health effects. Bisphenol A (BPA) and its chlorinated derivatives are some of these compounds. In this work, we propose a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine these compounds in human placental tissue samples. The method involves an extraction phase of the extracts from the samples using ethyl acetate, followed by a clean-up phase by centrifugation prior to their quantification by LC-MS/MS using an atmospheric pressure chemical ionization (APCI) interface in the negative mode. Deuterated Bisphenol A (BPA-d(16)) was used as internal standard. Found detection limits (DL) ranged from 0.2 to 0.6 ng g(-1) and quantification limits (QL) from 0.5 to 2.0 ng g(-1) for Bisphenol A and its chlorinated derivatives, while inter- and intra-day variability was under 8.1%. The method was validated using standard addition calibration and a spike recovery assay. Recovery rates for spiked samples ranged from 97% to 105%. This method was satisfactorily applied to the determination of BPA and its chlorinated derivatives in 49 placental tissue samples collected from women who live in the province of Granada (Spain).