Bisphenol A co-exposure effects: A key factor in understanding BPA’s complex mechanism and health outcomes

Abstract

Bisphenol A (BPA) is an environmental endocrine disrupting chemical widely used in the production of consumer products, such as polycarbonate plastics, epoxies and thermal receipt paper. Human exposure to BPA is ubiquitous due to its high-volume production and use. BPA exposure has been associated with obesity, diabetes, reproductive disorders and cancer. Yet, the molecular mechanisms or modes of action underlying these disease outcomes are poorly understood due to the pleiotropic effects induced by BPA. A further confounding factor in understanding BPA’s impact on human health is that co-exposure of BPA with endogenous and exogenous agents occurs during the course of daily life. Studies investigating BPA exposure effects and their relationship to adverse health outcomes often ignore interactions between BPA and other chemicals present in the environment. This review examines BPA co-exposure studies to highlight potentially unexplored mechanisms of action and their possible associations with the adverse health effects attributed to BPA. Importantly, both adverse and beneficial co-exposure effects are observed between BPA and natural chemicals or environmental stressors in in vitro and in vivo models. These interactions clearly influence cellular responses and impact endpoint measures and need to be considered when evaluating BPA exposures and their health effects.

Introduction

Bisphenol A (BPA) is a building block of polycarbonate plastics and is used heavily in the production of consumer products including food containers (both plastic containers and lining films), medical tubing, toys and water pipes. BPA is also found in epoxy resins and is an additive in thermal paper, commonly used in store receipts (Liao and Kannan 2011; Geens et al. 2012; Lu et al. 2013). Given this widespread use in consumer products, BPA is one of the highest volume chemicals produced worldwide with global volume consumption of 7.7 million metric tons of BPA in 2015 and projections of BPA consumption reaching 10.6 million metric tons by 2022 (Research and Markets 2016).

Initial concern for BPA exposure resulted from its endocrine disrupting properties and its ability to leach from polycarbonate plastics and epoxy resins lining cans into food products (reviewed in Vandenberg et al. 2007; Le et al. 2008; Geens et al. 2012). However, the increasing demands for BPA-containing products over past several decades have resulted in extensive environmental contamination and near continuous human exposure to BPA through ingestion, inhalation, and absorption (Vandenberg et al. 2012).

Biomonitoring of US populations have determined daily BPA intake of approximately 25 ng/kg body weight/day (b.w./day) (LaKind and Naiman 2015). The U.S. Food and Drug Administration (FDA) and the U.S. Environmental Protection Agency (EPA) have stated that a daily exposure dose of 50 µg/kg b.w./day is safe for human exposure (Welshons et al. 2006; USEPA 2010). However, numerous studies have demonstrated that gestational and lactational exposures in human and in rodent model at or below 50 µg/kg b.w./day show negative health effects such as birth defects, developmental and reproductive disorders, diabetes, obesity, cardiovascular disease, chronic kidney and respiratory diseases and cancer (reviewed in Rochester 2013; Rezg et al. 2014; Seachrist et al. 2016).

The difficulty in the acceptance of these studies and others showing negative health consequences of young adult and adult exposures is that variations in study design, measurements of exposures, and recording of other extrinsic factors, such as environment and lifestyle, confound interpretation of the observed adverse health effects. The National Institute of Environmental Health Sciences (NIEHS) and the FDA developed the Consortium Linking Academic and Regulatory Insights on BPA toxicity (CLARITY-BPA) project to encourage more robust study designs to better evaluate BPA exposure effects (Heindel et al. 2015). With these controls in place, newer studies have confirmed that BPA exposure effects ranging from developmental issues to carcinogenesis (Dolinoy et al. 2007; Hass et al. 2016; Mandrup et al. 2016; Vandenberg and Prins 2016). However, BPA exposure does not occur in isolation. Continuous BPA exposure for long periods of time is the reality of modern life, and this exposure is combined with other exposures to physical and chemical agents.

Many studies that investigate the relationship between exposures of endocrine disrupting chemicals (EDCs) and adverse effects ignore the interaction between different EDCs and other relevant environmental stressors. Mixture exposure studies are difficult to perform and interpret. However, they are critical to understanding co-exposure effects. Given the ubiquitous nature of BPA, co-exposure studies are essential for evaluating the impact of BPA exposure has on human health and understanding the BPA’s diverse and pleiotropic effects. This review examines the current literature investigating BPA co-exposures with chemicals and environmental stressors to highlight BPA mechanisms of action during co-exposures and to examine both the beneficial and adverse outcomes common co-exposures have on human health. Overall, this review reveals the landscape of current BPA co-exposure research and seeks to stimulate further investigation into these relevant human exposures to BPA.

Current monoexposure mechanisms of action

Studies of BPA exposure have revealed several different molecular mechanisms. BPA can stimulate receptor-mediated effects, induce DNA damage and epigenetic changes, alter oxidation-reduction balance, and induce mitochondrial dysfunction (extensively reviewed in Rochester 2013; Rezg et al. 2014; Gassman 2017).

Briefly, BPA is a well-known EDC with some affinity for binding estrogen receptors (ERs) (Gould et al. 1998; Kuiper et al. 1998). Although, BPA has been found to have lower affinity for the nuclear ERs than estradiol (Kuiper et al. 1998; Le Fol et al. 2017), its estrogenic potency for non-nuclear ERs is equal to estradiol with changes in cell function being observed at doses of 1 picomolar (pM) (Wozniak et al. 2005; Viñas et al. 2013). BPA has been also shown to antagonize estrogen action (Bonefeld-Jørgensen et al. 2007; Richter et al. 2007) and antagonize androgen action (Sohoni and Sumpter 1998; Lee HJ et al. 2003). BPA has also been shown to be a potent activator of non-classical estrogen receptors, like G-coupled protein receptors (GPER) and estrogen-related receptor γ (ERRγ), as well as have both agonistic and antagonistic effects on thyroid hormone (Moriyama et al. 2002; Wetherill et al. 2007; Alonso-Magdalena et al. 2012; Rochester 2013; Lee S et al. 2019). Beyond hormone receptors, BPA can activate glucocorticoid receptor and stimulate adipogenesis in 3T3-L1 cells (Sargis et al. 2010). BPA can also bind to aryl hydrocarbon receptor (AhR) and affect metabolism of xenobiotics as well as synthesis and metabolism of steroids (Bonefeld-Jørgensen et al. 2007; Krüger et al. 2008). Altered steroidogenesis also results from BPA activating protein kinase A and mitogen-activated protein kinase (MAPK), in particular p42/44^MAPK, which further induces expression of the nuclear transcription factor Nur77 in mouse Leydig cells (Song et al. 2002).

In addition to receptor mediated effects, BPA is known to damage DNA contributing to carcinogenesis and teratogenesis (reviewed in Seachrist et al. 2016). Direct interaction of BPA with DNA induces DNA adducts (Atkinson and Roy 1995a, 1995b; Izzotti et al. 2009), chromosomal aberrations (Keri et al. 2007; Allard and Colaiacovo 2010), and aneuploidy (George et al. 2008; Johnson and Parry 2008). BPA-induced DNA damage is also associated with increased reactive oxygen species (ROS) production leading to oxidative stress (reviewed in Gassman 2017). Alterations in the DNA also occur from epigenetic changes. Developmental exposure to BPA changes DNA methylation patterns altering the epigenome (reviewed in Mileva et al. 2014). In addition, BPA alters the gene expression of DNA methyltransferases (DNMTs) and methyl CpG binding protein 2 (MECP2) in developing hypothalamic cells (Warita et al. 2013). BPA exposure has been shown to affect histone methyltransferase EZH2 (Doherty et al. 2010) and chromatin structure (Gassman et al. 2015; Gassman et al. 2016).

Increased ROS from BPA exposure also induces antioxidant depletion, impacts mitochondrial function, alters in cell signaling pathways, and can induce cell death (reviewed in Gassman 2017). Both nanomolar and micromolar doses of BPA can accumulate in the mitochondria of cells, due to lipophilic nature of BPA, and induce mitochondrial dysfunction by increasing mitochondrial ROS and altering mitochondrial membrane potential (Ooe et al. 2005; Chepelev et al. 2013; Pfeifer et al. 2015).

This brief summary of the identified mechanisms of action of BPA highlight the complex and pleiotropic effects observed after exposure, which contributes to difficulty in linking adverse health risks with chronic low dose BPA exposure (Trasande et al. 2016; Vandenberg and Prins 2016). Further, tissue and developmental specific effects observed throughout the literature highlight that interactions may be occurring between BPA and the cellular environment, i.e., high levels of oxidative stress in reproductive tissues, increased energy production during development, or even cellular responses to environmental exposures, that enhance or mitigate BPA exposure effects. Therefore, understanding co-exposure effects of BPA may be essential to clarifying links between BPA exposure and human disease.

Co-exposure effects with BPA

Given the prevalence of BPA in our environment, interactions between BPA and other exogenous and endogenous exposures are highly possible. Humans and even experimental animals are exposed to multiple chemicals on a daily basis from their physical environments, hygienic routines, and their diets. Soy-based diets in particular expose mammals to EDCs in combination with BPA exposure. Studies have indicated that the effects observed with the low dose BPA exposure may be due to its additive effect with other estrogenic like molecules present (Soto et al. 1997; Rajapakse et al. 2002). Thus, understanding BPA co-exposure effects with other EDCs and other environmental agents is critical for understanding the “real world” risks associated with BPA exposure. Here, we review the published data on BPA co-exposure studies with various classes of relevant co-exposures in cell line models, laboratory animal models, and human subjects. Tables 1 and 2 presents BPA co-exposure studies characterize based on their class of chemicals, model system used, dosage and exposure duration, methods applied and the key findings.

Table 1.

Published papers reporting cellular effects of BPA co-exposure with different class of chemical compounds.

Chemical Class	Model System	Dose	Exposure duration	Methods	Key findings	References
Naturally occurring compounds
Eupatorium cannabinum ethanolic extract (EcEE)	HT29 (colon cancer cells)	4.4 µM of BPA; 0.5 to 50 µg/ml of EcEE	24 hr co-exposure	Cell viability, TUNEL assay	BPA co-exposure with EcEE decrease cell viability and increase mitotic disruption.	(Ribeiro-Varandas et al. 2014)
Ginsenosides (active component of ginseng)	15P-1 Sertoli cells	10–200 µM of BPA; 10–200 µg/ml of Ginsenosides	24 hr pre-treatment with ginseng followed by BPA exposure for 24 hr	MTT assay, Apoptosis analyses, Immunofluorescence analysis, Western blot, Determination of antioxidant enzymes	Pre-treatment with ginsenosides:	(Wang L et al. 2012)
					- Protects BPA-induced apoptosis and cytotoxicity.
					- Rescued BPA-induced collapse of the vimentin intermediate filament network.
					- Altered protein expression of ERK1/2 and apoptotic-related proteins (Bcl-2 and Bax).
					- Restored anti-oxidative function of 15P-1 sertoli cells
Melatonin	MCF-7 and T47D (breast cancer cells)	100 nM of BPA; 1nM of Melatonin	24 hr −48 hr exposure after serum starvation	MTT assay, Colony formation assay, Western blot	- Melatonin abolishes BPA-induced cell proliferation by decreasing p-ERK and p-AKT.	(Wang T et al. 2018)
					- Blocked steroid receptor coactivators (SRC-1 and SRC-3) and ERE activity.
Stigmasterol	GC-1spg (spermatogonial cell line)	10 nM of BPA; 10 µM of stigmasterol	Simultaneous treatment 12 hr. Pre-treatment BPA for 12 hr and then molecule for 12 hr.	Testis Explant Assay, RNA-Seq, RT-PCR	Alters sertoli cell homeostasis and neonatal germ cell physiology	(Sèdes et al. 2018)
Environmental chemicals
Cadmium	HepG2 (hepatocellular carcinoma)	0.01 to 100 µM of BPA; 0.001 to 10 µM of Cd	6 hr for oxidative stress assay and 24 hr for cytotoxicity and comet assays	MTT assay, ROS assay (DCFH-DA), Comet assay	- BPA co-exposure with Cd increases intracellular ROS generation.	(Li et al. 2017)
					- Higher MDA levels.
					- BPA co-exposure with 1 µM Cd enhanced genotoxicity measured by comet assay.
Cadmium	NIH 3T3 (established from primary MEFs)	2–50 µM of BPA; 5 and 10 µM of CdCl2	24 hr for both BPA and CdCl2	Cytotoxicity assay (CCK-8 and LDH release), ROS measurement (DCFH-DA), DNA damage (8-OHdG and yH2AX, comet assay), TUNEL, Western blot	- Cadmium pre-exposure promotes BPA-induced cytotoxicity.	(Chen ZY et al. 2016)
					- Increased ROS production and 8-oHdG formation.
					- Increased cell cycle arrest and apoptosis.
Dibutyl phthalate	HepG2 (hepatocellular carcinoma)	0.01 to 100 µM of BPA; 0.01 to 100 µM of DBP	6 h for oxidative stress assay and 24 hr for cytotoxicity and comet assays	MTT assay, ROS assay (DCFH-DA), Comet assay	- BPA co-exposure with 1 µM DBP increases cytotoxicity, higher MDA levels and lower SOD activity.	(Li et al. 2017)
					- Enhanced genotoxicity measured by comet assay.
Hydrogen peroxide	Swiss 3T3 fibroblast cells	0.1–100 nM for BPA; 30 µM for H2O2	30–45 days for BPA; 2 days for H2O2	Cytotoxicity and cell growth assay, RT-PCR, western blot, GTPγS binding assay (GPR30 activation detection)	10 nM BPA ameliorates H2O2 induced cell death by increasing bcl-2 expression and altered histone methylation mediated via GPR30.	(Nishimura et al. 2014)
PFOS/PFOA	Mouse ESCs, NIH 3T3 (established from primary MEFs)	0.5 to 64 µg/ml of BPA; 2.5 to 320 µg/ml of PFOS or PFOA	7 day exposure with renewal after 4 days for BPA, PFOS or PFOA.	MTT, myocardial differentiation, Q-PCR, embryotoxic potential	BPA co-exposure with PFOS in several combinations showed synergistic action on myocardial differentiation.	(Zhou et al. 2017)
Potassium Bromate	WT MEF and Ku70-deficient cells	150 µM for BPA; 20 mM KBrO3	4 hr and 24 hr for BPA; 1hr for KBrO3	Cytotoxicity assay, oxidative DNA lesion measurement, microarray assay, chromatin condensation, reduced glutathione assay, pH measurement	- BPA prevents KBrO3 induced cytotoxicity by altering cellular microenvironment and modulating DNA repair proteins	(Gassman et al. 2015; Gassman et al. 2016)
Therapeutics
Camptothecin	WT MEFs	150 µM for BPA; 80 nM for CPT	24 hr for BPA and CPT	Cytotoxicity assay, Slot Blot, western blot, immunofluorescence	- BPA co-exposure confers resistance to CPT induced cell death.	(Sonavane et al. 2018)
					- BPA reduced Top1-DNA adducts and increased Top1 protein expression levels.
					- BPA reduced nuclear volume and chromatin compaction, thus reducing the accessibility of DNA to Top1.
Cisplatin	T47D and MDA-MB-468 (breast cancer cells)	0.1–10 nM of BPA; 100–400 ng/mL Cis	120 hr for BPA and 96 hr for Cis	MTT assay	- BPA co-exposure protects T47D and MDA-MB-468 cells from Cis-induced cytotoxicity.	(Lapensee et al. 2009; Lapensee et al. 2010)
					- BPA co-exposure opposes Cis-induced anti-proliferation and pro-apoptosis by increasing Bcl-2 expression.
Doxorubicin	Hep-2 (human epithelial type 2) and MRC-5 (human lung fibroblasts)	0.44 nM- 4.4 µM of BPA; 4 µM for Dox	24 hr for BPA, followed by 24 hr of BPA and Dox	CellTiter-Blue, Immunofluorescence, Comet assay, Cytology analysis	- BPA co-exposure increases cellular viability in MRC-5 cells	(Ramos et al. 2019)
					- Reduction of Dox-induced DNA damage in both cells after BPA co-exposure.
Doxorubicin	HT29 colon cancer cells	4.4 µM of BPA; 0.04–4 µM for Dox	24 hr for BPA and Dox	qRT-PCR, CellTiter-Blue, TUNEL assay and DAPI staining	- Dox induced alterations in transcription levels are antagonizes by BPA co-exposure.	(Delgado and Ribeiro-Varandas 2015)
					- BPA co-exposure decreases Dox induced apoptotic bodies.
Doxorubicin	T47D and MDA-MB-468 (breast cancer cells)	0.1–10 nM of BPA; 5–150 ng/mL Dox	48 hr-120 hr for BPA and 24 hr- 96 hr for Dox	MTT assay, RT-PCR, Western blot	- Prevents Dox-induced cytotoxicity in T47D and MDA-MB-468 cells.	(Lapensee et al. 2009)
					- Alters antiapoptotic proteins (Bcl-2 and Bcl-xL)
Vinblastine	T47D and MDA-MB-468 (breast cancer cells)	0.1–10 nM of BPA; 1–25 ng/mL Vin	120 hr for BPA and 96 hr for Vin	MTT assay	- BPA co-exposure protects T47D and MDA-MB-468 cells from Vin-induced cytotoxicity	(Lapensee et al. 2009)
Electromagnetic radiation
X-rays	somatic cells of female mice	5–20 mg/kg b.w. of BPA; 0.05 and 0.10 Gy of X-rays	1–2 weeks	Comet assay, Micronucleus test	- Genotoxicity varies depending on tissue, assay and time.	(Gajowik et al. 2013)
					- At week 1, increase number of MN.
					- At week 2, significant reduction of MN in peripheral blood.
					- Comet assay showed higher DNA strand breaks in Lymphocytes of bone marrow and lower in lung cells.

Table 2.

Published papers reporting effects of BPA co-exposure with different chemical class on animal models and human subject.

Chemical Class	Model System	Dose	Exposure duration	Methods	Key findings	References
Dietary or naturally occurring compounds
Folic acid	Avy (Agouti gene in the viable yellow agouti) mouse	50 mg/kg diet of BPA; 4.3 mg/kg diet of folic acid	2 weeks before mating and throughout gestation and lactation	DNA Methylation assay	Maternal nutritional supplementation with folic acid negated the BPA-induced DNA hypomethylation in the offspring.	(Dolinoy et al. 2007)
Ginkgo biloba	male rat	40 mg/kg b.w. of BPA; 100 mg/kg b.w. of G. biloba extract (GBE)	orally and daily for 70 days	Liver function tests and activities of the antioxidant enzymes, levels of urea, creatinine, TBARS and GSH	- GBE ameliorates BPA-induced hepatotoxicity and oxidative stress by altering important biomarkers.	(Wahby et al. 2017)
					- Decreased hepatic and renal TNF-α level.
					-Improved liver and kidney tissues compared to BPA alone.
Genistein	Avy (Agouti gene in the viable yellow agouti) mouse	50 mg/kg diet of BPA; 250 mg/kg diet of genistein	2 weeks before mating and throughout gestation and lactation	DNA Methylation assay	Maternal nutritional supplementation with genistein counteracted DNA hypomethylating effect of BPA in the offspring.	(Dolinoy et al. 2007)
Ginseng	female adult albino rats	150 mg/kg/day of BPA; 200 mg/kg of ginseng	0–20 days (oral supplementation)	Measurement of serum hormone levels and mRNA transcripts	- Ginseng co-treatment reversed BPA induced decrease of sex hormone levels.	(Saadeldin et al. 2018)
					- Alter ovarian and placental mRNA transcript levels of steroidogenic enzymes (STAR, CYP17 and HSD), concomitantly related to AKT1 and PTEN expression.
High butterfat (HBF)	Female rat (DMBA-induced mammary carcinogenesis Sprague-Dawley (SD) rat model)	2.5–2500 µg/kg b.w./day of BPA; 39% kcal from butterfat for high-butterfat diet	During mating and throughout gestation	Q-PCR, Bisulfite sequencing analysis, TCGA survival analysis	- Maternal BPA co-exposure with HBF diet increases breast cancer incidence in offspring.	(Leung et al. 2017)
					- Increase in the number of terminal end buds.
					- Alters gene expression levels.
High sugar	Fruit fly Drosophila melanogaster	16 mM of BPA; 450 mM of high sugar (for both acute and chronic exposure)	48 hr (acute), from eggs to adults (chronic)	Gene expression analysis, Tissue perturbation	Up-regulation of ribosome-associated genes and enhancement of impaired testis-specific gene expression.	(Branco and Lemos 2014)
Melatonin	male rat	40 mg/kg b.w. of BPA; 10 mg/kg b.w. of melatonin	orally and daily for 70 days	Liver function tests and activities of the antioxidant enzymes, levels of urea, creatinine, TBARS and GSH	- Melatonin ameliorates BPA-induced hepatotoxicity and oxidative stress by altering important biomarkers.	(Wahby et al. 2017)
					- Decreased hepatic and renal TNF-α level.
					-Improved liver and kidney tissues compared to BPA alone.
Melatonin	adult male Sprague-Dawley (SD) rats	200 mg/kg b.w. per day of BPA; 10 mg/kg b.w. per day of Melatonin	10 consecutive days exposure (Intraperitoneal injection of melatonin 30 min before BPA; gavage exposure of BPA)	Sperm counts, TBARS and SOD analysis in testis, Comet assay, yH2AX foci analysis, Meiotic chromosomal spread and Flow cytometry analysis	- Melatonin pretreatment restored TBARS and SOD levels altered by BPA.	(Wu HJ et al. 2013)
					- Reduced BPA-induced DNA damage by comet assay and yH2AX foci analysis.
					- Increased testicular cell numbers that were decreased by BPA exposure.
Stigmasterol	mice and ex vivo model system (human adult testis explant)	50 mg/kg/day of BPA; 5 mg/kg/day of stigmasterol	From PC6.5 up to 5 days after birth	Testis Explant Assay, Histology, Immunohistochemistry, RNA-Seq, RT-PCR	- Co-exposure induces male fertility disorders, alters sertoli cell homeostasis and neonatal germ cell physiology.	(Sèdes et al. 2018)
					- Defects in human testis histology
Environmental chemicals
Arsenic	male mice and offspring	10 µg/kg b.w. of BPA; 10 ppb in drinking water of sodium arsenite	gestational day 10 through 18 (subcutaneous for BPA and oral for arsenic)	Glucose tolerance and insulin tolerance test, qPCR, NMR	- Co-exposure exacerbates metabolic changes induced by either chemical alone in male mice offspring.	(Wang D et al. 2018)
					- Influence both glucose and insulin tolerance, alter expression levels of genes involved in lipid and glucose homeostasis.
Dimethyl benzanthracene	Female mice, offspring	25–250 µg/kg of BPA; 1 mg/100 µl corn oil for DMBA	Daily, from postcoital day 8 until parturition for BPA; one dose each at 5 and 6 week (oral gavage)	Tumor Susceptibility, MCF-7 cell Xenografts, MKI67 immunostaining	- BPA exposure increase in susceptibility to DMBA-induced mammary tumorigenesis, primary through its estrogenic properties.	(Weber Lozada and Keri 2011)
					- BPA-treated females formed tumors by 7 week post injection by Xenograft experiment.
Dimethyl benzanthracene	Female Sprague Dawley rats	25–250 µg/kg bw/day of BPA; 30 mg/kg bw/day of DMBA	during lactation (intragastrically gavage) for BPA; single gavage for DMBA	Tumorigenesis, Hormone and protein concentration assays, Apoptosis assay.	- Reduced tumor latency and increased tumor multiplicity in offspring when BPA exposed to rats during lactation.	(Jenkins et al. 2009; Betancourt et al. 2010)
					- Increased cell proliferation and decreased apoptosis.
					-Increased expression of erbB3 SRCs, and Akt.
nano-TiO2	zebrafish	2–20 µg/L of BPA; 100 µg/L of TiO2	4 months	Measurement of Thyroid Hormone contents, qRT-PCR, western blot, neurotransmitter levels, AChE activity, Quantification of BPA and n-TiO2	- Parental co-exposure of BPA and n-TiO2 enhance bioaccumulation in adults and increase transfer to their progeny.	(Guo et al. 2019)
					- Disrupts thyroid hormones and facilitates developmental neurotoxicity in larval offspring.
					- Lethargic swimming behavior.
Nonylphenol	zebrafish embryos	0.1–1000 µg/L of BPA; 0.1–100 µg/L of NP	4–168 hpf (hour post fertilization)	Measurement of oxidative stress indices, antioxidant parameters and alkaline phosphatase	- Combined exposure causes increase of hydroxyl radical and MDA.	(Wu M et al. 2011)
					- Decrease of antioxidant responses and alkaline phosphatase.
Polystyrene nanoplastics	zebrafish	1 µg/L of BPA; 1 mg/L of NPPs	0 hr, 6 hr, 24 hr, 48 hr, 72 hr	Bioaccumulation assay, Gene transcription analyses, Protein expression by western blot.	- Bioaccumulation potential of BPA significantly increased in the head and viscera when co-exposed with NPPs.	(Chen Q et al. 2017)
					- Upregulation of CNS biomarkers (myelin and tubulin protein/gene expression), dopamine content, and the mRNA expression of mesencephalic astrocyte derived neurotrophic factor (MANF).
Electromagnetic radiation and radioactive substances
137-Cesium	male mice	25 µg/kg b.w. of BPA; 4000 and 8000 Bq 137 Cs/kg b.w.	2 months (Single subcutaneous dose)	Urinary and serum parameters, CYP1A2 gene expression, Levels of 8-OHdG	- Decreased urea levels and LDH activity, Higher excretion of uric acid and GGT, NAG activity restored to control.	(Esplugas et al. 2018)
					- Increase in 8-iso-PGF2alpha excretion compared to BPA and Cs8000 alone, BPA exposure decreased CYP1A2 gene expression, BPA co-exposure decrease levels of 8-OHdG in liver.
X-rays	male mice	5–40 mg/kg b.w. of BPA; 0.05 and 0.10 Gy of X-rays	2 weeks	Sperm count, motility, morphology assessment, Comet assay	- BPA co-exposure with X-rays decreases testes weight and sperm motilities.	(Dobrzynska and Radzikowska 2013)
					- Increased abnormal spermatozoa.
					- Prevents DNA damage in somatic and germ cells of mice.
X-rays	male adult and pubescent mice	5–20 mg/kg b.w. of BPA; 0.05 Gy of X-rays	24 h - 8 weeks	Sperm count, motility, morphology assessment, Comet assay	- Combined BPA exposure with X-rays enhanced the harmful effect of BPA in male germ cells.	(Dobrzynska et al. 2014)
					- Increased abnormal spermatozoa and decreased sperm count.
					- Prevents DNA damage in pubescent males.
Others
Hypoxia	zebrafish larvae	0.01 to 100 µg/L of BPA; (2.0 +/− 0.5 mg/L O2) for hypoxia	1 hpf to 72–96 hpf (hour post fertilization)	Measurement of cardiovascular functions and developmental effects	- Severe bradycardia and reduced cardiac output	(Cypher et al. 2015; Cypher et al. 2018)
					- At later development: slower arterial and venous RBC velocity.
Environmental chemicals
nonylphenol	Human subjects (pregnant women cancer free, age 18–45, at gestational week 27–38; healthy fetuses)	Geometric mean of maternal urinary exposure: 1.62 ng/mL of BPA and 2.80 ng/mL of NP	Urinary exposure	Distribution of urinary NP and BPA levels and oxidative stress biomarkers in the mother, Measurement of fetal reproductive indices (penis length and anogenital distance).	- Maternal co-exposure to NP and BPA enhance 8-OHdG.	(Huang et al. 2018)
					- Reduce penis length in the high 8-isoPF2α group.

BPA dosing used for the co-exposure can be highly variable, so dose, route and time of exposure are also noted for clarity in the text and in Tables 1 and 2 to assist in the BPA co-exposure evaluation with different chemicals.

Co-exposure with dietary or naturally occurring compounds

In the literature, nine dietary or naturally occurring chemicals have been used to study BPA co-exposure effects. Ameliorative effects of dietary or natural chemicals have been reported with melatonin, Ginkgo biloba extract (GBE), folic acid, and genistein in cell line models, as well as in animal models.

Melatonin is an important endogenous hormone that regulates circadian rhythm; and studies have shown that BPA co-exposure with melatonin significantly reduces BPA-induced cell proliferation and BPA-induced oxidative stress (Wu HJ et al. 2013; Wahby et al. 2017; Wang T et al. 2018). Breast cancer cell lines co-exposed with BPA and melatonin showed inhibition of BPA-elevated levels of steroid receptor activators and estrogen response element (ERE) activity and downregulation of phosphorylated extracellular-regulated kinase (ERK) and serine/threonine-specific protein kinase (AKT) (Wang T et al. 2018). Co-treatment of BPA and melatonin also increased melatonin receptor 1 (MT1) and upregulation of cell cycle progression blocker, p21, in breast cancer cell models, highlighting MT1’s involvement in preventing BPA-induced cell proliferation (Wang T et al. 2018).

In vivo melatonin co-exposure with BPA also reduced BPA-induced hepatotoxicity and nephrotoxicity in male rats. Similar effects were also observed with GBE co-exposure in male rats (Wahby et al. 2017). GBE is one of the most widely used herbal supplements in Chinese traditional medicine having immunomodulation, antioxidant, anti-inflammatory and anti-tumor activities (Yan et al. 2015). Both melatonin and GBE co-exposures showed a significant reduction in the activities of aspartate transaminase (AST) and alanine transaminase (ALT) (liver damage indicators) and hepatic thiobarbituric acid reactive substances (TBARs) (lipid peroxidation end product) and a significant increase in anti-oxidative enzymes activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) along with increases in glutathione (GSH)). Renal toxicity was also reduced during co-exposure of GBE or melatonin with BPA though a reduction in urea and creatinine levels and a reduction in renal TBARs. Increasing GSH levels and activities of SOD, glutathione S-transferase (GST) and catalase (CAT) were also observed for both GBE and melatonin (Wahby et al. 2017).

Such alleviation of the increase levels of TBARS and decrease GSH levels induced by BPA exposure was also observed when same dose of melatonin was used to pretreat adult male rats prior to co-exposure with BPA (Wu HJ et al. 2013). In vivo exposure of adult male rats with melatonin and BPA also reduced DNA damage induced by BPA, with decreased in DNA strand breaks observed by comet assay and reduced phosphorylation of H2AX (γH2AX) foci in spermatocytes. Testicular cell number, particularly 4-C cells, was dramatically increased when rat germ cells were pre-treated with melatonin (Wu HJ et al. 2013).

Ginseng, another naturally occurring chemical reported to be beneficial against urinary BPA levels and di(2-ethylhexyl) phthalate (DEHP) toxicity (El-Drieny et al. 2009; Yang M et al. 2014). Pre-treatment with ginsenosides, the active component of ginseng, showed protective effects against BPA-induced Sertoli cell damage (Wang L. et al. 2012). Ginsenosides decreased ERK1/2 phosphorylation, increased Bcl-2 and decreased BAX protein expression levels, which inhibited BPA-induced apoptosis in 15P-1 Sertoli cells. Ginsenosides application prevented the damaging effect of BPA on the structure of vimentin intermediate filaments, an important Sertoli cell cytoskeletal component. Further, Sertoli cells pre-treated with ginsenosides decreased malondialdehyde (MDA) levels and increased total antioxidant capacity with activities of SOD, GPx, glutathione reductase (GSR) increased along with increased GSH (Wang L et al. 2012).

Co-exposure of ginseng with BPA was also reported to ameliorate reproductive toxicity induced by BPA in pregnant rats (Saadeldin et al. 2018). Ginseng reversed decreased serum progesterone, estrogen and testosterone levels induced by BPA exposure in rats. On pregnancy day 10, co-exposure with ginseng increased CYP17 expression and decreased AKT and PTEN expression in ovary and placenta. Similar effect was also observed on day 20 of pregnant rats after ginseng and BPA co-treatment showing decreased PTEN expression in both ovary and placental and reduced AKT expression only in placenta, restoring the reduced progesterone level in rats treated with BPA. The effect of ginseng on steroid synthesis can be explained by modulation of PTEN/AKT pathway and inactivation of the PI3K/AKT signaling (Yang Y et al. 2017). Moreover, ginseng co-treatment reduced placental STAR and HSD expression and decreased ovarian STAR expression. However, co-exposure of BPA with ginseng elevated HSD expression in ovary, reversing the reduced serum estradiol and testosterone levels induced by BPA (Saadeldin et al. 2018).

Along with increased cell proliferation and oxidative stress, monoexposure to BPA has also been linked to epigenetic alterations (Newbold et al. 2006). Dolinoy et al. (2007), first reported that BPA-exposed mouse offspring had significantly decreased DNA methylation across nine CpG sites, in the cryptic promoter region of the Agouti gene in the viable yellow agouti (A^vy) mouse allele resulted from the insertion of a murine intracisternal A particle (IAP), shifting the coat color toward the yellow coat color phenotype. However, co-exposure of male mice with BPA and folic acid for 2 weeks before mating restored the coat color of the mouse offspring to that of control litter. Interestingly, the study showed BPA-induced hypomethylation was also counteracted by genistein (Dolinoy et al. 2007), even though it is not a methyl-donating compound like folic acid.

Dietary supplements or natural chemicals can also enhance adverse side effects of BPA when co-exposed. Four studies have been published in the literature that demonstrate adverse outcomes when dietary or naturally occurring compounds are co-exposed with BPA (Branco and Lemos 2014; Ribeiro-Varandas et al. 2014; Leung et al. 2017; Sèdes et al. 2018).

Eupatorium cannabinum L ethanolic extract (EcEE), a chemical demonstrated to have anti-inflammatory, anti-parasitic, anti-proliferative, as well as anti-tumorigenic activities in several cell lines (Sulsen et al. 2007; Chen JJ et al. 2011; Forgo et al. 2012). Co-exposure of EcEE with BPA strongly increased levels of abnormal mitosis in human colon cancer (HT29) cells (Ribeiro-Varandas et al. 2014). BPA is an aneugenic chemical (Johnson and Parry. 2008) that can interfere with cell division mechanisms at very low concentrations (Ribeiro-Varandas et al. 2013). Co-exposure of EcEE with BPA increased the aneugenic effects of BPA (Ribeiro-Varandes et al. 2014).

In vivo, maternal consumption of high butter fat (HBF) with BPA significantly delayed the onset of puberty, altered mammary gland morphology, and increased mammary tumor incidence in offspring of female Sprague-Dawley (SD) rats (Leung et al. 2017). Increased numbers of terminal end buds (TEBs) in the mammary glands altered mammary gland development. Increased tumor incidence resulted in shorter tumor-free survival time during co-exposures. Gestational BPA exposure with HBF also dysregulated early cancer-related gene expression in mammary glands of offspring through changes in DNA methylation (Leung et al. 2017). It should be noted that the BPA doses which elicited highest mammary tumor incidence in the study were lower than the US EPA’s reference dose for human exposure, i.e., 50 µg/kg-b.w./day (USEPA 2010). While HBF or potent estrogenic EDCs (ethinylestradiol) supplemented maternal diet has been previously associated with changes in DNA methylation machinery and methylation patterns in mammary tissue (de Assis et al. 2012), co-exposure of HBF and BPA increased these effects allowing a permissive environment for mammary cancer risk in offspring (Leung et al. 2017).

Additionally, BPA co-exposure with a high sugar diet significantly intensified BPA-exposure effects in Drosophila melanogaster. BPA co-exposure with high-sugar diet disrupted elements of the translational machinery. A more pronounced disruption of gene expression coding for small and large components of the ribosome was observed with combined exposure, modulating BPA’s sensitivity via ribosome-mediated processes (Branco and Lemos 2014). BPA and high sugar diet co-exposure also significantly impaired catabolic gene expression in the midgut perturbing the physiology of the gut tissue. R1 and R2 retrotransposons were also induced which is an indicator of nucleolar stress and may indicate genotoxicity during BPA and high sugar diet co-exposure (Branco and Lemos 2014).

Finally, stigmasterol is a potent antagonist of the critical nuclear receptor bile acid sensor FXR (farnesoid X receptor, NRIH4) that aids regulation of essential liver gene expression (Carter et al. 2007). Co-exposure of BPA with stigmasterol decreased accumulation of FXRα mRNA (Sèdes et al. 2018). BPA-stigmasterol co-exposed male mice from post-coitum day 6.5 (PC 6.5) up to 5 days after birth showed impaired fertility of adult male mice originated from altered spermatogenesis, resulting in lower number of Sertoli cells. Six-month-old Fxrα+/+ male mice exposed to BPA and stigmasterol decreased number of undifferentiated spermatogonia per total seminiferous tubules with increased Dnmt31 mRNA accumulation, a known regulator of promyelocytic leukemia zinc finger (PLZF), a specific marker of undifferentiated spermatogonia. Using an ex vivo model system, stigmasterol co-exposed with BPA significantly decreased Sertoli cell number, showing the additive effect of both molecules on testis function. However, defects of spermatogenesis and lower sperm production were almost unaffected in Fxrα deficient male mice, suggesting an involvement of FXRα in the effects of BPA co-exposure (Sèdes et al. 2018). Based on in vitro data in GC1-spg cells, BPA might act in part as a downregulator of Fxrα expression, and stigmasterol as direct antagonist of FXRα in GC1-spg cells, similarly observed in vivo that could explain the synergistic impact of BPA-stigmasterol exposure (Sèdes et al. 2018).

Co-exposure with environmental chemicals

BPA, cadmium (Cd) and dibutyl phthalate (DBP) are known EDCs, co-occurring in aquatic environments due to their increased use in modern agriculture and industry. Hepatocellular carcinoma (HepG2) cells co-exposed with BPA and DBP increased cytotoxicity with severe DNA damage and enhanced lipid peroxidation levels (Li et al. 2017). Similar synergistic effects were also observed in HepG2 cells co-treated with BPA and Cd with significant increased levels of ROS (Li et al. 2017). Cd pre-treatment prior to BPA exposure in MEF cell line (NIH3T3) increased BPA-induced cytotoxicity with increased lactate dehydrogenase (LDH) release, ROS generation, DNA damage and cell cycle arrest observed compared to BPA treatment alone (Chen ZY et al. 2016). It was proposed that inefficient repair of the oxidatively induced DNA damage from Cd and BPA resulted in observed DNA strand breaks, cell cycle arrest, apoptosis and mitochondrial dysfunction (Chen ZY et al. 2016). Cd’s inhibition of 8-Oxoguanine DNA Glycosylase (OGG1) expression likely drives these effects, since OGG1 is critical to the removal of oxidative base lesions within the DNA (Potts et al. 2003; Youn et al. 2005).

Other well know EDCs such perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), which are known for their developmental toxicities, were also co-exposed with BPA to understand BPA co-exposure effects on myocardial differentiation of mouse embryonic stem cell (Zhou et al. 2017). BPA and PFOS co-exposure demonstrated synergistic effects on myocardial differentiation and reduced mRNA levels of ectoderm, mesoderm and endoderm markers altering embryoid body development. An additive effect on myocardial differentiation with BPA and PFOA was also observed (Zhou et al. 2017).

Two other environmental chemicals with oxidizing properties, hydrogen peroxide (H₂O₂) and potassium bromate (KBrO₃), were also co-exposed with BPA to assess the susceptibility of mouse embryonic fibroblasts (MEFs) against oxidative stress (Nishimura et al. 2014; Gassman et al. 2015; Gassman et al. 2016). Co-exposure of Swiss 3T3 fibroblasts to 1 nM BPA for 30–45 days showed resistance to 30 µM H₂O₂-induced cell death. BPA-induced resistance to H₂O₂ was mediated via GPR30 receptor. BPA activation of GPR30 receptor showed increased cell viability, which further increased the levels of anti-apoptotic protein, Bcl-2, and decreased histone H3 methylation levels (Nishimura et al. 2014). A strong pro-survival effect in MEFs was also observed when high dose BPA was co-exposed with KBrO₃ (Gassman et al. 2015). Combined exposure to BPA and KBrO₃ showed an increase in ROS production leading to oxidatively induced DNA lesions (Gassman et al. 2015; Gassman et al. 2016). The high micromolar dose of BPA (150 µM) promoted the transient compaction of chromatin and downregulated key DNA repair proteins within 4 hr of co-exposure. At 24 hr post-damage induction, BPA co-exposure promoted cell survival by reducing chromatin condensation and up-regulation of DNA repair proteins (Gassman et al. 2016).

In vivo and early development studies of BPA and arsenic co-exposures have also been performed. While BPA and arsenic have several distinct modes of action, there are overlapping mechanisms and health outcomes for these agents and both chemicals can interact with estrogen receptor (Davey et al. 2007; Vandenberg et al. 2009). Pregnant mice co-exposed to BPA and arsenic during gestation had male offspring with metabolic changes at 4-weeks of age with elevated branch chain amino acids (leucine, isoleucine and valine) and reduced TCA cycle intermediates (succinate and citrate) found in serum profiles. Metabolic profile of 12-weeks old mice co-exposed to BPA and arsenic during gestation showed more extreme metabolic dysfunction with altered expression levels of genes involved in glucose (Pepck, Glut2 and Gys2) and lipid (Acaca, Cpt1α, Fat and Fatp2) homeostasis (Wang D et al. 2018). The observed changes in glucose tolerance and insulin sensitivity by combined exposure to BPA and arsenic may be due to the upregulation of enzymes or genes involved in gluconeogenesis (Gys2) and glucose uptake (Glut2), as insulin is known to activate glucose transport and gluconeogenesis in skeletal muscle (Samuel and Shulman 2018). Significant metabolic disruptions in the adult mice (12-weeks old) rather than in juvenile mice (4-weeks old), indicated age-dependent metabolic effects in offspring by combined exposure, which could mediate long term health effects of BPA and may be associated with an altered age-related DNA methylation pattern observed in paired mouse tail tissues (Kochmanski et al. 2017).

BPA co-exposure with dimethylbenzanthracene (DMBA) increased breast cancer risks in animal models (Jenkins et al. 2009; Betancourt et al. 2010; Weber Lozada and Keri 2011). The combination of DMBA with lactational BPA exposure resulted in a dose-dependent increased susceptibility to mammary carcinogenesis with an increased cell proliferation and decreased apoptosis in terminal end buds (Jenkins et al. 2009; Betancourt et al. 2010). BPA co-exposure with DMBA increased steroid receptor coactivators (SRCs), erbB3 and Akt and phosphorylated Akt protein levels, suggesting decreased apoptosis via Akt signaling pathway (Jenkins et al. 2009). Similar increased in tumor growth was observed in female mice prenatally exposed to BPA and DMBA (Weber Lozada and Keri 2011). Early onset of puberty in mice was observed at both low (25 µg/kg/day) and high doses of BPA (250 µg/kg/day) exposure, with no morphological differences in the mammary gland. In MCF-7 cell xenografts model of estrogen-dependent breast cancer, BPA co-exposure with DMBA increased tumor formation, mediated via estrogen receptor (Weber Lozada and Keri 2011).

Aquatic models have also been used to examine BPA co-exposure with environmental chemicals. Danio rerio (zebrafish) has been used to examine the effects on bioaccumulation of chemicals, neurotoxicity, oxidative stress and vascular abnormalities (Wu M et al. 2011; Cypher et al. 2015; Chen Q et al. 2017; Cypher et al. 2018; Huang et al. 2018; Guo et al. 2019). Combined exposure of BPA with nano-sized titanium dioxide (n-TiO2) and nano-sized plastic particles (NPPs) increased bioaccumulation of BPA leading to developmental effects (Chen Q et al. 2017; Guo et al. 2019). BPA co-exposed with NPPs increased BPA’s uptake in viscera and head of zebrafish and increased mRNA expression levels of genes expressed in the nervous system, i.e. mbp gene on day 1 and α1-tubulin gene on day 3 (Chen Q et al. 2017). Dopamine levels and the dopaminergic neurons regulatory gene manf were also significantly increased in zebrafish on day 1 of the BPA and NPPs exposure (Chen Q et al. 2017), highlighting a potential contribution to a dopaminergic neurotoxic effect. Decreased dopamine and serotonin levels in the brain were also observed in zebrafish exposed to BPA and n-TiO2 (Guo et al. 2019). Acetylcholinesterase activity was also found to be reduced in zebrafish co-exposed to BPA and nano-sized materials (Chen Q et al. 2017; Guo et al. 2019). Guo et al. demonstrated thyroid disrupting effects of BPA in zebrafish with decreased plasma T4 or T3 levels in adult zebrafish co-exposed to BPA and n-TiO2. Reduced behavioral activity is a sensitive indicator of developmental neurotoxicity, which was observed to be reduced in the offspring of the zebrafish exposed to BPA and n-TiO2 (Guo et al. 2019). The levels of Mbp and α1-tubulin proteins were downregulated in zebrafish co-exposed to BPA and n-TiO2 (Guo et al. 2019), which was upregulated when zebrafish were exposed to BPA and NPPs (Chen Q et al. 2017). In both studies, BPA co-exposure induced adverse effects on brain architecture and function. However, differences observed in protein expression in these studies may be due to the difference in BPA doses used for combined exposure (Table 2).

Zebrafish have also been co-exposed to BPA and nonylphenol (NP), which resulted in a concentration-dependent increase of MDA, a marker of lipid peroxidation. High doses of BPA-NP induced oxidative stress response in zebrafish embryos and effected skeletal development of adult zebrafish through strong inhibition of antioxidant capacity through changes in expression and activity of SOD, CAT, GR, Gpx, GST, reduced glutathione-oxidized glutathione (GSH-GSSH) ratio and decreased of alkaline phosphatase activity (Wu M et al. 2011).

As presented in Table 2, there has been only one study published in the literature that measured BPA and nonylphenol (NP) co-exposure effects and their association of oxidative stress in human subjects (Huang et al. 2018). Maternal co-exposure to BPA and NP increased oxidatively induced DNA damage as measured by 8-hydroxydeoxyguanosine (8-OHdG), indicating enhanced oxidative stress in pregnant women (Huang et al. 2018). Increased in 8-OHdG levels are found to be mediated through ROS signaling (Chitra et al. 2003; Aly et al. 2012), and that NP may enhance the effect on ROS generation (Okai et al. 2004), as observed in zebrafish co-exposed embryos, which may be dominated by NP and not BPA (Wu M et al. 2011). Inverse relationship was observed with penis length in the high 8-isoPF2α group prenatally exposed to NP and BPA (Huang et al. 2018).

Co-exposure with therapeutics

In the literature, only five studies have been published so far that examined co-exposure effects of BPA with different chemotherapeutic agents. Lapensee et al. published one of the first BPA co-exposure study with chemotherapeutic agents that showed chemoresistance by BPA co-exposure in human breast cancer cell lines. Co-exposure with low nanomolar BPA concentration were able to diminish cytotoxic effects of three different chemotherapy treatments, cisplatin, doxorubicin (Dox) and vinblastine, all of which are known to induce cell death through different mechanism (Lapensee et al. 2009; Lapensee et al. 2010). BPA induced chemoresistance to doxorubicin in these cell lines may be due to the alteration of antiapoptotic proteins such as Bcl-2, Bcl-xL and survivin (Lapensee et al. 2009). The observed BPA effects were mediated irrespective of ER status with ER-positive (T47D) and ER-negative (MDA-MB-468) human breast cancer cell lines showing effects. The effects are likely mediated by GPR30-BPA interaction, which was later confirmed in a BPA co-exposure study that demonstrated GPR30 activation with BPA at the concentrations that showed increased cell viability in 3T3 cells (Nishimura et al. 2014). However, BPA is also known to bind strongly to ERRγ (Matsushima et al. 2007), which also makes ERRγ another potential candidate for mediating BPA-induced protective effects against chemotherapeutics.

Similar chemoresistance was observed with nanomolar and micromolar dose of BPA co-exposed with Dox in colon adenocarcinoma (HT29), human epithelial type 2 (Hep-2) and human lung fibroblasts (MRC-5) cells (Delgado and Ribeiro-Varandas 2015; Ramos et al. 2019). The observed increase in the bcl-xl transcript levels by BPA exposure was significantly reduced in HT29 cells co-exposed with BPA and Dox (Delgado and Ribeiro-Varandas 2015). Dox alone induced transcription changes in cancer-associated genes (c-fos, AURKA, p21 and CLU) in HT29 cells, and these effects were reversed upon pre-treated with 4 µM BPA for 24 hr followed by 24 hr combined exposure with BPA and Dox (4 µM) (Delgado and Ribeiro-Varandas 2015). Co-exposure of low and high dose BPA with Dox also increased viability of MRC-5 cells in a non-monotonic manner, though this effect was not observed in Hep-2 cells. Increased in viability of MRC-5 was associated with decreased DNA damage, while an increase in DNA damage was observed in the HepG2. Ramos et al. also reported different profiles of oxidative damage between the MRC-5 and Hep-2 cell lines induced by BPA and Dox co-exposure. Further analysis revealed MRC-5 expressed GPER, suggesting cell line specific mechanism of action of BPA-ER dependent and independent, as previously reported (Iso et al. 2006; Pfeifer et al. 2015).

High dose BPA was also able to confer resistance to camptothecin (CPT), in MEFs (Sonavane et al. 2018). Here, CPT exhibits its cytotoxic effects differently than the previously studied chemotherapeutic agents (Pommier 2006). BPA co-exposure prevented CPT-induced cytotoxicity by compacting chromatin and decreasing Top1-DNA crosslinks. This reduced cytotoxic crosslinks was observed despite an increased in the protein levels of Top1 by BPA (Sonavane et al. 2018).

Co-exposure with electromagnetic radiation or radioactive substances

Ionizing radiation (IR) like X-rays and BPA are widely present in the environment and may act together, particularly at low doses. For X-rays, about 60% of the damage occurs are due to indirect effects (Barcellos-Hoff et al. 2005). In somatic cells of female mice treated with BPA and X-rays, BPA enhanced genotoxic effect of X-rays at 1 week, as indicated by the increased number of micronucleus (MN) (Gajowik et al. 2013). Similar results were observed in their previous study (Radzikowska et al. 2012), suggesting low doses of BPA diminished sensitivity of the genetic material to damage induction. It is important to note that similar dose of BPA and X-rays were able to show both harmful and ameliorative effects in somatic cells of male mice, which may be due to the differences in the exposure time. Using COMET assay, the authors reported higher strand breaks in bone marrow lymphocytes of females exposed to X-rays and BPA (Gajowik et al. 2013). On the contrary, male mice exposed to combine treatment for 2 weeks showed reduced DNA damage (Dobrzynska and Radzikowska 2013). Combined exposure of spermatids and spermatozoa to BPA and X-rays decreased sperm motility as compared to effects observed after BPA exposure alone (Dobrzynska and Radzikowska 2013). Significant reduction of testis weight and sperm count was also observed in adult and pubescent male mice exposed to BPA and X-rays (Dobrzynska et al. 2014). After 2 weeks, combined exposure to BPA and X-rays significantly reduced DNA strand breaks in somatic and germ cells (Dobrzynska and Radzikowska 2013). Similar effects were also observed in younger males 24 hr post end of combined exposure (Dobrzynska et al. 2014). However, BPA exposure alone enhanced DNA strand breaks and showed genotoxic and reproductive toxic effects (Dobrzynska and Radzikowska 2013).

Mice co-exposed to low dose BPA and 137-Cesium (¹³⁷Cs) reduced urinary volume and increased urinary excretion of uric acid (Esplugas et al. 2018), indicative of renal injury as suggested in previous studies (Prowle et al. 2011; Abdelrahman 2018). The combined exposure of mice with Cs4000 and BPA lowered metabolic enzyme activity associated with renal injury that showed elevated levels of γ-glutamil transferase (GGT) and decreased LDH activity. Also, mice co-administered with BPA and ¹³⁷Cs decreased urinary N-acetyl-β-D-glucosaminidase (NAG) levels back to control. Decreased hepatic CYP1A2 expression was also reported in mice exposed to BPA alone and in combination with ¹³⁷Cs. Increased levels of 8-iso-PGF2α were also observed in mice exposed to BPA and ¹³⁷Cs, suggesting increased lipid peroxidation. However, co-exposure resulted in decrease 8-OHdG in liver demonstrating tissue specificity of BPA effects and co-exposure effects (Esplugas et al. 2018). Together the study suggests that co-exposure show compensatory mechanism, which may reverse the damage caused by chemical agents alone.

Co-exposure with other stressors

Exposure to multiple stressors such as hypoxia is one of the norms in natural environment, particular for wildlife management. BPA exposure during hypoxia resulted in severe bradycardia and reduced cardiac output (heart rate) in zebrafish larvae, compared to normoxia condition (Cypher et al. 2015). Vascular parameters were the mostly affected at the lowest concentration of BPA (0.01 µg/L) that showed decreased arterial RBC velocity by 25% during and after the exposure. Furthermore, the larvae were more affected in later developmental stages, causing 51% and 55% slower arterial and venous RBC velocity, with no effect on developmental parameters (Cypher et al. 2018). However, no effects on cardiovascular parameters were observed with BPA exposure alone, suggesting minimal role of BPA associated toxicity. Together these data suggest that co-occurrence of environmental stressors such as hypoxia and BPA, clearly has a more potential effect on cardiovascular function as compared to individual exposure.

Conclusions

The present review provides an overview of literature examining BPA co-exposures with different environmental stressors. Despite an increase in the number of BPA exposure studies during the last two decades, only thirty-six studies have been published that highlights BPA co-exposure effects and its potential health outcomes as presented in Table 1 and 2. Although limited, these co-exposure studies provide previously unexplored mechanisms and effects of BPA co-exposure in presence of natural chemicals and environmental stressors. Therefore, while assessing the health effects of BPA, closer attention should be paid to the impact of concurrent exposures. Here, model systems, dose, route and time of exposure are noted in Table 1 and 2 to assist in the BPA co-exposure evaluation with different chemicals.

Naturally occurring compounds or dietary supplements are known to be beneficial in overcoming the deleterious effects of estrogenic EDCs such as BPA on different endpoints such as oxidative stress biomarkers, sex hormone levels, organ morphology, DNA damage and epigenetic modification. These changes may alter the adult phenotype or impact offspring, and even contribute to organ toxicity and carcinogenesis (Dolinoy et al. 2007; Wu HJ et al. 2013; Wahby et al. 2017; Saadeldin et al. 2018). However, there can be deleterious effects with some dietary supplements, including increased aneuploidy, endocrine dysfunction, and wide spread changes in epigenetic modifications and gene expression changes (Branco and Lemos 2014; Ribeiro-Varandas et al. 2014; Leung et al. 2017; Sèdes et al. 2018). These studies highlight that the toxicity may not be the most detrimental effect induced by BPA co-exposure. Epigenetic modifications and gene expression changes could have long-term effects on disease susceptibility, i.e., providing a permissive environment for breast cancer in the first-generation offspring (Leung et al. 2017).

BPA co-exposure with industrial chemicals and heavy metals enhanced BPA-induced metabolic dysfunction, mammary tumorigenesis, genotoxicity and cytotoxicity in NIH 3T3 and HepG2 cells and mice models (Jenkins et al. 2009; Betancourt et al. 2010; Weber Lozada and Keri 2011; Chen ZY et al. 2016; Li et al. 2017; Wang D et al. 2018). While co-exposure with agents that causes oxidative stress prevented BPA-induced cytotoxicity in mouse fibroblasts, though did not reduce the induction of potentially mutagenic oxidative DNA damage (Nishimura et al. 2014; Gassman et al. 2015; Gassman et al. 2016). Co-exposure studies also provided evidence that combined exposure to BPA and NP enhanced oxidative stress and impaired human reproductive indices (Wu M et al. 2011; Huang et al. 2018).

Epidemiological studies on zebrafish suggest that BPA co-exposure with nanoparticles can impact development and induce neurotoxicity by increasing bioaccumulation of BPA (Chen Q et al. 2017; Guo et al. 2019). It is difficult to predict the exact relationship between the organic pollutant like BPA and nano-sized particles without further studies in human cell models and animals, but contamination of the environment with nano-sized particles is increasing and co-exposures studies may be critical to understand adverse health outcomes.

Altogether, the synergistic interactions and other additive or antagonistic effects reviewed here highlight that the mechanisms of interactions between BPA and environmental stressors are poorly defined and much more work is needed in this area in order to understand how these co-exposures contribute to the oxidative stress, reproductive toxicity, carcinogenicity, and metabolic dysfunction observed in some human BPA monoexposure studies (reviewed in Rochester 2013; Rezg et al. 2014; Seachrist et al. 2016).

Chemotherapy drugs can be packaged in BPA-containing polycarbonate materials. BPA co-exposure data with chemotherapeutics suggests that low and high doses of BPA exposure altered the activity of these agents in tissue specific manners (Lapensee et al. 2009; Lapensee et al. 2010; Delgado and Ribeiro-Varandas 2015; Sonavane et al. 2018). Conferring resistance to different chemotherapy treatment that acts through different mechanism further provides information on the carcinogenic potential of BPA and highlights previously unexplored mechanism by which BPA could alter the metabolism, induction of DNA damage, and potential DNA repair of chemotherapeutic agents.

Even physical exposures to X-rays were altered in the presence of BPA. Co-exposure enhanced the harmful effects of X-rays, particularly in male germ cells (Dobrzynska and Radzikowska 2013; Dobrzynska et al. 2014), and in bone marrow lymphocytes of female mice (Gajowik et al. 2013). However, compensatory effects were also observed when male mice were co-exposed with BPA and ¹³⁷Cs (Esplugas et al. 2018) and when BPA co-exposed with X-rays (Dobrzynska and Radzikowska 2013). This may be due to the stimulation of DNA repair and removal of damage DNA after low doses of X-rays exposure, though these mechanisms require more investigation.

In summary, BPA co-exposure with some natural chemicals and environmental stressors can enhance BPA-induced oxidative stress biomarkers, induce metabolic changes, alter gene and protein expression levels, promote organ toxicity, increase carcinogenesis and promote the development of chemoresistance. All of these outcomes would adversely affect the health and longevity of the population. However, BPA co-exposure with some dietary supplements (Folic acid, GBE, genistein and melatonin) can actually ameliorate the effects induced by BPA exposure alone. Although, these studies span a wide range of concentration, exposure durations and model system, the data collected are sufficiently robust to raise concerns about the potentially adverse effects of BPA co-exposures, particularly during development.

More work is needed to understand how BPA interacts with environmental stressors, dietary supplements, and chemotherapeutics in order to better delineate adverse health effects of BPA on the population. Additionally, just as in monoexposure studies caution is needed in extrapolating evidence from in vitro studies to animal studies and further to humans due to the differences in the sensitivity observed among cells, tissues, species and strains in number of toxicological endpoints. However, two recurring mechanisms that emerge from both mono- and co-exposure studies are epigenetic modifications and alterations in antioxidant cofactors and enzymes. Epigenetic modifications can have long term and heritable consequence, and alterations in the oxidation-reduction capacity of cells can numerous consequences, including metabolic dysfunction and aging. Therefore, co-exposure studies incorporating robust study designs like those used in CLARITY are essential to investigating BPA co-exposure effects and identifying methods for ameliorating or remediating these effects, which would be especially relevant for addressing the currently ubiquitously exposed population.