Mutation Research 774 (2015) 33–39
Contents lists available at ScienceDirect
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres
Bisphenol A and congenital developmental defects in humans Maurizio Guida a , Jacopo Troisi a,∗ , Carla Ciccone b , Giovanni Granozio a , Cosimo Cosimato a , Attilio Di Spiezio Sardo c , Cinzia Ferrara c , Marco Guida d , Carmine Nappi c , Fulvio Zullo a , Costantino Di Carlo c a
Department of Medicine, University of Salerno, Italy “G. Moscati” Hospital Avellino, Italy Department of Medicine, “Federico II”, University of Naples, Italy d Department of Biology, “Federico II”, University of Naples, Italy b c
a r t i c l e
i n f o
Article history: Received 30 May 2014 Received in revised form 12 February 2015 Accepted 26 February 2015 Available online 6 March 2015 Keywords: Bisphenol A Fetal malformations Endocrine disruptors
a b s t r a c t Over 50% of the causes of fetal malformations in humans are still unknown. Recent evidence suggests the relationship between environmental exposure to endocrine disruptors and fetal malformations. Our study aims to establish the role of Bisphenol A (BPA), if any, in altering human reproduction. We enrolled 151 pregnant women who were divided into two groups: case group (CS, n = 101), women with established diagnosis of developmental defect, and control group (CL, n = 50), pregnant women with normally developed fetus. Total, free and conjugated BPA were measured in their blood using GC–MS with isotopic dilution. The results show a correlation between environmental exposure to BPA and the genesis of fetal malformations. Conjugated BPA, which was higher in the CL, casts light on the hypothesis that a reduced ability to metabolize the chemical in the mother can concur to the occurrence of malformation. In a more detailed manner, in case of chromosomal malformations, the average value of free BPA appears to be nearly three times greater than that of the controls. Similarly, in case of central and peripheral nervous system non-chromosomal malformations, the value of free BPA is nearly two times greater than that of the controls. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Over the last decade, an increasing amount of evidence has focused attention on how the exposure, in utero or in the early periods of extra uterine life, to industrial chemicals may interfere with the programming of complex endocrine pathways [50,60,27]. During intrauterine life, these endocrine pathways are involved with the various stages of embryo-fetal development, particularly the neurological, endocrine and immunological ones [58]. The chemicals that interact with the endocrine system are called exogenous endocrine disruptors (EDs) or xeno hormones, for their ability to mimic or antagonize the biological activity of several hormones through interaction, as agonists or antagonists, with specific hormone receptors [9]. Bisphenol A (BPA) is one of the most widespread endocrine disruptors: it is an ubiquitous organic compound, essential in plastic (polycarbonates and epoxy resins) and
∗ Corresponding author at: Department of Medicine, University of Salerno, LargoCittà di Ippocrate, 84131 Salerno, Italy. Tel.: +39 089 671111. E-mail address: [email protected] (J. Troisi). http://dx.doi.org/10.1016/j.mrfmmm.2015.02.007 0027-5107/© 2015 Elsevier B.V. All rights reserved.
plastic additive synthesis, whose production has steadily spread around the world [59]. Polycarbonate has got many applications, including the production of bottles, dishes, compact discs, paper used in some purchase receipts, self-adhesive labels, paper de fax and dental sealants [13,28]. Epoxy resins, however, are used as the internal linings of metal products (for example, cans for food and beverages) and in the water pipes. Despite the spread of the compound, it is thought that food is the main source of human exposure [62], with the contamination of food and beverages contained in polycarbonate bottles and cans which are internally coated with epoxy resin [20], following phenomena of “migration” of BPA unbound monomers [18]. Indeed, prolonged contact between plastic containers containing BPA and acid or base, or even exposure to high temperatures (especially with microwave heating) favor the phenomenon of migration [61,26]. In the body, BPA can be found in two forms: free, i.e. the proportion with endocrine-like activity and conjugated, which is free of xeno hormonal activity. Studies on BPA pharmacokinetics (Matsumoto et al., 2002) [31,10] have shown that the BPA metabolism mainly occurs via hepatic glucuronosyltransferase (GT), with the formation of BPA-glucuronide, which is water-soluble and, therefore, eliminated by the kidney. To
34
M. Guida et al. / Mutation Research 774 (2015) 33–39
a lesser percentage, the breakdown is performed by a sulfotransferase, with the formation of BPA-sulfate. The two metabolites, BPA-glucuronide and BPA-sulfate, do not interfere with the biological processes of the body. The results of Matsumoto et al. (2002) [31] have shown that GT activity is age-dependent and, in particular, much lower in fetal and neonatal age. In contrast, in fetal and neonatal life, the major route of BPA metabolism would seem to be that of sulfation [5]. Indeed, a number of enzymes involved in BPA metabolism to BPA-sulfate are known and have already been shown to be active during intrauterine life [49,17]. In pregnant women, Bisphenol A can be detected in blood, amniotic fluid, placental tissue and umbilical cord blood [41,12,25]. Thus, the exposure of an organism to BPA, and EDs in general, can indirectly occur as early as during intrauterine life, when hundreds of toxic chemicals can reach the embryo/fetus through the placental-blood barrier [58]. In this phase, called the “critical window of development” the product of conception appears extremely sensitive to chemicals with hormone-like activity, the effects of which will be much more pronounced, starting at much lower concentrations than those which would be harmful in adulthood [40]. This increased susceptibility is mainly due to the immaturity of all the systems of “protection” available to the adult, including: enzymatic mechanisms of DNA repair, the above-cited hepatic, but not only, enzymatic mechanisms of detoxification and metabolism, a competent immune system, the impenetrability of the blood–brain barrier, and so on [58]. Due to its estrogen-like properties, which mimic the hormone 17--estradiol, BPA can bind to the estrogen receptors (ER␣, and ER), triggering activation through atypical pathway. However, the National Toxicology Program – U.S. Department of Health and Human Services considers it appropriate not to exclusively refer to its action mediated by estrogen receptors ␣ and  binding [48]; a growing number of in vitro studies, in fact, suggest that it would be too simplistic to merely attribute to BPA the classical mechanism of estrogen action. In addition to binding nuclear ER␣-Er, bisphenol A has the ability to interact with “non-classical membrane estrogen receptors” (ncmER) [48,37,2], a seven transmembrane domain receptor (GPR30) [53], an “estrogen-related receptor-␥” (ERR-␥), an orphan nuclear receptor [32,1], the aryl hydrocarbon receptor (AhR) [3,23], the androgen receptor, acting as an antagonist [3,42,52,35]. Most of the information about the role of BPA in the genesis of malformations derives from studies on animal models, but the results in this regard are still fragmentary and conflicting. A clear influence of BPA on the development processes was inferred from studies on laboratory mice subjected to high doses of BPA during pregnancy. Exposure to BPA has shown to be capable of reducing the embryo-fetal survival [35] (Tyl et al., 2002) [56], the birth weight and the growth of the offspring in the first years of life [35,56,57]. In addition to these “high dose” effects on survival and growth, other studies have shown a variety of effects induced by much lower doses of BPA, i.e. doses below the established NOAEL of 50 g/kg/day, which reproduce better the actual human exposure. The adverse effects induced by a “low dose” of BPA include neuronal and behavioral alterations: documented effects of prenatal exposure to BPA are abnormal development of the neocortex in terms of differentiation and neuronal migration [19,38], aberrant positions and connections between thalamus and cortex [39], inhibition of the proliferation of neural progenitor cells [21], loss of sexual dimorphism in terms of brain structure and behavior [24], increased anxiety and cognitive deficits [45,54]. Part of these results are justified by the strong affinity of BPA to the dopamine receptor, the estrogen-like receptor- type [8] and the estrogen-like receptor-␥ type present in hippocampal neurons [51]. In all these hypotheses the normal synaptic communication was altered. Moreover, other documented effects concern potentially pre-cancerous lesions of prostate and mammary gland [36]; malformation of male urinary tract (decreased anogenital distance)
(Gupta et al., 2000) [14,55,7], and vagina [30]. In contrast to the harmful effects induced by high doses of BPA, there is a dispute about the interpretation of the effects induced by low doses, for which the evidence is limited and conflicting, even in laboratory animals. As far as studies on humans are concerned, effects of maternal exposure to BPA and birth weight [34,6] and behavior [4,43,64] are still limited and conflicting. In the light of this knowledge, this study aims at investigating the correlation between maternal blood BPA (representing an esteem of maternal exposure) and fetal malformations.
2. Materials and methods 2.1. Patients The samples were collected during years 2011–2013 in three Italian hospitals (University of Naples “Federico II”, University of Salerno and Hospital “Moscati” of Avellino). One hundred seventy healthy pregnant women, who either visited the hospital for voluntary interruption of pregnancy due to diagnosis of fetal malformation (case group: CS), or visited the hospital for routine analyses during their normally conducted pregnancy (control group: CL), and accepted to participate in the study, were enrolled. Human tissue collection strictly adhered to the guidelines outlined in the Declaration of Helsinki and the relevant ethic committees approved the study. Subjects completed a questionnaire addressing physical characteristics and health and pregnancy-related features and were subjected to a complete gynecological visit. Blood samples of the CS group were collected before surgery for pregnancy interruption and before any drug administration using a BD vacutainer (BPA free) (Becton Dickinson UK Limited, Oxfordshire, UK) blood collection red tube (with no additives) and immediately freezed to −30 ◦ C until analysis. Blood samples of the CL group were collected during the routine control visits of mothers with normally developed fetuses. All patients were asked to respect a 12 h fast before blood collection. Diagnostic suspect of fetal malformation subsequent to US examination or amniocentesis was confirmed by autopic post explant fetal examination. Inclusion/exclusion criteria used were different for the two groups. In the CS group, we included patients meeting the following inclusion criteria: age 40 years (risk factor for chromosomal malformation) [47] and women with known etiology of fetopathy were excluded from the CS group. In the CL group we included patients meeting the following inclusion criteria: age 191.20 m/z for BPA and 370.50 > 73.10 m/z for the d16-BPA. The event time for the SCAN mode was set to 0.07 s (corresponding to a scan speed of 10,000 amu/s, the maximum obtainable using the TQ-8030). Calibration was carried out using nine standard solutions and a blank. The standard ranged from 0.01 g/L up to 100 g/L. Each standard was analyzed in triplicate. Two recovery tests were conducted at 0.5
35
and at 80 g/L, showing a recovery of 79% at low concentration level and 85% at high concentration, according to [22]. The results were corrected according to the nearest recovery value. Quantization was conducted with the isotopic dilution method using d16-BPA as an internal standard. Every 10 samples, a blank sample and two calibration solutions: 25 g/L, and 0.01 g/L were analyzed. The tolerance criteria adopted were
Contents lists available at ScienceDirect
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres
Bisphenol A and congenital developmental defects in humans Maurizio Guida a , Jacopo Troisi a,∗ , Carla Ciccone b , Giovanni Granozio a , Cosimo Cosimato a , Attilio Di Spiezio Sardo c , Cinzia Ferrara c , Marco Guida d , Carmine Nappi c , Fulvio Zullo a , Costantino Di Carlo c a
Department of Medicine, University of Salerno, Italy “G. Moscati” Hospital Avellino, Italy Department of Medicine, “Federico II”, University of Naples, Italy d Department of Biology, “Federico II”, University of Naples, Italy b c
a r t i c l e
i n f o
Article history: Received 30 May 2014 Received in revised form 12 February 2015 Accepted 26 February 2015 Available online 6 March 2015 Keywords: Bisphenol A Fetal malformations Endocrine disruptors
a b s t r a c t Over 50% of the causes of fetal malformations in humans are still unknown. Recent evidence suggests the relationship between environmental exposure to endocrine disruptors and fetal malformations. Our study aims to establish the role of Bisphenol A (BPA), if any, in altering human reproduction. We enrolled 151 pregnant women who were divided into two groups: case group (CS, n = 101), women with established diagnosis of developmental defect, and control group (CL, n = 50), pregnant women with normally developed fetus. Total, free and conjugated BPA were measured in their blood using GC–MS with isotopic dilution. The results show a correlation between environmental exposure to BPA and the genesis of fetal malformations. Conjugated BPA, which was higher in the CL, casts light on the hypothesis that a reduced ability to metabolize the chemical in the mother can concur to the occurrence of malformation. In a more detailed manner, in case of chromosomal malformations, the average value of free BPA appears to be nearly three times greater than that of the controls. Similarly, in case of central and peripheral nervous system non-chromosomal malformations, the value of free BPA is nearly two times greater than that of the controls. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Over the last decade, an increasing amount of evidence has focused attention on how the exposure, in utero or in the early periods of extra uterine life, to industrial chemicals may interfere with the programming of complex endocrine pathways [50,60,27]. During intrauterine life, these endocrine pathways are involved with the various stages of embryo-fetal development, particularly the neurological, endocrine and immunological ones [58]. The chemicals that interact with the endocrine system are called exogenous endocrine disruptors (EDs) or xeno hormones, for their ability to mimic or antagonize the biological activity of several hormones through interaction, as agonists or antagonists, with specific hormone receptors [9]. Bisphenol A (BPA) is one of the most widespread endocrine disruptors: it is an ubiquitous organic compound, essential in plastic (polycarbonates and epoxy resins) and
∗ Corresponding author at: Department of Medicine, University of Salerno, LargoCittà di Ippocrate, 84131 Salerno, Italy. Tel.: +39 089 671111. E-mail address: [email protected] (J. Troisi). http://dx.doi.org/10.1016/j.mrfmmm.2015.02.007 0027-5107/© 2015 Elsevier B.V. All rights reserved.
plastic additive synthesis, whose production has steadily spread around the world [59]. Polycarbonate has got many applications, including the production of bottles, dishes, compact discs, paper used in some purchase receipts, self-adhesive labels, paper de fax and dental sealants [13,28]. Epoxy resins, however, are used as the internal linings of metal products (for example, cans for food and beverages) and in the water pipes. Despite the spread of the compound, it is thought that food is the main source of human exposure [62], with the contamination of food and beverages contained in polycarbonate bottles and cans which are internally coated with epoxy resin [20], following phenomena of “migration” of BPA unbound monomers [18]. Indeed, prolonged contact between plastic containers containing BPA and acid or base, or even exposure to high temperatures (especially with microwave heating) favor the phenomenon of migration [61,26]. In the body, BPA can be found in two forms: free, i.e. the proportion with endocrine-like activity and conjugated, which is free of xeno hormonal activity. Studies on BPA pharmacokinetics (Matsumoto et al., 2002) [31,10] have shown that the BPA metabolism mainly occurs via hepatic glucuronosyltransferase (GT), with the formation of BPA-glucuronide, which is water-soluble and, therefore, eliminated by the kidney. To
34
M. Guida et al. / Mutation Research 774 (2015) 33–39
a lesser percentage, the breakdown is performed by a sulfotransferase, with the formation of BPA-sulfate. The two metabolites, BPA-glucuronide and BPA-sulfate, do not interfere with the biological processes of the body. The results of Matsumoto et al. (2002) [31] have shown that GT activity is age-dependent and, in particular, much lower in fetal and neonatal age. In contrast, in fetal and neonatal life, the major route of BPA metabolism would seem to be that of sulfation [5]. Indeed, a number of enzymes involved in BPA metabolism to BPA-sulfate are known and have already been shown to be active during intrauterine life [49,17]. In pregnant women, Bisphenol A can be detected in blood, amniotic fluid, placental tissue and umbilical cord blood [41,12,25]. Thus, the exposure of an organism to BPA, and EDs in general, can indirectly occur as early as during intrauterine life, when hundreds of toxic chemicals can reach the embryo/fetus through the placental-blood barrier [58]. In this phase, called the “critical window of development” the product of conception appears extremely sensitive to chemicals with hormone-like activity, the effects of which will be much more pronounced, starting at much lower concentrations than those which would be harmful in adulthood [40]. This increased susceptibility is mainly due to the immaturity of all the systems of “protection” available to the adult, including: enzymatic mechanisms of DNA repair, the above-cited hepatic, but not only, enzymatic mechanisms of detoxification and metabolism, a competent immune system, the impenetrability of the blood–brain barrier, and so on [58]. Due to its estrogen-like properties, which mimic the hormone 17--estradiol, BPA can bind to the estrogen receptors (ER␣, and ER), triggering activation through atypical pathway. However, the National Toxicology Program – U.S. Department of Health and Human Services considers it appropriate not to exclusively refer to its action mediated by estrogen receptors ␣ and  binding [48]; a growing number of in vitro studies, in fact, suggest that it would be too simplistic to merely attribute to BPA the classical mechanism of estrogen action. In addition to binding nuclear ER␣-Er, bisphenol A has the ability to interact with “non-classical membrane estrogen receptors” (ncmER) [48,37,2], a seven transmembrane domain receptor (GPR30) [53], an “estrogen-related receptor-␥” (ERR-␥), an orphan nuclear receptor [32,1], the aryl hydrocarbon receptor (AhR) [3,23], the androgen receptor, acting as an antagonist [3,42,52,35]. Most of the information about the role of BPA in the genesis of malformations derives from studies on animal models, but the results in this regard are still fragmentary and conflicting. A clear influence of BPA on the development processes was inferred from studies on laboratory mice subjected to high doses of BPA during pregnancy. Exposure to BPA has shown to be capable of reducing the embryo-fetal survival [35] (Tyl et al., 2002) [56], the birth weight and the growth of the offspring in the first years of life [35,56,57]. In addition to these “high dose” effects on survival and growth, other studies have shown a variety of effects induced by much lower doses of BPA, i.e. doses below the established NOAEL of 50 g/kg/day, which reproduce better the actual human exposure. The adverse effects induced by a “low dose” of BPA include neuronal and behavioral alterations: documented effects of prenatal exposure to BPA are abnormal development of the neocortex in terms of differentiation and neuronal migration [19,38], aberrant positions and connections between thalamus and cortex [39], inhibition of the proliferation of neural progenitor cells [21], loss of sexual dimorphism in terms of brain structure and behavior [24], increased anxiety and cognitive deficits [45,54]. Part of these results are justified by the strong affinity of BPA to the dopamine receptor, the estrogen-like receptor- type [8] and the estrogen-like receptor-␥ type present in hippocampal neurons [51]. In all these hypotheses the normal synaptic communication was altered. Moreover, other documented effects concern potentially pre-cancerous lesions of prostate and mammary gland [36]; malformation of male urinary tract (decreased anogenital distance)
(Gupta et al., 2000) [14,55,7], and vagina [30]. In contrast to the harmful effects induced by high doses of BPA, there is a dispute about the interpretation of the effects induced by low doses, for which the evidence is limited and conflicting, even in laboratory animals. As far as studies on humans are concerned, effects of maternal exposure to BPA and birth weight [34,6] and behavior [4,43,64] are still limited and conflicting. In the light of this knowledge, this study aims at investigating the correlation between maternal blood BPA (representing an esteem of maternal exposure) and fetal malformations.
2. Materials and methods 2.1. Patients The samples were collected during years 2011–2013 in three Italian hospitals (University of Naples “Federico II”, University of Salerno and Hospital “Moscati” of Avellino). One hundred seventy healthy pregnant women, who either visited the hospital for voluntary interruption of pregnancy due to diagnosis of fetal malformation (case group: CS), or visited the hospital for routine analyses during their normally conducted pregnancy (control group: CL), and accepted to participate in the study, were enrolled. Human tissue collection strictly adhered to the guidelines outlined in the Declaration of Helsinki and the relevant ethic committees approved the study. Subjects completed a questionnaire addressing physical characteristics and health and pregnancy-related features and were subjected to a complete gynecological visit. Blood samples of the CS group were collected before surgery for pregnancy interruption and before any drug administration using a BD vacutainer (BPA free) (Becton Dickinson UK Limited, Oxfordshire, UK) blood collection red tube (with no additives) and immediately freezed to −30 ◦ C until analysis. Blood samples of the CL group were collected during the routine control visits of mothers with normally developed fetuses. All patients were asked to respect a 12 h fast before blood collection. Diagnostic suspect of fetal malformation subsequent to US examination or amniocentesis was confirmed by autopic post explant fetal examination. Inclusion/exclusion criteria used were different for the two groups. In the CS group, we included patients meeting the following inclusion criteria: age 40 years (risk factor for chromosomal malformation) [47] and women with known etiology of fetopathy were excluded from the CS group. In the CL group we included patients meeting the following inclusion criteria: age 191.20 m/z for BPA and 370.50 > 73.10 m/z for the d16-BPA. The event time for the SCAN mode was set to 0.07 s (corresponding to a scan speed of 10,000 amu/s, the maximum obtainable using the TQ-8030). Calibration was carried out using nine standard solutions and a blank. The standard ranged from 0.01 g/L up to 100 g/L. Each standard was analyzed in triplicate. Two recovery tests were conducted at 0.5
35
and at 80 g/L, showing a recovery of 79% at low concentration level and 85% at high concentration, according to [22]. The results were corrected according to the nearest recovery value. Quantization was conducted with the isotopic dilution method using d16-BPA as an internal standard. Every 10 samples, a blank sample and two calibration solutions: 25 g/L, and 0.01 g/L were analyzed. The tolerance criteria adopted were
