Ecotoxicology and Environmental Safety 101 (2014) 7–13
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Bisphenol A affects larval growth and advances the onset of metamorphosis in Drosophila melanogaster A.K. Weiner a, A. Ramirez a, T. Zintel a, R.W. Rose a, E. Wolff a, A.L. Parker a, K. Bennett a, K. Johndreau a, C. Rachfalski a, J. Zhou b, S.T. Smith a,n a b
Arcadia University, 450 South Easton Road, Glenside, PA 19038, USA Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming, Yunnan Province 650223, China
art ic l e i nf o
a b s t r a c t
Article history: Received 8 August 2013 Received in revised form 11 December 2013 Accepted 16 December 2013 Available online 7 January 2014
Exposure to Bisphenol A (BPA) has been reported to dysregulate endocrine pathways in a wide array of vertebrate species. The effects of BPA on invertebrate species are less well understood. We tested the effects of BPA on growth and development in Drosophila as these processes are governed by well-studied endocrine pathways. In this study, we tested the effects of three concentrations of BPA (0.1 mg/L, 1 mg/L or 10 mg/L) and found a statistically significant increase in larval growth for the low dose treatment group (0.1 mg/L), but not statistically significant for the high dose treatment group (10 mg/L). BPA exposure resulted in an increased body size in treated animals at 48, 72 and 96 h after egg laying (AEL). This finding reflects a non-monotonic dose–response that has been observed for an increasing number of endocrine disrupting compounds. The increase in growth rate found for all treatment groups was associated with a statistically significant increase in food intake observed at 72 h AEL. Furthermore, we observed that the increased growth rate was coupled with an earlier onset of pupariation consistent with previously reported phenotypes resulting from increased activity of insulin/insulin growth factor signaling (IIS) in Drosophila. Since the timing of the onset of pupariation in Drosophila is controlled through the complex interaction of the IIS and the ecdysone signaling pathways, our findings suggest that BPA exerts its effects through disruption of endocrine signaling in Drosophila. & 2013 Elsevier Inc. All rights reserved.
Keywords: Endocrine Non-monotonic dose–response curve Body size Toxicant Pupariation Development
1. Introduction Bisphenol A (BPA) is a high-production industrial monomer used world-wide in the manufacture of polycarbonate plastics and epoxy resins found in food packaging, thermal paper, dental sealants, medical devices, flame retardant materials and other products (reviewed in (Staples et al., 1998)). Human exposure to BPA through dietary and non-dietary sources has been well-documented (reviewed in (Geens et al., 2011)). The effects of BPA have been investigated in a diverse array of vertebrate species, where BPA has been reported to function as a potent endocrine disruptor at environmentally relevant levels, affecting reproductive processes, including gonadal function and sex determination (reviewed in (Crain et al., 2007)). BPA is classified as a xenoestrogen that is thought to exert its effects through direct binding to estrogen receptors (ER) in a wide range of species (Andersen et al., 1999). In vertebrates, both agonistic and antagonistic activities of BPA
n
Corresponding author. Fax: þ 1 215 881 8758. E-mail address: [email protected] (S.T. Smith).
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.12.008
have been reported and were dependent on ER isoform-specific interactions (Hiroi et al., 1999). The mechanism of BPA-induced reproductive toxicity in invertebrates is not as well understood as compared to vertebrate species. However, a growing number of invertebrate studies have reported reproductive abnormalities, including reduced fecundity in Daphnia magna and Ceriodaphnia dubia (Tsui et al., 2005), Drosophila melanogaster (Atli and Ünlü, 2012), Chironomus riparius (Watts et al., 2001) and the terrestrial isopod, Porcellio scaber (Lemos et al., 2010). BPA exposure was also found to induce germ cell production in the nematode, Caenorhabditis elegans (Hoshi et al., 2003), as well as increased egg production in the snail, Potamppyrgus antipodarum (Duft et al., 2003) and the copepod, Acartia tonsa (Andersen et al., 1999). Furthermore, BPA exposure resulted in the suppression of testes formation in Hydra oligactis (Fukuhori et al., 2005) and gonad resorption in the mussel, Mytilus edulis (Ortiz-Zarragoitia and Cajaraville, 2006). In addition to playing a central role in reproductive processes, endocrine signaling is critical to an organism0 s growth and development. In vertebrates, BPA treatment that exceeds environmentally relevant levels has been associated with teratogenic effects including abnormal growth, cephalic malformations, and vertebral defects (reviewed in (Crain et al., 2007)). Exposure to BPA in invertebrates
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has resulted in reduced growth rate in the freshwater sponge, Eunapius fragilis (Hill et al., 2002), delayed time of emergence in the housefly, Musca domestica (Izumi et al., 2008), delayed completion of the naupliar stages in the copepod, Tigriopus japonicus, (Marcial et al., 2003) and molting defects in the insect, C. riparius (Watts et al., 2001; Segner et al., 2003). The focus of this study was to assess the effects of BPA on growth, development and maturation in D. melanogaster, a holometabolous insect. In Drosophila, the larval stage is characterized by feeding and growth in which larvae proceed through a series of three instars. The duration of each instar is determined by the activity of the steroid hormone ecdysone and its active metabolite, 20-hydroxyecdysone (20E) (Thummel, 1996). In insects, final body size is controlled by a size-monitoring mechanism that dictates when growth should cease, and occurs during the period in larval development when growth rate is at its highest (Davidowitz et al., 2003). In Drosophila, the prothoracic gland (PG) is thought to act as the size assessment tissue that is responsive to activities of the highly conserved insulin/insulin-like growth factor signaling pathway (IIS) (Colombani et al., 2005; Mirth et al., 2005; Caldwell et al., 2005). The growth of the PG, in response to insulin signaling, therefore specifies when the critical weight has been attained and through the synthesis and release of ecdysteroids, the PG determines the timing of the onset of metamorphosis. Given the growing evidence that BPA exposure results in changes in growth and development in a number of invertebrate systems that share highly conserved pathways that regulate these processes, we hypothesized that BPA exposure in Drosophila would result in alterations in growth rate that would impact animal size and the timing of pupariation. To test this, we treated Drosophila larvae with increasing concentrations of BPA and used imaging software (ImageJ) to monitor growth rate and food intake at 48, 72, 96 and 120 h AEL. Because growth rate can affect the timing of onset of pupariation, we also examined the puparia of BPA-treated and untreated animals at 168 h to assess pupal stages. Furthermore, we conducted an experiment to assess potential differences in the onset of pupariation in BPA-treated and untreated animals.
2. Materials and methods 2.1. Drosophila stocks Experiments were carried out in Oregon-R-modENCODE (25211), obtained from the Bloomington Stock Center, Bloomington, IN. Oregon-R-modENCODE is a wild-type strain of D. melanogaster that is used by modENCODE contributors (http://www.modencode.org/publications/about/index.shtml).
2.2. BPA treatment Collection bottles with D. melanogaster flies were kept at 25 1C, in an incubator with 60–65% relative humidity, and reared on a 12 h light/12 h dark cycle. One-hour embryo collections were carried out on standard grape-agar plates (Genesee Scientific, San Diego, CA) supplemented with yeast paste. Embryos were allowed to hatch into first instar larvae and 50 animals were transferred to fresh plates prior to feeding. Three concentrations of BPA were introduced into larval food as follows: BPA (10 mg) was first dissolved in 1 mL acetone and then brought to 1 L with distilled water (10 mg/L BPA, 0.1% acetone). Ten fold dilutions (1 mg/L and 0.1 mg/L) were made, adjusting acetone concentration to 0.1%. The solvent control solution (hereafter referred to as “control”) contained 0.1% acetone in distilled water. Active, dry yeast (0.2 g) was added to 800 μL of each BPA solution and the control. To each of these formulations, 5 μL of food coloring (green) was added and mixed thoroughly. Food was dyed in order to monitor food consumption among control and treatment groups. Yeast preparations containing control and BPA-supplemented solutions were prepared freshly each day, and a 50 μL volume was applied to the grape-agar plate every 24 h beginning at first instar and continued until animals were collected for imaging analysis at the appropriate time point.
2.3. Growth assay Fifty newly-hatched first instar larvae were transferred to fresh agar plates (35 mm) and fed 50 μL of yeast-paste formulations containing either no BPA (control), 0.1 mg/L BPA, 1 mg/L BPA, or 10 mg/L BPA. Four sets of plates were prepared for each treatment group in order to image larvae at 48, 72, 96 and 120 h AEL. Freshly prepared yeast-paste formulations were administered once every twenty-four hour period or until larvae were collected for imaging analysis. Larvae ready for imaging analysis were collected into a wire mesh basket, washed with 1 Phosphate Buffered Saline (PBS) and placed at 20 1C for 3–5 min to decrease larval movement for imaging. Images were taken using a Leica FireCam V 3.2.0 (Leica Microsystems, Inc., Buffalo Grove, IL, USA) and the area of each individual larva was determined using ImageJ software (Schneider et al., 2012). Fig. 1f illustrates an example of larvae imaged at the 72 h time point. Pupal area was analyzed using ImageJ tools and assessment of pupal stages was conducted according to Bainbridge and Bownes (1981). 2.4. Analysis of food intake Larval images were converted to grayscale and a threshold value was set that corresponded to the darkest pixels where food was present in the gut. Selections of specific areas of the gut tube were made using the ImageJ wand tool and mean gray values for the selections were determined. The mean gray values for each control and treatment groups were averaged and compared for each time point: 48, 72, 96 and 120 h AEL. 2.5. Statistical analyses Statistical analyses were carried out using IBM SPSS Statistics for Windows, Version 19.0 (released 2010), IBM Corp., Armonk, NY. With the exception of the staging analysis, one factor Welsh ANOVA followed by Games-Howell post-hoc comparisons were used to compare control and treatment groups. The staging analysis was conducted using Kruskal Wallis analysis of variance by ranks followed by Bonferonni-adjusted Mann–Whitney post-hoc comparisons. Statistical data for larval/pupal area analyses can be found in Supplementary data Tables 1 and 2. Food intake was analyzed using a one-way ANOVA followed by a Tukey post-hoc analysis. The significance level for all statistical analyses was set at p o0.05.
3. Results 3.1. BPA treatment affects larval growth We tested the effects of BPA on the growth of Drosophila larvae through direct administration in food as described in Section 2. Fig. 1a–c shows that beginning at 48 h and continuing through 96 h, larvae exposed to the lowest concentration treatment (0.1 mg/L) were found to be significantly larger in mean area than the non-treated control group. At 72 h, the two lowest treatment groups (1 mg/L and 0.1 mg/L) were found to be significantly larger in area (Fig. 1b). At 120 h, there was no significant difference in area among the control and BPA-treated groups (Fig. 1d). We hypothesized that the decrease in area in the 0.1 mg/L treatment group observed from the 96 h to 120 h time point may reflect a shortening of the larval body in preparation for pupariation. Analysis of growth over time shows a significant increase in growth rate between 72 h and 96 h AEL, with the lowest concentration, 0.1 mg/L, showing the highest growth rate and the 1 mg/L and 10 mg/L treatment groups showing a similar growth rate to the control (Fig. 1e). Interestingly, the lowest concentration treatment group, 0.1 mg/L, showed an attenuation in growth from 96 h to 120 h, while the 1 mg/L, 10 mg/L and control groups continued to show an increase in growth rate during this interval. 3.2. Effects of BPA on food intake We observed an increase in larval body size in response to BPA treatment for all treatment groups at 48, 72, and 96 h AEL. However, only the low dose treatment (0.1 mg/L) group was found to be statistically greater in area. In order to determine whether increased food consumption accompanied the observed increase
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Fig. 1. BPA treatment affects larval growth. Larvae from control and treatment groups were collected at 48, 72, 96 and 120 h after egg laying (AEL), and larvae were imaged. The area of each individual larva was calculated using ImageJ and the mean larval area (mm2) was plotted against treatment concentration for the group. Means 7SEMs are indicated. p valueso 0.01 or o0.05 are indicated as nn and n, respectively. Larval area analysis was conducted on combined data from two independent experiments using a one-way Welsh ANOVA followed by Games-Howell post-hoc analysis. The significance level for all statistical analyses was set at 0.05. (a–d). All p value indicators represent comparison of treatment groups to the control group. Growth rate analysis of mean larval area for all groups plotted against time of development is shown (e). An example of ImageJ analysis for larvae collected at 72 h after egg laying (AEL) is shown. All larvae were imaged using the same magnification (as shown by the underlying grid). Larval images were traced in yellow using ImageJ tools and a mean larval area (mm2) was calculated for each of the groups (mean). Corresponding statistical data for Fig. 1 are found in Supplemental Table 1. The actual area of the larvae shown in Fig. 1f is indicated (actual) and approximates the mean for the group (f).
in body size, we employed ImageJ tools to approximate food intake. The food that was administered each 24 h period was dyed green and therefore visible in the gut of the larvae, whose bodies are opaque to translucent. In order to assess the approximation of food intake, we examined the images used to assess body size as described above in Sections 2, 2.3, and 3.1 (Fig. 2a). A threshold value was set in order to compare images between control and treatment groups for each time point AEL, and mean gray values were determined for each larva as described in Section 2.4 (Fig. 2b). The mean values within each control or treatment group were averaged and compared using a one-way ANOVA followed by a Tukey post-hoc analysis. BPA-treated larvae at the 72 h time point AEL were found to have selections from the gut tube that
were more optically dense than those from the control group (p o0.01 for all treatment groups) (Fig. 2c). 3.3. Effects of BPA on pupal size We conducted an analysis of pupal area at 168 h AEL and found that pupae from all treatment groups were similar in area to the control pupae (Fig. 3a), and was consistent with the observation that larvae from all treatment groups and the control group were similar in size at the 120 h time point. We hypothesized that the attenuation in growth rate observed between 96 and 120 h of development in the 0.1 mg/L BPA treatment group may be associated with an early onset of pupariation. In order to test this
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Fig. 2. BPA exposure is associated with an increased consumption of food. Larval images from the growth analysis described in Section 3.1 were further analyzed for potential differences in optical density in the gut tube as an approximation of food intake. Fig. 2a1–4 is a representative example of a larva from the 96 h time point. Larval images were first converted to grayscale (a1 and a2) and a threshold value was set that corresponded to the darkest pixels where food was present in the gut. Pixels falling below the thresholded value appear blue, while less optically dense pixels remain in grayscale (a3). For each larva the blue area was specifically selected using the ImageJ wand tool (a4) and the mean gray value for that selection was determined as shown in the histogram in (b). The mean gray values for each control and treatment groups were averaged and compared at each time point: 48, 72, 96 and 120 h AEL. A one-way ANOVA was performed followed by a Tukey post-hoc analysis. The significance level for all statistical analyses was set at 0.05. n Indicates a statistically significant p value (p o 0.01) comparing the BPA treated group with respect to the control group (c).
hypothesis, we examined the pupal images of the control and treatment groups at 168 h and estimated the pupal stage according to guidelines for staging metamorphosis in Drosophila, outlined by Bainbridge and Bownes (1981). Our analysis confirmed that pupae from the BPA treatment groups were advanced in metamorphosis compared to pupae from the control group, with the lowest treatment group (0.1 mg/L) showing a statistically significant advancement (Fig. 3b).
pupariation, indicated by the lowest concentration treatment group experiencing the highest larval growth rate (Fig. 1e), as well as, an observed peak pupariation 24 h before other treatment and control groups (Fig. 3c).
4. Discussion 4.1. BPA effects on growth and food intake
3.4. BPA treatment affects rate of entry into pupariation In order to confirm and extend the results of the observed pupariation staging (described above), we conducted an experiment to determine the number pre-pupae/pupae formed at 144, 168 and 192 h AEL following treatment with 0.1 mg/L, 1 mg/L and 10 mg/L BPA or no treatment (control), as outlined in Section 2.2. At 144 AEL, the lowest concentration treatment group (0.1 mg/L) entered into pupariation at the fastest rate (Fig. 3c), with 62% of this treatment group entering into pupariation compared to 34% of the control group at this time point. In addition, at 144 h AEL, 48% of the 1 mg/L treatment group and 43% of the 10 mg/L treatment groups had already entered pupariation. The peak pupariation time for the control group was not observed until 168 h AEL, with 56% of animals pupariated at this time point (Fig. 3c). These data reveal that larval growth rate is highly correlated with the rate of onset of
In this work we undertook a detailed analysis of the effects of a direct administration of BPA on growth and development in the invertebrate model organism, D. melanogaster. We found an increase in growth rate during larval development, most significantly in the lowest concentration treatment group (0.1 mg/L), with the highest growth rate observed between 72 and 96 h of development. The dose–response observed at the 48, 72, 96 and 120 h time points reflects a non-monotonic response curve that has been reported for a number of endocrine disrupting chemicals (Vandenberg, et al., 2012). A more comprehensive study conducted at the 72 h time point showed that 0.001 mg/L and 0.01 mg/L did not result in larvae that were statistically larger in area than control larvae (data not shown). While negative effects of BPA on growth rate have been reported for the invertebrate freshwater sponge, E. fragilis (Hill et al., 2002), a recent study in the housefly, M. domestica (Izumi et al., 2008),
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Fig. 3. BPA affects the rate of entry into metamorphosis. Pupal images were taken at 168 h AEL and analyzed using ImageJ tools. Means 7SEMs are indicated with error bars. The pupal area analysis was conducted using combined data from two independent experiments, each with three trials, using a one-way Welsh ANOVA followed by GamesHowell post-hoc analysis (a). Pupal images were examined and staged according to guidelines established by Bainbridge and Bownes (1981). Means 7SEMs are indicated with error bars. The staging analysis from combined data from three trials was conducted using Kruskal Wallis analysis of variance by ranks followed by Bonferonni-adjusted Mann–Whitney post-hoc comparisons. The significance level for all statistical analyses was set at 0.05. n indicates a statistically significant p value comparing BPA treated group and control group (b). The % of pupae was calculated by the number of new pupae observed (the total number of pupae observed on a given day minus pupae counted on the previous day(s))/total number of animals, at 144, 168 and 192 h AEL. The percentage of animals that had entered metamorphosis was plotted against sampling times (c). Corresponding statistical data for Fig. 3 are found in Supplemental Table 2.
found an increase in pupal weight in response to BPA treatment. Further, Michail et al., (2012) recently reported on the effects of BPA in the moth, Sesamia nonagrioides, and found an increase in weight in larvae exposed to a 100 mg/L dosage, the same as the lowest dosage used in our studies (0.1 mg/L) that produced a significant increase in growth. In Drosophila, larval size is affected, in part, by the growth rate, which is governed by the insulin/insulin growth factor signaling (IIS) pathway. During the feeding phase in Drosophila, insulin-like peptides (DILPs) are synthesized and released from insulinproducing cells (IPC) located in the Prothoracic Gland (PG) of the larval brain (Colombani et al., 2005); a size-assessing tissue that monitors its own growth (Mirth et al., 2005). Indeed, blocking the Protein Kinase A (PKA) pathway in IPCs has been associated with increased insulin signaling in the PG and an increased growth rate in Drosophila (Walkiewicz and Stern, 2009). A number of mammalian studies have also reported a link between BPA exposure, insulin signaling and body size. The Drosophila and mammalian insulin signaling pathways are evolutionarily conserved in function (Oldham et al., 2000; Garofalo, 2002) and proper functioning of the insulinproducing ß cells of the pancreas is dependent, in part, on endocrine (estrogen) signaling (Nadal et al., 1998). Importantly, knockout mice that do not express estrogen receptor-alpha (ER-α), one of two human estrogen-specific receptors, shows an increase in insulin resistance and adiposity (Heine et al., 2000). Similarly, insulin resistance in mice that were treated with the xenoestrogen BPA was shown to occur in an ER-dependent manner (Alonso-Magdalena
et al., 2006), and increased body weight has also been reported in rats exposed perinatally to BPA (Rubin et al., 2001). Finally, a growing number of human epidemiological studies have also suggested that BPA, acting as a xenoestrogen, may interfere with the body0 s natural mechanisms to control weight (Wang et al., 2012; Trasande et al., 2012). Our findings show that BPA can induce growth abnormalities during development that are consistent with those caused by dysregulation of the insulin/insulin growth factor signaling (IIS) pathway (Walkiewicz and Stern, 2009). In this study, we also approximated the amount of food intake by calculating the density of the material in the gut tube and found that there was a statistically significant increase in the amount material in the gut tube of treated versus non-treated larvae at the 72 h time point AEL. This time point also marks the beginning of the highest growth rate for the control and treatment groups in this study, and the time point when all treatment groups are most significantly larger than the control group. There are several possible explanations for the increase in food intake during this period. As foraging is continuous through the third instar, one possibility is that BPA-induced growth may allow for greater food consumption because of increased tissue size. Another possibility is that metabolic and/or digestive processes may be altered among treated versus non-treated control groups, leading to an increased amount of food observed in treated larvae at the 72 h time point. While our studies examined food intake during development, a recent study by Angle et al. (2013) showed that male mice
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offspring treated with BPA in utero displayed a statistically significant increase in food intake for a 2–3 week period shortly after weaning.
Future studies should offer valuable insight into the mechanisms of BPA toxicity through the interplay between endocrine signaling pathways in flies and humans.
4.2. BPA effects on precocious pupariation Acknowledgments The life cycle of Drosophila and other holometabolous insects includes a transition from an immature juvenile to a sexually mature adult. In Drosophila, a high titer of ecdysone, relative to the titers produced during the instar transformations, at the end of the third-instar larval stage initiates the onset of pupariation/metamorphosis (Thummel, 2001). The time required to initiate pupariation is strongly influenced by the insulin/insulin growth factor signaling (IIS), which affects both the growth rate and metamorphic timing through the control of synthesis and release of ecdysone (Rulifson et al., 2002; Shingleton et al., 2005). In the present study, we found that all test concentrations of BPA resulted in a greater number of animals entering pupariation at the 144 h time point than was found with the non-treated control group. These data are not consistent with a recent study by Atli and Ünlü (2012) where it was reported that the mean pupariation time was delayed several hours in Drosophila exposed to 0.1 mg/L, 1 mg/L or 10 mg/L concentrations of BPA. There were two important distinctions in experimental design between the Atli and Ünlü (2012) study and the present study. First, the method of BPA administration, via wetting papers, was indirect in the Atli and Ünlü study, and importantly, BPA was not administered until larvae reached the third instar larval stage. In the present study, BPA was administered directly beginning at the first instar and supplemented every 24 h until pupariation was reached. Our results indicate the effects of BPA are first apparent at 48 h of development. The timing and method of exposure may account for differences in the onset of pupariation due to BPA exposure in the two studies. Our findings were similar to those of Walkiewicz and Stern (2009) who found that increased insulin signaling in the PG of Drosophila advances the onset of metamorphosis. Walkiewicz and Stern (2009) proposed that under high growth rate conditions, larvae proceeded through a rapid development and early pupariation at the expense of forming excessively large pupae or adults, which may account for the attenuation of growth in the 0.1 mg/L treatment group, beginning at 96 h. Both agonists and antagonists of the IIS have been shown to affect downstream activation of the ecdysone signaling pathway. Synthesis and release of ecdysone is a necessary step in the transition from the larval to pupal state (Caldwell et al., 2005; Walkiewicz and Stern, 2009). A study by Planello et al. (2008) has reported a BPA-induced increase in the expression of Ecdysone receptor (EcR) in the insect C. riparius (Planello et al., 2008). While the potential effects of BPA on EcR expression are yet to be determined, a study by Zhou and Riddiford (2002) demonstrated that misexpression of the Z3 isoform of broad, a downstream target of EcR, in the second instar suppresses the third-instar molt, and after a feeding period, induced the onset of pupariation. In addition to potential indirect effects of BPA on premature ecdysone signaling through activation of the insulin/insulin growth factor signaling (IIS) pathway, the possibility exists that BPA could directly affect ecdysteroid signaling through interaction with EcR, as a weak interaction has been reported in cultured cells (Dinan et al., 2001). The action of BPA through IIS and EcR signaling in flies would be consistent with a growing number of studies that suggest that BPA acts through multiple endocrine pathways in vertebrates (reviewed in (Rubin, 2011)). Although the mechanism of BPA-mediated endocrine disruption in flies and other invertebrate species has yet to be fully explored, this study demonstrates that D. melanogaster can serve as an excellent model system for studying BPA-induced growth defects.
We would like to thank Dr. Alexander Mazo for critical comments to this manuscript. This work was supported by a research grant from the Stacy Anne Vitetta Professorship Award, Arcadia University.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Bisphenol A affects larval growth and advances the onset of metamorphosis in Drosophila melanogaster A.K. Weiner a, A. Ramirez a, T. Zintel a, R.W. Rose a, E. Wolff a, A.L. Parker a, K. Bennett a, K. Johndreau a, C. Rachfalski a, J. Zhou b, S.T. Smith a,n a b
Arcadia University, 450 South Easton Road, Glenside, PA 19038, USA Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming, Yunnan Province 650223, China
art ic l e i nf o
a b s t r a c t
Article history: Received 8 August 2013 Received in revised form 11 December 2013 Accepted 16 December 2013 Available online 7 January 2014
Exposure to Bisphenol A (BPA) has been reported to dysregulate endocrine pathways in a wide array of vertebrate species. The effects of BPA on invertebrate species are less well understood. We tested the effects of BPA on growth and development in Drosophila as these processes are governed by well-studied endocrine pathways. In this study, we tested the effects of three concentrations of BPA (0.1 mg/L, 1 mg/L or 10 mg/L) and found a statistically significant increase in larval growth for the low dose treatment group (0.1 mg/L), but not statistically significant for the high dose treatment group (10 mg/L). BPA exposure resulted in an increased body size in treated animals at 48, 72 and 96 h after egg laying (AEL). This finding reflects a non-monotonic dose–response that has been observed for an increasing number of endocrine disrupting compounds. The increase in growth rate found for all treatment groups was associated with a statistically significant increase in food intake observed at 72 h AEL. Furthermore, we observed that the increased growth rate was coupled with an earlier onset of pupariation consistent with previously reported phenotypes resulting from increased activity of insulin/insulin growth factor signaling (IIS) in Drosophila. Since the timing of the onset of pupariation in Drosophila is controlled through the complex interaction of the IIS and the ecdysone signaling pathways, our findings suggest that BPA exerts its effects through disruption of endocrine signaling in Drosophila. & 2013 Elsevier Inc. All rights reserved.
Keywords: Endocrine Non-monotonic dose–response curve Body size Toxicant Pupariation Development
1. Introduction Bisphenol A (BPA) is a high-production industrial monomer used world-wide in the manufacture of polycarbonate plastics and epoxy resins found in food packaging, thermal paper, dental sealants, medical devices, flame retardant materials and other products (reviewed in (Staples et al., 1998)). Human exposure to BPA through dietary and non-dietary sources has been well-documented (reviewed in (Geens et al., 2011)). The effects of BPA have been investigated in a diverse array of vertebrate species, where BPA has been reported to function as a potent endocrine disruptor at environmentally relevant levels, affecting reproductive processes, including gonadal function and sex determination (reviewed in (Crain et al., 2007)). BPA is classified as a xenoestrogen that is thought to exert its effects through direct binding to estrogen receptors (ER) in a wide range of species (Andersen et al., 1999). In vertebrates, both agonistic and antagonistic activities of BPA
n
Corresponding author. Fax: þ 1 215 881 8758. E-mail address: [email protected] (S.T. Smith).
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.12.008
have been reported and were dependent on ER isoform-specific interactions (Hiroi et al., 1999). The mechanism of BPA-induced reproductive toxicity in invertebrates is not as well understood as compared to vertebrate species. However, a growing number of invertebrate studies have reported reproductive abnormalities, including reduced fecundity in Daphnia magna and Ceriodaphnia dubia (Tsui et al., 2005), Drosophila melanogaster (Atli and Ünlü, 2012), Chironomus riparius (Watts et al., 2001) and the terrestrial isopod, Porcellio scaber (Lemos et al., 2010). BPA exposure was also found to induce germ cell production in the nematode, Caenorhabditis elegans (Hoshi et al., 2003), as well as increased egg production in the snail, Potamppyrgus antipodarum (Duft et al., 2003) and the copepod, Acartia tonsa (Andersen et al., 1999). Furthermore, BPA exposure resulted in the suppression of testes formation in Hydra oligactis (Fukuhori et al., 2005) and gonad resorption in the mussel, Mytilus edulis (Ortiz-Zarragoitia and Cajaraville, 2006). In addition to playing a central role in reproductive processes, endocrine signaling is critical to an organism0 s growth and development. In vertebrates, BPA treatment that exceeds environmentally relevant levels has been associated with teratogenic effects including abnormal growth, cephalic malformations, and vertebral defects (reviewed in (Crain et al., 2007)). Exposure to BPA in invertebrates
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has resulted in reduced growth rate in the freshwater sponge, Eunapius fragilis (Hill et al., 2002), delayed time of emergence in the housefly, Musca domestica (Izumi et al., 2008), delayed completion of the naupliar stages in the copepod, Tigriopus japonicus, (Marcial et al., 2003) and molting defects in the insect, C. riparius (Watts et al., 2001; Segner et al., 2003). The focus of this study was to assess the effects of BPA on growth, development and maturation in D. melanogaster, a holometabolous insect. In Drosophila, the larval stage is characterized by feeding and growth in which larvae proceed through a series of three instars. The duration of each instar is determined by the activity of the steroid hormone ecdysone and its active metabolite, 20-hydroxyecdysone (20E) (Thummel, 1996). In insects, final body size is controlled by a size-monitoring mechanism that dictates when growth should cease, and occurs during the period in larval development when growth rate is at its highest (Davidowitz et al., 2003). In Drosophila, the prothoracic gland (PG) is thought to act as the size assessment tissue that is responsive to activities of the highly conserved insulin/insulin-like growth factor signaling pathway (IIS) (Colombani et al., 2005; Mirth et al., 2005; Caldwell et al., 2005). The growth of the PG, in response to insulin signaling, therefore specifies when the critical weight has been attained and through the synthesis and release of ecdysteroids, the PG determines the timing of the onset of metamorphosis. Given the growing evidence that BPA exposure results in changes in growth and development in a number of invertebrate systems that share highly conserved pathways that regulate these processes, we hypothesized that BPA exposure in Drosophila would result in alterations in growth rate that would impact animal size and the timing of pupariation. To test this, we treated Drosophila larvae with increasing concentrations of BPA and used imaging software (ImageJ) to monitor growth rate and food intake at 48, 72, 96 and 120 h AEL. Because growth rate can affect the timing of onset of pupariation, we also examined the puparia of BPA-treated and untreated animals at 168 h to assess pupal stages. Furthermore, we conducted an experiment to assess potential differences in the onset of pupariation in BPA-treated and untreated animals.
2. Materials and methods 2.1. Drosophila stocks Experiments were carried out in Oregon-R-modENCODE (25211), obtained from the Bloomington Stock Center, Bloomington, IN. Oregon-R-modENCODE is a wild-type strain of D. melanogaster that is used by modENCODE contributors (http://www.modencode.org/publications/about/index.shtml).
2.2. BPA treatment Collection bottles with D. melanogaster flies were kept at 25 1C, in an incubator with 60–65% relative humidity, and reared on a 12 h light/12 h dark cycle. One-hour embryo collections were carried out on standard grape-agar plates (Genesee Scientific, San Diego, CA) supplemented with yeast paste. Embryos were allowed to hatch into first instar larvae and 50 animals were transferred to fresh plates prior to feeding. Three concentrations of BPA were introduced into larval food as follows: BPA (10 mg) was first dissolved in 1 mL acetone and then brought to 1 L with distilled water (10 mg/L BPA, 0.1% acetone). Ten fold dilutions (1 mg/L and 0.1 mg/L) were made, adjusting acetone concentration to 0.1%. The solvent control solution (hereafter referred to as “control”) contained 0.1% acetone in distilled water. Active, dry yeast (0.2 g) was added to 800 μL of each BPA solution and the control. To each of these formulations, 5 μL of food coloring (green) was added and mixed thoroughly. Food was dyed in order to monitor food consumption among control and treatment groups. Yeast preparations containing control and BPA-supplemented solutions were prepared freshly each day, and a 50 μL volume was applied to the grape-agar plate every 24 h beginning at first instar and continued until animals were collected for imaging analysis at the appropriate time point.
2.3. Growth assay Fifty newly-hatched first instar larvae were transferred to fresh agar plates (35 mm) and fed 50 μL of yeast-paste formulations containing either no BPA (control), 0.1 mg/L BPA, 1 mg/L BPA, or 10 mg/L BPA. Four sets of plates were prepared for each treatment group in order to image larvae at 48, 72, 96 and 120 h AEL. Freshly prepared yeast-paste formulations were administered once every twenty-four hour period or until larvae were collected for imaging analysis. Larvae ready for imaging analysis were collected into a wire mesh basket, washed with 1 Phosphate Buffered Saline (PBS) and placed at 20 1C for 3–5 min to decrease larval movement for imaging. Images were taken using a Leica FireCam V 3.2.0 (Leica Microsystems, Inc., Buffalo Grove, IL, USA) and the area of each individual larva was determined using ImageJ software (Schneider et al., 2012). Fig. 1f illustrates an example of larvae imaged at the 72 h time point. Pupal area was analyzed using ImageJ tools and assessment of pupal stages was conducted according to Bainbridge and Bownes (1981). 2.4. Analysis of food intake Larval images were converted to grayscale and a threshold value was set that corresponded to the darkest pixels where food was present in the gut. Selections of specific areas of the gut tube were made using the ImageJ wand tool and mean gray values for the selections were determined. The mean gray values for each control and treatment groups were averaged and compared for each time point: 48, 72, 96 and 120 h AEL. 2.5. Statistical analyses Statistical analyses were carried out using IBM SPSS Statistics for Windows, Version 19.0 (released 2010), IBM Corp., Armonk, NY. With the exception of the staging analysis, one factor Welsh ANOVA followed by Games-Howell post-hoc comparisons were used to compare control and treatment groups. The staging analysis was conducted using Kruskal Wallis analysis of variance by ranks followed by Bonferonni-adjusted Mann–Whitney post-hoc comparisons. Statistical data for larval/pupal area analyses can be found in Supplementary data Tables 1 and 2. Food intake was analyzed using a one-way ANOVA followed by a Tukey post-hoc analysis. The significance level for all statistical analyses was set at p o0.05.
3. Results 3.1. BPA treatment affects larval growth We tested the effects of BPA on the growth of Drosophila larvae through direct administration in food as described in Section 2. Fig. 1a–c shows that beginning at 48 h and continuing through 96 h, larvae exposed to the lowest concentration treatment (0.1 mg/L) were found to be significantly larger in mean area than the non-treated control group. At 72 h, the two lowest treatment groups (1 mg/L and 0.1 mg/L) were found to be significantly larger in area (Fig. 1b). At 120 h, there was no significant difference in area among the control and BPA-treated groups (Fig. 1d). We hypothesized that the decrease in area in the 0.1 mg/L treatment group observed from the 96 h to 120 h time point may reflect a shortening of the larval body in preparation for pupariation. Analysis of growth over time shows a significant increase in growth rate between 72 h and 96 h AEL, with the lowest concentration, 0.1 mg/L, showing the highest growth rate and the 1 mg/L and 10 mg/L treatment groups showing a similar growth rate to the control (Fig. 1e). Interestingly, the lowest concentration treatment group, 0.1 mg/L, showed an attenuation in growth from 96 h to 120 h, while the 1 mg/L, 10 mg/L and control groups continued to show an increase in growth rate during this interval. 3.2. Effects of BPA on food intake We observed an increase in larval body size in response to BPA treatment for all treatment groups at 48, 72, and 96 h AEL. However, only the low dose treatment (0.1 mg/L) group was found to be statistically greater in area. In order to determine whether increased food consumption accompanied the observed increase
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Fig. 1. BPA treatment affects larval growth. Larvae from control and treatment groups were collected at 48, 72, 96 and 120 h after egg laying (AEL), and larvae were imaged. The area of each individual larva was calculated using ImageJ and the mean larval area (mm2) was plotted against treatment concentration for the group. Means 7SEMs are indicated. p valueso 0.01 or o0.05 are indicated as nn and n, respectively. Larval area analysis was conducted on combined data from two independent experiments using a one-way Welsh ANOVA followed by Games-Howell post-hoc analysis. The significance level for all statistical analyses was set at 0.05. (a–d). All p value indicators represent comparison of treatment groups to the control group. Growth rate analysis of mean larval area for all groups plotted against time of development is shown (e). An example of ImageJ analysis for larvae collected at 72 h after egg laying (AEL) is shown. All larvae were imaged using the same magnification (as shown by the underlying grid). Larval images were traced in yellow using ImageJ tools and a mean larval area (mm2) was calculated for each of the groups (mean). Corresponding statistical data for Fig. 1 are found in Supplemental Table 1. The actual area of the larvae shown in Fig. 1f is indicated (actual) and approximates the mean for the group (f).
in body size, we employed ImageJ tools to approximate food intake. The food that was administered each 24 h period was dyed green and therefore visible in the gut of the larvae, whose bodies are opaque to translucent. In order to assess the approximation of food intake, we examined the images used to assess body size as described above in Sections 2, 2.3, and 3.1 (Fig. 2a). A threshold value was set in order to compare images between control and treatment groups for each time point AEL, and mean gray values were determined for each larva as described in Section 2.4 (Fig. 2b). The mean values within each control or treatment group were averaged and compared using a one-way ANOVA followed by a Tukey post-hoc analysis. BPA-treated larvae at the 72 h time point AEL were found to have selections from the gut tube that
were more optically dense than those from the control group (p o0.01 for all treatment groups) (Fig. 2c). 3.3. Effects of BPA on pupal size We conducted an analysis of pupal area at 168 h AEL and found that pupae from all treatment groups were similar in area to the control pupae (Fig. 3a), and was consistent with the observation that larvae from all treatment groups and the control group were similar in size at the 120 h time point. We hypothesized that the attenuation in growth rate observed between 96 and 120 h of development in the 0.1 mg/L BPA treatment group may be associated with an early onset of pupariation. In order to test this
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Fig. 2. BPA exposure is associated with an increased consumption of food. Larval images from the growth analysis described in Section 3.1 were further analyzed for potential differences in optical density in the gut tube as an approximation of food intake. Fig. 2a1–4 is a representative example of a larva from the 96 h time point. Larval images were first converted to grayscale (a1 and a2) and a threshold value was set that corresponded to the darkest pixels where food was present in the gut. Pixels falling below the thresholded value appear blue, while less optically dense pixels remain in grayscale (a3). For each larva the blue area was specifically selected using the ImageJ wand tool (a4) and the mean gray value for that selection was determined as shown in the histogram in (b). The mean gray values for each control and treatment groups were averaged and compared at each time point: 48, 72, 96 and 120 h AEL. A one-way ANOVA was performed followed by a Tukey post-hoc analysis. The significance level for all statistical analyses was set at 0.05. n Indicates a statistically significant p value (p o 0.01) comparing the BPA treated group with respect to the control group (c).
hypothesis, we examined the pupal images of the control and treatment groups at 168 h and estimated the pupal stage according to guidelines for staging metamorphosis in Drosophila, outlined by Bainbridge and Bownes (1981). Our analysis confirmed that pupae from the BPA treatment groups were advanced in metamorphosis compared to pupae from the control group, with the lowest treatment group (0.1 mg/L) showing a statistically significant advancement (Fig. 3b).
pupariation, indicated by the lowest concentration treatment group experiencing the highest larval growth rate (Fig. 1e), as well as, an observed peak pupariation 24 h before other treatment and control groups (Fig. 3c).
4. Discussion 4.1. BPA effects on growth and food intake
3.4. BPA treatment affects rate of entry into pupariation In order to confirm and extend the results of the observed pupariation staging (described above), we conducted an experiment to determine the number pre-pupae/pupae formed at 144, 168 and 192 h AEL following treatment with 0.1 mg/L, 1 mg/L and 10 mg/L BPA or no treatment (control), as outlined in Section 2.2. At 144 AEL, the lowest concentration treatment group (0.1 mg/L) entered into pupariation at the fastest rate (Fig. 3c), with 62% of this treatment group entering into pupariation compared to 34% of the control group at this time point. In addition, at 144 h AEL, 48% of the 1 mg/L treatment group and 43% of the 10 mg/L treatment groups had already entered pupariation. The peak pupariation time for the control group was not observed until 168 h AEL, with 56% of animals pupariated at this time point (Fig. 3c). These data reveal that larval growth rate is highly correlated with the rate of onset of
In this work we undertook a detailed analysis of the effects of a direct administration of BPA on growth and development in the invertebrate model organism, D. melanogaster. We found an increase in growth rate during larval development, most significantly in the lowest concentration treatment group (0.1 mg/L), with the highest growth rate observed between 72 and 96 h of development. The dose–response observed at the 48, 72, 96 and 120 h time points reflects a non-monotonic response curve that has been reported for a number of endocrine disrupting chemicals (Vandenberg, et al., 2012). A more comprehensive study conducted at the 72 h time point showed that 0.001 mg/L and 0.01 mg/L did not result in larvae that were statistically larger in area than control larvae (data not shown). While negative effects of BPA on growth rate have been reported for the invertebrate freshwater sponge, E. fragilis (Hill et al., 2002), a recent study in the housefly, M. domestica (Izumi et al., 2008),
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Fig. 3. BPA affects the rate of entry into metamorphosis. Pupal images were taken at 168 h AEL and analyzed using ImageJ tools. Means 7SEMs are indicated with error bars. The pupal area analysis was conducted using combined data from two independent experiments, each with three trials, using a one-way Welsh ANOVA followed by GamesHowell post-hoc analysis (a). Pupal images were examined and staged according to guidelines established by Bainbridge and Bownes (1981). Means 7SEMs are indicated with error bars. The staging analysis from combined data from three trials was conducted using Kruskal Wallis analysis of variance by ranks followed by Bonferonni-adjusted Mann–Whitney post-hoc comparisons. The significance level for all statistical analyses was set at 0.05. n indicates a statistically significant p value comparing BPA treated group and control group (b). The % of pupae was calculated by the number of new pupae observed (the total number of pupae observed on a given day minus pupae counted on the previous day(s))/total number of animals, at 144, 168 and 192 h AEL. The percentage of animals that had entered metamorphosis was plotted against sampling times (c). Corresponding statistical data for Fig. 3 are found in Supplemental Table 2.
found an increase in pupal weight in response to BPA treatment. Further, Michail et al., (2012) recently reported on the effects of BPA in the moth, Sesamia nonagrioides, and found an increase in weight in larvae exposed to a 100 mg/L dosage, the same as the lowest dosage used in our studies (0.1 mg/L) that produced a significant increase in growth. In Drosophila, larval size is affected, in part, by the growth rate, which is governed by the insulin/insulin growth factor signaling (IIS) pathway. During the feeding phase in Drosophila, insulin-like peptides (DILPs) are synthesized and released from insulinproducing cells (IPC) located in the Prothoracic Gland (PG) of the larval brain (Colombani et al., 2005); a size-assessing tissue that monitors its own growth (Mirth et al., 2005). Indeed, blocking the Protein Kinase A (PKA) pathway in IPCs has been associated with increased insulin signaling in the PG and an increased growth rate in Drosophila (Walkiewicz and Stern, 2009). A number of mammalian studies have also reported a link between BPA exposure, insulin signaling and body size. The Drosophila and mammalian insulin signaling pathways are evolutionarily conserved in function (Oldham et al., 2000; Garofalo, 2002) and proper functioning of the insulinproducing ß cells of the pancreas is dependent, in part, on endocrine (estrogen) signaling (Nadal et al., 1998). Importantly, knockout mice that do not express estrogen receptor-alpha (ER-α), one of two human estrogen-specific receptors, shows an increase in insulin resistance and adiposity (Heine et al., 2000). Similarly, insulin resistance in mice that were treated with the xenoestrogen BPA was shown to occur in an ER-dependent manner (Alonso-Magdalena
et al., 2006), and increased body weight has also been reported in rats exposed perinatally to BPA (Rubin et al., 2001). Finally, a growing number of human epidemiological studies have also suggested that BPA, acting as a xenoestrogen, may interfere with the body0 s natural mechanisms to control weight (Wang et al., 2012; Trasande et al., 2012). Our findings show that BPA can induce growth abnormalities during development that are consistent with those caused by dysregulation of the insulin/insulin growth factor signaling (IIS) pathway (Walkiewicz and Stern, 2009). In this study, we also approximated the amount of food intake by calculating the density of the material in the gut tube and found that there was a statistically significant increase in the amount material in the gut tube of treated versus non-treated larvae at the 72 h time point AEL. This time point also marks the beginning of the highest growth rate for the control and treatment groups in this study, and the time point when all treatment groups are most significantly larger than the control group. There are several possible explanations for the increase in food intake during this period. As foraging is continuous through the third instar, one possibility is that BPA-induced growth may allow for greater food consumption because of increased tissue size. Another possibility is that metabolic and/or digestive processes may be altered among treated versus non-treated control groups, leading to an increased amount of food observed in treated larvae at the 72 h time point. While our studies examined food intake during development, a recent study by Angle et al. (2013) showed that male mice
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offspring treated with BPA in utero displayed a statistically significant increase in food intake for a 2–3 week period shortly after weaning.
Future studies should offer valuable insight into the mechanisms of BPA toxicity through the interplay between endocrine signaling pathways in flies and humans.
4.2. BPA effects on precocious pupariation Acknowledgments The life cycle of Drosophila and other holometabolous insects includes a transition from an immature juvenile to a sexually mature adult. In Drosophila, a high titer of ecdysone, relative to the titers produced during the instar transformations, at the end of the third-instar larval stage initiates the onset of pupariation/metamorphosis (Thummel, 2001). The time required to initiate pupariation is strongly influenced by the insulin/insulin growth factor signaling (IIS), which affects both the growth rate and metamorphic timing through the control of synthesis and release of ecdysone (Rulifson et al., 2002; Shingleton et al., 2005). In the present study, we found that all test concentrations of BPA resulted in a greater number of animals entering pupariation at the 144 h time point than was found with the non-treated control group. These data are not consistent with a recent study by Atli and Ünlü (2012) where it was reported that the mean pupariation time was delayed several hours in Drosophila exposed to 0.1 mg/L, 1 mg/L or 10 mg/L concentrations of BPA. There were two important distinctions in experimental design between the Atli and Ünlü (2012) study and the present study. First, the method of BPA administration, via wetting papers, was indirect in the Atli and Ünlü study, and importantly, BPA was not administered until larvae reached the third instar larval stage. In the present study, BPA was administered directly beginning at the first instar and supplemented every 24 h until pupariation was reached. Our results indicate the effects of BPA are first apparent at 48 h of development. The timing and method of exposure may account for differences in the onset of pupariation due to BPA exposure in the two studies. Our findings were similar to those of Walkiewicz and Stern (2009) who found that increased insulin signaling in the PG of Drosophila advances the onset of metamorphosis. Walkiewicz and Stern (2009) proposed that under high growth rate conditions, larvae proceeded through a rapid development and early pupariation at the expense of forming excessively large pupae or adults, which may account for the attenuation of growth in the 0.1 mg/L treatment group, beginning at 96 h. Both agonists and antagonists of the IIS have been shown to affect downstream activation of the ecdysone signaling pathway. Synthesis and release of ecdysone is a necessary step in the transition from the larval to pupal state (Caldwell et al., 2005; Walkiewicz and Stern, 2009). A study by Planello et al. (2008) has reported a BPA-induced increase in the expression of Ecdysone receptor (EcR) in the insect C. riparius (Planello et al., 2008). While the potential effects of BPA on EcR expression are yet to be determined, a study by Zhou and Riddiford (2002) demonstrated that misexpression of the Z3 isoform of broad, a downstream target of EcR, in the second instar suppresses the third-instar molt, and after a feeding period, induced the onset of pupariation. In addition to potential indirect effects of BPA on premature ecdysone signaling through activation of the insulin/insulin growth factor signaling (IIS) pathway, the possibility exists that BPA could directly affect ecdysteroid signaling through interaction with EcR, as a weak interaction has been reported in cultured cells (Dinan et al., 2001). The action of BPA through IIS and EcR signaling in flies would be consistent with a growing number of studies that suggest that BPA acts through multiple endocrine pathways in vertebrates (reviewed in (Rubin, 2011)). Although the mechanism of BPA-mediated endocrine disruption in flies and other invertebrate species has yet to be fully explored, this study demonstrates that D. melanogaster can serve as an excellent model system for studying BPA-induced growth defects.
We would like to thank Dr. Alexander Mazo for critical comments to this manuscript. This work was supported by a research grant from the Stacy Anne Vitetta Professorship Award, Arcadia University.
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