Comparative Biochemistry and Physiology, Part C 174–175 (2015) 39–45

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Bisphenol A alters the cardiovascular response to hypoxia in Danio rerio embryos Alysha D. Cypher ⁎, Jessica R. Ickes, Brian Bagatto Department of Biology, Program in Integrated Bioscience, The University of Akron, Akron, OH, USA

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Article history: Received 16 March 2015 Received in revised form 15 June 2015 Accepted 17 June 2015 Available online 24 June 2015 Keywords: Endocrine disruption Bisphenol A Hypoxia Zebrafish Cardiovascular Co-exposure HIF-1α

a b s t r a c t The purpose of this study was to determine if the cardiovascular response to hypoxia was altered by the presence of bisphenol A (BPA) in Danio rerio embryos. It was expected that BPA exposure would affect cardiovascular parameters during hypoxia more than normoxia due to an interaction between BPA and the hypoxia-inducible factor (HIF-1α) pathway. We demonstrate that BPA exposure has a minimal effect during normoxia but can severely affect the cardiovascular system during a hypoxic event. Cardiovascular response was measured in vivo using video microscopy and digital motion analysis. RBC density increased 35% in hypoxia alone but decreased 48% with addition of 0.25 mg/L BPA. Tissue vascularization (% coverage) was unaffected by hypoxia alone but decreased 37% with addition of 0.25 mg/L BPA. The diameter and RBC velocity of arteries were more sensitive than veins to BPA exposure during both normoxia and hypoxia. Arterial RBC velocity decreased 42% during normoxia and 52% during hypoxia with 1 mg/L BPA. This decrease in velocity may in part be due to the 86% decrease in heart rate (ƒH) observed during co-exposure to hypoxia and 5 mg/L BPA. While stroke volume (SV) was unaffected by treatment, cardiac output (Q) decreased by 69% with co-exposure. ƒH and Q were not affected by BPA exposure during normoxia. Development ultimately slowed by 146% and mortality rates were 95% during hypoxia when exposed to 5 mg/L BPA. Our results show for the first time that BPA exposure alters the cardiovascular system during hypoxia more so than normoxia. © 2015 Elsevier Inc. All rights reserved.

1. Introduction The teleost cardiovascular system is one of the first organ systems to be fully functional and is capable of responding to changes in environmental oxygen concentration even prior to the development of oxygen sensing cells (Fritsche et al., 2000; Jacob et al., 2002; Pelster et al., 2005). For teleosts, surviving hypoxia, a common event in aquatic ecosystems, requires a balance between oxygen availability and oxygen demand which can be achieved through a variety of physiological responses (Farrell, 2007). Danio rerio larvae, for example can meet oxygen demand by increasing gill ventilation, heart rate, and rapid swimming to avoid hypoxic conditions (Jonz and Nurse, 2005). During a chronic or developmental exposure however, larvae are more likely to decrease oxygen demand via responses like bradycardia, shunting blood to more vital organs, and decreasing metabolic rate (Barrionuevo and Burggren, 1999; Pelster, 2003; Barrionuevo et al., 2010). Erythropoiesis and angiogenesis are more energetically costly options that increase the efficiency of convective oxygen transport by increasing the density of oxygen carriers and vascular surface area, respectively (Iyer et al., 1998; Hirota and Semenza, 2006). Though these cardiovascular changes ⁎ Corresponding author at: Department of Biology, Program in Integrated Bioscience, The University of Akron, Akron, OH 44325, USA. Tel.: +1 724 272 8061. E-mail address: [email protected] (A.D. Cypher).

http://dx.doi.org/10.1016/j.cbpc.2015.06.006 1532-0456/© 2015 Elsevier Inc. All rights reserved.

are documented in early development, the importance of convective oxygen transport during early stages is debated. Larvae of D. rerio can perfuse tissue through bulk diffusion alone without the assistance of convective oxygen transport until 42 days post fertilization (dpf) in normoxia (Pelster and Burggren, 1996; Jacob et al., 2002). However during hypoxia, convective oxygen transport may assist in the delivery of diffused oxygen to tissue and acquisition of oxygen at the gill arches prior to lamellae development (Kimmel et al., 1995; Rombough, 2002, 2004, 2007; Jonz and Nurse, 2005; Rombough and Drader, 2009; Yaqoob and Schwerte, 2010). Therefore, a properly functioning cardiovascular system may be important during stressful events like hypoxia during early development. It may be even more important when hypoxia occurs in combination with other environmental variables like endocrine disruptors. Bisphenol A, a common contaminant in surface water, is a polycarbonate synthesizer known for its ability to mimic estrogen (Vandenberg et al., 2009). In addition to its effects on reproduction, it has been documented to alter calcium handling in cardiomyocytes and nitric oxide signaling (Papandreou et al., 2006; Gao and Wang, 2014). BPA also disrupts the hypoxia-inducible factor-1 α (HIF-1α), a key transcription factor that elicits erythropoiesis, angiogenesis, and upregulation of glycolytic pathways during hypoxia, by binding a heat shock protein, HSP90, destabilizing HIF-1α and thereby promoting its proteasomal degradation (Kubo et al., 2004; Brahimi-Horn and Pouyssegur, 2009; Pant et al., 2011).

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While there is clearly an interaction between BPA and HIF-1α in vitro, there is little research concerning the in vivo cardiovascular effects of co-exposure to BPA and hypoxia. This study was conducted to determine how hypoxia and BPA co-exposure affects cardiovascular function during early development. Because BPA affects HIF-1α and cardiac function, we expected to see a decrease in red blood cell density, tissue vascularization, heart rate (ƒH) and cardiac output (Q) in co-exposed embryos. Lastly, we expected vascular parameters, development, and mortality to be affected more by hypoxia and BPA in combination than singly. 2. Methods 2.1. Animals In stock populations of adult long-finned gold zebrafish (D. rerio) at The University of Akron Research Vivarium were used as breeding stock for the embryos in this study. Long-finned gold zebrafish were appropriate for this study because they are semi-transparent, allowing for easy viewing of the developing blood vessels and heart. Standard husbandry procedures were maintained and adult fish were housed in acrylic tanks (75 L) with a 14L:10D light cycle and temperature of 26 ± 0.5 °C. Breeding boxes (1 L) were introduced to mixed gender pooled tanks with up to 30 adults an hour before the start of the light cycle and embryos were collected about 30 min post fertilization. Eggs were incubated with a 14L:10D light cycle at 28 °C for the duration of the exposure. Experiments were conducted on days where breeding resulted in more than 300 embryos which were evenly distributed amongst the treatment groups. 2.2. Chemicals Bisphenol A (BPA) (96%) was purchased from Sigma Aldrich (133027). BPA was heat dissolved in dechlorinated and carbon filtered tap water without the use of solvent in order to obtain exposure concentrations, 0, 0.25, 1, and 5 mg/L. Concentrations were based on previous studies and demonstrate environmentally relevant concentrations (Staples et al., 1998; Crain et al., 2007; Lam et al., 2011; Wu et al., 2011; Huang et al., 2012). Non-toxic control solutions were dechlorinated, carbon filtered tap water. 2.3. Treatment Hypoxia (1.0 ± 0.5 mg O2/L) was created and maintained in BPA solutions by bubbling N2 while normoxic treatments (N6.0 mg O2/L) were maintained by bubbling compressed air. Both hypoxic and normoxic treatment flasks were sealed with stoppers to minimize drift in oxygen concentration. Despite sealing, oxygen concentration decreased about 0.5 mg/L every 12 h and therefore were bubbled with compressed air twice a day and the oxygen concentration was measured in order to maintain a range of 1.0 ± 0.5 mg O2/L. Oxygen concentrations were monitored utilizing a YSI Model 55 handheld dissolved oxygen meter. Embryos were exposed to BPA from 1 hour post fertilization (hpf) until late hatching when jaw protrusion occurs, approximately 72 hpf for embryos exposed to normoxia at 28 °C. Fresh BPA solutions were created every 2 days in order to minimize reoxygenation while maintaining fresh exposure. Embryos were checked for hatching and staged for jaw protrusion every 24 h as described in Kimmel et al. (1995). Video was taken when greater than 50% of the embryos in a flask reached late hatching stage. Hypoxia exposed embryos generally take up to 4–5 days post fertilization (dpf) to reach this stage. 2.4. Measurements After exposure, embryos were placed in 1 mL wells on a temperature controlled stage (Harvard Apparatus) on an inverted microscope (Leica DMIRB) and allowed to acclimate for 5 min in normoxic de-chlorinated

tap water. A Red Lake MASD high speed video camera (Morgan Hill, CA) was used to record video of embryos at 125 frames per second. Image Pro Software (Silver Spring, MD) was used to measure vascular and cardiac parameters including caudal vessel diameters, RBC velocity and density, tissue vascularization, heart rate (ƒH), stroke volume (SV), and cardiac output (Q). In order to measure vascular parameters, 10 s of video was recorded of trunk vessels at 10× magnification. Next, digital motion analysis was used as described by Schwerte and Pelster (2000) to create a cast of the vessels by overlaying differential images for 500 frames. In this cast, areas of movement appear whiter than areas where movement is absent. From this cast, arterial and venous diameters were measured by drawing a line across the caudal artery and vein five times per embryo as described by Bagatto (2005). This cast was also used to measure tissue vascularization (% coverage) which is the ratio of vessel area to body area. The area of vessel was determined by outlining areas within a gray scale range of 160 ± 10 to 255 using a count/size feature. Body area was determined by drawing a polygon around the tail region within 1 μm posterior to the anus. RBC velocity was determined by taking the differential of each trunk vessel video and measuring distance traveled by RBCs in 1/125 of a second. This was done seven times for both the artery and vein at peak velocity during systole. The differential was also used to measure RBC density or the number of blood cells per nanoliter as an indicator for erythropoiesis (Schwerte et al., 2003). A 1 μm transect is drawn over the artery just posterior to the anus. Within this transect, the number of red blood cells is counted at three different systoles per embryo. RBC density is calculated by dividing the number of cells by the volume of each vessel, assumed to be a cylinder. Next, ƒH and SV were measured from video of the heart for each embryo. ƒH was measured by counting the number of frames between each systole for three heart beats. SV was determined by measuring the area and major axis length of the ventricle at end systole and end diastole for 3 heart beats. Area was measured by drawing a perimeter around the ventricle. Ventricular volume was measured using the following formula for a prolate spheroid as described by Bagatto and Burggren (2006): Volume ¼

8A2 3πL

ð1Þ

where A is the area of the ventricle at end systole or end diastole and L is major axis length. The mean volumes for end systole (ESV) and end diastole (EDV) were used to calculate SV. Sv was then multiplied with ƒH to get Q. Lastly, in order to assess the progression of development during each exposure eye area was obtained for each embryo (Parichy et al., 2009; Wang et al., 2009). For each embryo, a perimeter was drawn around one eye. Survival rates and the numbers of days to reach 50% hatching were also recorded for each treatment. 2.5. Statistics A nested two-way analysis of variance (ANOVA) was used to compare cardiovascular parameters (vessel diameters, RBC velocities, Q, ƒH, ESV, EDV, SV) and developmental parameters (eye area, time to 50% hatching, mortality) between treatments. BPA concentration, oxygen concentration, and their interaction were included as sources of variation. A statistical interaction between hypoxia and BPA was used to determine if co-exposure had a synergistic effect. A nested approach was used in order to account for variation between flasks which were the unit of replication with at least 20 embryos nested within flasks (n = 10). When significant effects were noted, Tukey's multiple comparison test was conducted post hoc to investigate specific between treatment differences. Statistics were performed using JMP Pro 11 (SAS Institute) with alpha set at p b 0.01. All results are presented as mean ± SEM. Non-toxic controls for normoxia and hypoxia were used

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to compare co-exposure treatments and exhibited a response similar to previous studies (Pelster et al., 2005; Bagatto and Burggren, 2006; Moore et al., 2006). 3. Results 3.1. Vascular parameters RBC density increased 35% with hypoxia alone (Fig. 1, p b 0.0001) while vascularization was unaffected (Fig. 2, p = 0.2). Interestingly, BPA exposure during hypoxia had the reverse effect on RBC density by decreasing 48% in the 0.25 mg/L treatment (Fig. 1, p b 0.0001). Conversely, RBC density was not affected by BPA during normoxia. BPA and oxygen concentration had a synergistic effect on RBC density that could not be predicted by individual exposure (p b 0.0001). Tissue vascularization on the other hand decreased with BPA exposure during both normoxia and hypoxia. Vascularization decreased 21.4% (p b 0.01) during normoxia and 37% during hypoxia with 0.25 mg/L BPA (p b 0.001). Vessel diameter and red blood cell velocity were measured as indicators of vascular resistance and arterial pressure, respectively. Veins decreased 24% in diameter with hypoxia exposure alone (p b 0.001) but were unaffected by BPA during normoxia or hypoxia (Fig. 3B). Artery diameter was unaffected by hypoxia alone (p = 0.8) nor BPA during normoxia. BPA exposure during hypoxia however did result in a 20% decrease in arterial diameter in the 1 mg/L BPA treatment (p b 0.01). Arterial (Fig. 3C) and venous RBC velocity (Fig. 3D) decreased by 36% and 28%, respectively with hypoxia alone (p b 0.001, p b 0.01). Unexpectedly, arterial RBC velocity slowed by as much as 42% with BPA treatment during normoxia (p b 0.0001). Simultaneous exposure however caused a 52% and 48%, decrease for arterial and venous RBC velocities, respectively at 1 mg/L BPA (p b 0.0001, p b 0.0001). Overall, vascular parameters decreased the most with BPA exposure during hypoxia and arteries were more affected than veins. 3.2. Cardiac parameters Cardiac parameters also decreased more significantly with coexposure than BPA and hypoxia individually. ƒH was unaffected by hypoxia alone (p = 0.1, Fig. 4A) but decreased with 5 mg/L of BPA during normoxia by 17% (p b 0.01). More significantly, BPA and hypoxia synergized to decrease ƒH by 48 and 86% with 1 mg/L and 5 mg/L BPA,

Fig. 2. Tissue vascularization (% coverage) of embryos exposed to BPA during normoxia and hypoxia. Percent coverage was unaffected by hypoxia treatment alone, but decreased with BPA in both normoxia and hypoxia. Data is presented as mean ± SEM. Asterisks denote p b 0.01 between treatments and their respective control. Double asterisks denote p b 0.01 between hypoxia and normoxia at same BPA concentration.

respectively (p b 0.0001, p b 0.0001). A statistical interaction between oxygen and BPA concentration suggests that co-exposure has a synergistic effect to decrease ƒH (p b 0.0001). ESV and EDV (Supp Figs. 1 and 2) were unaffected by hypoxia, BPA, or their interaction (p N 0.01). Though SV (Fig. 4B) is a function of ESV and EDV, it was unaffected by hypoxia exposure (p = 0.09), BPA (p = 0.2), or their interaction (p = 0.8). Q, a function of ƒH and Sv, was unaffected by hypoxia alone (p = 0.4) nor BPA during normoxia (Fig. 4C). Exposure to BPA during hypoxia, however resulted in a 30, 50 and 69% decrease in Q with 0.25, 1 and 5 mg/L BPA, respectively (p b 0.01, p b 0.0001, p b 0.001). Overall, cardiac parameters were unaffected by hypoxia or BPA individually, but decreased significantly with co-exposure. 3.3. Developmental parameters Normoxia treated embryos reached late hatching at 3 dpf despite BPA concentration which is typical for embryos raised at 28 °C (Kimmel et al., 1995). While embryos exposed to hypoxia alone took 86% longer, about 5 days, co-exposed embryos at 5 mg/L BPA took 146% longer, approximately 8 days. Eye area, which was used as an indicator for developmental progress, was unaffected by BPA during normoxia but decreased 21% with hypoxia alone (p = b 0.0001, Fig. 5), indicating that hypoxia treated embryos were underdeveloped despite controlling for developmental stage. BPA exposure during hypoxia resulted in an additional 38% decrease in eye area at 5 mg/L BPA (p b 0.01). A statistical interaction between oxygen and BPA concentration suggests that co-exposure has a synergistic effect to decrease eye area (p b 0.01).Lastly, mortality rates (Table 1) also varied between treatments with control treatments having a relatively low mortality of 22% while hypoxia exposure alone caused 55% mortality. Exposure to BPA increased mortality in both normoxia and hypoxia. BPA exposure led to 43% in normoxia and 95% mortality in hypoxia at the highest concentration. Replication in the 5 mg/L BPA and hypoxia treatment was affected by high mortality and therefore the 1 mg/L concentration is described for many comparisons. 4. Discussion

Fig. 1. Red blood cell (RBC) density of embryos exposed to BPA during normoxia and hypoxia. RBC density increased with hypoxia exposure alone, but decreased with the addition of 0.25 mg/L of BPA. Data is presented as mean ± SEM. Asterisks denote p b 0.01 between treatments and their respective control. Double asterisks denote p b 0.01 between hypoxia and normoxia at same BPA concentration.

The cardiovascular system of zebrafish embryos is more susceptible to hypoxia and BPA in combination than individually during early development. Every cardiovascular parameter except venous diameter

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Fig. 3. Caudal vessel diameters (A—arterial; B—venous) and peak red blood cell (C—arterial; D—venous) velocity of embryos exposed to BPA during normoxia and hypoxia. (A) Arterial diameter only decreased with BPA and hypoxia in combination at 1 mg/L BPA. (B) Venous diameter decreased with hypoxia alone and when in combination with 0.25 and 1 mg/L BPA. (C) Arterial RBC velocity decreased more with BPA and hypoxia in combination than individually at 1 mg/L and 5 mg/L BPA. (D) Venous RBC velocity decreased with hypoxia alone and in combination with 1 mg/L BPA. Data is presented as mean ± SEM. Asterisks denote p b 0.01 between treatments and their respective control. Double asterisks denote p b 0.01 between hypoxia and normoxia at same BPA concentration.

decreased more during co-exposure than to BPA and hypoxia alone. While most parameters decreased merely because of the addition of BPA and hypoxia's individual effects, some parameters were synergistically affected by co-exposure which was confirmed by a statistical interaction between the two exposures. Because this is an environmentally relevant co-exposure, these data indicate that early teleost cardiovascular development may be more susceptible to the combination of BPA and hypoxia than to each variable individually.

resistance, increased with hypoxia treatment and may have contributed to the decrease in velocity. Higher RBC density, however is likely advantageous if it increases the efficiency of oxygen binding and transport. Lastly, hypoxia treated embryos had smaller eye area and slower developmental rate which can be a means to lessen oxygen demand or a constraint of inadequate perfusion (Shang and Wu, 2004; Barrionuevo et al., 2010; Navas and Carvalho, 2010). Overall, embryos had a typical response to developmental hypoxia in non-toxic controls.

4.1. Hypoxia exposure

4.2. BPA exposure

Embryos had a classic response to developmental hypoxia as compared with other studies including increased RBC density, decreased venous diameter, and decreased RBC velocity (Bagatto, 2005; Pelster et al., 2005; Moore et al., 2006). In addition, no changes were seen in percent coverage of vessels or cardiac parameters which is not abnormal. RBC velocity, which is proportional to blood pressure, is most readily influenced by changes in total peripheral resistance and ƒH. Since ƒH was unchanged by hypoxia alone, it is unlikely that it was a contributing factor to the decrease in velocity with hypoxia treatment. However, a 24% decrease in venous diameter likely increased peripheral resistance slowing RBC velocity. In addition to reduced vein size, RBC density (Fig. 1), which is proportional to blood viscosity and therefore

BPA alone can alter cardiac contractility and vascular tone through many potential mechanisms including disruption of calcium handling, decreasing nitric oxide concentration, and increasing oxidative stress (Gao and Wang, 2014). Therefore it was not surprising that arterial RBC velocity and ƒH decreased with BPA exposure in this study (Figs. 3C and 4A). Unexpectedly, percent coverage of vessels also decreased with BPA exposure during normoxia (Fig. 2). This may in part be due to lower video contrast which can occur with slower RBC velocity. It may also be due to some effect of BPA on angiogenesis or vascular function during normoxia. While cardiovascular function appears to be affected by BPA during normoxia, eye area and mortality were not affected by this exposure. This may mean that BPA's effect

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4.3. Hypoxia and BPA interaction BPA exposure during hypoxia caused a decrease in RBC density, percent coverage, ƒH, Q, and eye area that was less significant or absent during normoxia (Figs. 1, 2, 4A, C and 5). The synergistic effect of coexposure on these parameters suggests an interaction that is greater than the cumulative effects of BPA and hypoxia. This interaction may involve the HIF-1α pathway. Because BPA exposure can cause the proteasomal degradation of HIF-1α, exposure to BPA may interfere with the cardiovascular system's ability to respond to hypoxia via angiogenesis, erythropoiesis, and other cellular changes mediated by this pathway (Iyer et al., 1998; Kubo et al., 2004). This was supported by a greater decrease in RBC density and percent coverage (Figs. 1 and 2) during hypoxia than normoxia with BPA exposure. In addition to inducing angiogenesis and erythropoiesis, HIF-1α plays an important role in mediating nitric oxide synthase expression, energy metabolism, and cellular function (Iyer et al., 1998; Tekin and Dursin, 2010). Therefore, BPA exposure has the potential to interrupt these important cellular functions further constraining oxygen supply. The higher mortality rates during hypoxia than normoxia with BPA exposure (Table 1) indicate that this co-exposure may cause neither diffusion nor convective oxygen transport to be able to meet oxygen demands. 4.4. Cardiovascular consequences Maintaining adequate perfusion until reoxygenation requires a dynamic cardiorespiratory response which appears to be compromised in hypoxia and BPA-exposed embryos. Because the cardiovascular system is responsive to intrasystemic changes, the large decreases in RBC density, percent coverage, ƒH, and Q may have contributed to the other effects on RBC velocity, diameter, and ultimately eye area and mortality. A reduction in RBC density and percent coverage could decrease the efficiency of convective oxygen transport by decreasing oxygen binding and transport. Though embryos at this stage of development mainly rely on bulk diffusion for oxygen, convective oxygen transport may be important during hypoxia by transporting diffused oxygen (Rombough, 2002, 2007). Therefore, decreased RBC density and percent coverage may have further constrained oxygen supply in co-exposure treatments. This ultimately could have contributed to the large decrease in ƒH, since cardiomyocytes require large stores of oxygen to maintain contractility. Inadequate oxygen concentration in cardiomyocytes leads to a longer refractory period between contractions. As ƒH decreased, RBC velocity also decreased in co-exposure treatments due to the lower rate of propulsion. RBC velocity may have been further slowed by an increase total peripheral resistance caused by a decrease in artery diameter by 20% in the 1 mg/L and hypoxia treatment (Fig. 3A). Since vascular resistance increases to the fourth power as vessel radius decreases, this was likely a significant factor in decreasing velocity. In turn, a reduction in RBC velocity can further slow ƒH by increasing the filling time and decreasing filling pressure. Overall, perfusion was more greatly affected by BPA exposure during hypoxia than either exposure individually which likely contributed to the effects on development and mortality. 4.5. Development and mortality Fig. 4. Heart rate (ƒH), stroke volume (SV), and cardiac output (Q) of embryos exposed to BPA during normoxia and hypoxia. (A) ƒH was un affected by hypoxia alone, but decreased 17% with 5 mg/L BPA and normoxia, and decreased 86% with 5 mg/L BPA and hypoxia. (B) SV was unaffected by hypoxia, BPA, or their combination. (C) Q was unaffected by hypoxia or BPA alone, but decreased 69% at 5 mg/L BPA. Data is presented as mean ± SEM. Asterisks denote p b 0.01 between treatments and their respective control. Double asterisks denote p b 0.01 between hypoxia and normoxia at same BPA concentration.

on cardiac function does not interfere with development or oxygen demand. BPA exposure during hypoxia, however does appear to affect the balance between oxygen demand and availability.

Development during a hypoxic event is clearly hinged on the availability of oxygen and whether or not it can be transported efficiently to tissues (Driedzic, 1988; Padilla and Roth, 2001; Barrionuevo et al., 2010). While embryos exposed to BPA during normoxia did not experience an interruption in development, those exposed during hypoxia developed up to 146% slower. Therefore, some interaction between BPA and hypoxia delays development. Because exposure to BPA alone did not affect development, this interaction may involve constraints on oxygen rather than toxicity. It is possible however that factors like oxidative stress contributed to the effects on development and mortality in this study (Wu et al., 2011; Gao and Wang, 2014). Oxygen

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Acknowledgments This work was supported by an internal grant, The Buchtel College of Arts and Sciences Student Research/Scholarship Awards at The University of Akron (UA). The funding source had no involvement in the design of this study. References

Fig. 5. Eye area of embryos exposed to BPA during normoxia and hypoxia. Eye area was unaffected by BPA alone, but decreased with hypoxia alone and in combination with 0.25, 1, and 5 mg/L BPA. Data is presented as mean ± SEM. Asterisks denote p b 0.01 between treatments and their respective control. Double asterisks denote p b 0.01 between hypoxia and normoxia at same BPA concentration.

constraints would explain why many co-exposed embryos were observed entering a suspended animation where ƒH and RBC transport came to a stop but later recovered (Padilla and Roth, 2001). It is also unclear if oxygen constraints, toxicity, delayed development, or some combination contributed to the higher mortality rates in co-exposure treatments. More research is needed to better understand developmental constraints and survival during hypoxia when in combination with other stressors.

4.6. Conclusions Our data clearly shows that cardiovascular function is altered by BPA exposure during hypoxia in a way that is not merely the addition of their individual effects. The ubiquitous contamination of waterways with novel compounds like BPA presents a new exposure regime when in combination with historically common stressors like hypoxia. Though the highest concentrations of BPA are not environmentally relevant, hypoxic events and BPA production are on the rise (Huang et al., 2012; Rocha et al., 2012). More importantly, continuous exposure to BPA allows for some bioaccumulation in tissue despite its short halflife (Wade et al., 2006). Therefore, aquatic concentrations of BPA may be less important than tissue concentrations. More research is needed to explore the role of the HIF-1α pathway in the interaction between hypoxia and BPA in vivo and to determine whether or not these effects are pertinent to cardiovascular disease. BPA exposure has been correlated with heart disease, and therefore may affect cardiac function during myocardial ischemia in humans (Melzer et al., 2010). Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbpc.2015.06.006.

Table 1 Mortality rates (%) were highest for embryos exposed to BPA during hypoxia. Data is presented as mean ± SEM. BPA concentration (mg/L)

Normoxia (N6.0 mg O2/L)

Hypoxia (1.0 ± 0.5 mg O2/L)

0 0.25 1.0 5.0

21.8 ± 8.4 12.6 ± 4.3 22.2 ± 12.3 43.3 ± 30.0

54.9 ± 21.8 37.3 ± 9.9 84.1 ± 10.5 95.2 ± 4.8

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