Microbially Mediated O-Methylation of Bisphenol a Results in

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Microbially Mediated O-Methylation of Bisphenol a Results in Metabolites with Increased Toxicity to the Developing Zebrafish (Danio rerio) Embryo Jessica M. McCormick, Theo Van Es, Keith R. Cooper, Lori A. White, and Max M. H€aggblom* Department of Biochemistry and Microbiology, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, 76 Lipman Drive, New Brunswick, New Jersey 08901, United States ABSTRACT: Bisphenol A (BPA) is used in the manufacture of plastics, and has been identified in various environmental matrices, including human serum and breast milk. The prevalence of BPA in the environment and the potential exposure to humans underscores the need to more fully understand the fate of BPA in the environment and the resulting effects and toxicity to humans and other organisms. Here we demonstrate that Mycobacterium species, including Mycobacterium vanbaalenii strain PYR-1, are able to O-methylate BPA to its mono- and dimethyl ether derivatives (BPA MME and BPA DME, respectively). The O-methylation of BPA results in metabolites with increased toxicity as shown from differences in survival and occurrence of developmental lesions in developing zebrafish embryos exposed to BPA, BPA MME, and BPA DME. The mono- and dimethyl ether derivatives were more toxic than BPA, resulting in increased mortality at 5 (LC50 = 0.66 and 1.2 mg L
1) and 28 (LC50 = 0.38, 98% purity). Bisphenol A dimethyl ether (BPA DME, 4,40 isopropylidenediphenol dimethyl ether, CAS# 1568
83
8), purity >99% by GC-MS) was obtained from Acros Organics. To synthesize the O-methylated derivatives, bisphenol A was refluxed overnight in an excess of methyl iodide and potassium carbonate buffer.24 The resulting mixture was approximately 50% BPA MME (4-hydroxy-40 -methoxy-2,2-diphenylpropane) and 50% BPA DME (as analyzed by gas chromatography-mass spectrometry, GC-MS), with a trace remaining amount of BPA (data not shown). The reaction product was then evaporated to dryness and dissolved in benzene. BPA MME and BPA DME were purified in a silica gel column by first eluting BPA DME with 100% benzene, followed by 5
10% acetone in benzene to elute BPA MME. Fractions were collected, evaporated to dryness and screened by thin layer chromatography for purity. The BPA DME fractions were discarded and the BPA MME-containing fractions were combined and analyzed by GC-MS to confirm purity (>99%). Bacterial Strains and Growth Medium. Mycobacterium vanbaalenii strain PYR-125 was obtained from Dr. Carl Cerniglia (National Center for Toxicological Research, U.S. Food and Drug Administration) and was grown in Brain Heart Infusion (BHI) broth at 28 °C with shaking at 60 rpm. Mycobacterium chlorophenolicum strain PCP-126 and Mycobacterium fortuitum CG-227 were grown in R2A broth at 28 °C with shaking at 60 rpm. When isolated colonies were needed, cultures were grown on R2A (Difco) agar plates and incubated at 28 °C. Mycobacterium smegmatis strain mc2155, obtained from Dr. Nancy Connell (University of Medicine and Dentistry of New

Jersey - New Jersey Medical School), was grown in Middlebrook 7H9 (Difco) broth supplemented with albumin, dextrose and sodium chloride with shaking at 100 rpm at 37 °C. Zebrafish Strains and Husbandry. The AB strain of zebrafish (Danio rerio) was used for all experiments, and was obtained from the Zebrafish International Resource Center (ZIRC). Zebrafish, aged 3 months to 1 year, were bred and maintained in a recirculating Aquatic Habitat System at 28 °C utilizing a light: dark cycle of 14:10 h, respectively. All experiments were conducted according to a protocol approved by the Rutgers University Animal Care and Facilities Committee. O-Methylation of bisphenol A by Mycobacterium species. M. vanbaalenii PYR-1, M. fortuitum CG-2, M. chlorophenolicum PCP-1 and M. smegmatis 155 were inoculated into 5 mL of brain heart infusion broth (BHI) and grown at 28 °C with aeration for 48 h. Once the cultures reached an OD680 of 0.6 they were diluted 1:10 and grown to an OD680 of 1.0 at 28 °C with aeration. Experiments were prepared in triplicate, with one mL of culture inoculated into a 7 mL sterile glass vial with Teflon cap liners, and spiked with 100 μM of BPA. To test whether O-methylation was inducible or constitutively expressed, cultures pregrown on BHI were incubated with and without 100 μL of 1 mg mL
1 of chloramphenicol to inhibit protein synthesis. Triplicate cultures were incubated at room temperature with shaking. At 0, 1, 3, 6, 8, 9, and 14 days, one vial per condition was stored at
20 °C for further processing. The samples were thawed and then acidifed with hydrochloric acid and 20 μM 2,4,6-tribromophenol was added as an internal standard followed by extraction with 1 mL hexane for one hour. The hexane phase was removed and analyzed by GC-MS. Analytical Methods. GC-MS was conducted using an Agilent Series 6890 gas chromatograph with a 5973N mass selective detector equipped with a splitless injector, and a HP-5MS 6568

dx.doi.org/10.1021/es200588w |Environ. Sci. Technol. 2011, 45, 6567–6574

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Figure 2. Mass spectra of BPA, and its two metabolites, BPA MME and BPA DME. The m/z of 228, 242, and 256 correspond to the respective molecular ions. All spectra show the typical fragmentation of M-15.

column (30 m length  0.25 mm diameter  0.25 μm i.d.). The injector temperature was 250 °C with an injection volume of 1 μL, the oven temperature program started at 70 °C, ramped by 15 °C min
1 to 300 °C, and held for 5 min. The mass selective detector was operated in full scan mode (32 to 600 m/z) for identification of metabolites. Selected Ion Monitoring (SIM) was used to detect and quantify BPA (213 and 228 m z
1), BPA MME (227 and 241 m z
1) and BPA DME (241 and 256 m z
1). Collection and Treatment of Zebrafish Embryos. Zebrafish embryos were collected at approximately 3 h post fertilization (hpf) and were exposed in 4 mL glass vials to BPA (1, 2.5, 5, 10, and 20 mg L
1), BPA MME (0.25, 0.5, 1, 2.5, and 5 mg L
1), BPA DME (0.5, 1, 2.5, 5, and 10 mg L
1) or dimethyl sulfoxide (0.15%),28 as the vehicle control, in sterile water. A nonvehicle control was included to confirm that the dimethyl sulfoxide concentration used had no effect on zebrafish development. A static, nonrenewal approach was used. Experimental doses were determined after preliminary dose response studies to obtain the optimal doses for exposure, and concentrations were similar to those used in previous zebrafish studies.29
32 One embryo was placed into each vial and observed every 24 h for 7 days. Studies were conducted with 25 embryos per dose and all studies were repeated at least three times. Developmental lesions, including edema and hemorrhage, as well as death and date of hatching, were recorded daily. After 7 days, larvae were removed from vials and placed into a beaker containing water from the zebrafish aquatic system and boiled wheat seeds. They were fed paramecium culture and AP100 (Aquatic Habitats) for the next 21 days. At 28 days post fertilization (dpf) all remaining live larvae were counted and euthanized using an overdose of Tricaine Methane Sulfonate (MS-222, Sigma). The LC50, which is the dose required to cause mortality in 50% of the test population, and the standard error were determined using the probit method as described.33 Statistical Analyses. Sigma Stat Version 10.1 was used to determine the normality of the data, the power and the appropriate statistical test. The Mann
Whitney Rank Sum test was used to examine the hatching data. Chi square analysis was used for the lesion and mortality data. All data referred to as significant are p e 0.05.

’ RESULTS O-Methylation of Bisphenol A. To determine whether microorganisms could O-methylate BPA, we utilized Mycobacterium

Table 1. Comparison of BPA O-Methylation by Mycobacterium Speciesa BPA MME (%) species and strain

1 week

2 weeks

BPA DME (%) 1 week

2 weeks

M. vanbaalenii PYR1

7.5

8

0.4

0.4

M. chlorophenolicum PCP1

4.4

12

0.2

0.5

M. fortuitum CG2

0.46

0.91

0

0

M. smegmatis 155

1.1

1.2

0

0

a Late log phase cultures were incubated at 28 °C in the presence of BPA. Samples were taken at one and two weeks, acidified and extracted with hexane for analysis by GC-MS for the presence of the mono- and dimethyl BPA metabolites. Metabolite concentrations are % of BPA substrate converted.

strains known to O-methylate other hydroxylated aromatic compounds, such as polyaromatic hydrocarbons, 2,4,6-tribromophenol and tetrabromobisphenol A (TBBPA).34 M. vanbaalenii PYR1 cultures incubated with 100 μM BPA produced two new metabolites (Figure 1) identified by their mass spectra as monomethyl ether (BPA MME) and dimethyl ether (BPA DME) (Figure 2). To monitor the rate and extent of BPA O-methylation, late log phase (OD = 1.0) cultures of Mycobacterium strains PYR-1, PCP-1, CG-2, and 155 were exposed to 100 μM BPA and analyzed for the production of the monomethyl and dimethyl ether metabolites. All Mycobacterium strains tested were able to O-methylate BPA, albeit at different rates and to different extent (Table 1). M. vanbaalenii PYR1 and M. chlorophenolicum PCP1 converted approximately 10% of BPA to BPA MME and less than 1% to BPA DME over the two week incubation period (Table 1). To test whether O-methylation was an inducible or constitutively expressed transformation, cultures of M. vanbaalenii PYR-1 pregrown on BHI (without prior exposure to BPA) were spiked with BPA, with or without chloramphenicol to inhibit protein synthesis. The rate of BPA transformation in the cultures with or without chloramphenicol was near equal, resulting in 5
7% transformation of BPA to BPA MME and BPA DME after 9 days (Figure 3). These data indicate that the O-methyltransferase responsible for the transformation of BPA to the BPA MME and BPA DME is constitutively expressed in M. vanbaalenii PYR-1. Relative Toxicity of BPA, BPA MME, and BPA DME to the Developing Zebrafish Embryo. To evaluate the relative toxicity of BPA and its biotransformation products, developing zebrafish 6569

dx.doi.org/10.1021/es200588w |Environ. Sci. Technol. 2011, 45, 6567–6574

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embryos were exposed to BPA, BPA MME, and BPA DME and examined for differences in survival and developmental lesions. The LC50 values for BPA, BPA MME, and BPA DME exposure were determined by the percent mortality after 5 and 28 dpf in order to rank these compounds in order of increasing toxicity. These time points were chosen because they represent the embryonic (short-term survival) and larval (long-term survival) periods.35 At 5 dpf, the BPA MME was the most potent, followed by BPA DME and by BPA (Table 2). In contrast, at 28 dpf, BPA MME and BPA DME had similar LC50 values, but we consider BPA DMA to be more toxic to juveniles because the percent mortality of juveniles at 28 dpf was higher for those exposed to BPA DME. Thus, BPA-DME was more potent over the juvenile period of zebrafish development, while BPA MME was more toxic than BPA in embryonic zebrafish (Table 2). The lowest observable adverse effect level (LOAEL, the dose needed to detect significant developmental malformations compared to control) was used to rank the toxicity of the compounds as well as to indicate levels of exposure at which development is affected. The LOAEL was lower in BPA MME and BPA DME

than in BPA exposed animals (Table 2). Again, these data illustrate that the O-methylated metabolites of BPA are more toxic than BPA. Zebrafish Mortality Following Exposure to BPA, BPA MME, or BPA DME. BPA DME exposure was more acutely toxic than BPA, resulting in 100% mortality in embryos exposed to 2.5, 5, or 10 mg L
1 by 4 dpf (Figure 4C). BPA MME was also more toxic than BPA, resulting in 100% mortality of 5 mg L
1 exposed embryos by 2 dpf, and the 2.5 mg L
1 dose by 5 dpf (Figure 4B). BPA exposure resulted in 100% mortality at the 20 mg L
1 dose by day 2 and by day 5 for the 10 mg L
1 dose, but these doses are higher in comparison to the doses used for BPA MME and BPA DME (Figure 4A). Additionally, BPA exposure at 5 mg L
1 resulted in significant mortality (60%) by 5 dpf as compared to control (Figure 4A). The impact of BPA, BPA MME, or BPA DME exposure on juvenile mortality was assessed. At 28 dpf, BPA DME exposure resulted in increased mortality, relative to control, at both the 1 and 0.5 mg L
1 doses with survival at 12 and 24%, respectively (Figure 4C). BPA MME exposure also resulted in significant increase in mortality in the 1, 0.5, and 0.25 mg L
1 doses as compared to control, resulting in embryo survival of 24, 52, and 60%, respectively (Figure 4B). BPA exposure resulted in an increase in juvenile mortality for the 5 and 2.5 mg L
1 doses as compared to control, but not at the 1 mg L
1dose (Figure 4A). These data show that BPA MME and BPA DME were more toxic to zebrafish embryos and juveniles than BPA. Lesion Occurrence in Exposed Embryos. Exposure to BPA, BPA MME, and BPA DME resulted in developmental lesions in the zebrafish embryo. BPA exposure resulted in significant pericardial (PC) and yolk sac (YS) edema, hemorrhage and curved tail at 10 mg L
1, and PC edema at 5 mg L
1 (Table 3). Lesions detected at other doses of BPA exposure did not differ from control. BPA MME exposure (0.5, 1, and 2.5 mg L
1) resulted in significant PC edema, with the 1 and 2.5 mg L
1 doses also exhibiting significant YS edema and hemorrhage (Table 3). BPA MME (1 mg L
1) exposed embryos had an increase in curved tails as compared to control embryos. BPA DME exposed embryos exhibited significant PC and YS edema as compared to control embryos. Additionally, BPA DME exposed embryos had an increase in hemorrhage. Curved tails were significant in BPA DME exposed embryos at 1 and 2.5 mg L
1. These data show that exposure to O-methylated metabolites results in developmental lesions at a lower dose than BPA exposure. Hatching Success Following Exposure to BPA, BPA MME, or BPA DME. The time to hatch was measured in embryos exposed to BPA, BPA MME or BPA DME and compared to control embryos. BPA exposed embryos at 1, 2.5, and 5 mg L
1

Figure 3. O-methylation of BPA by M. vanbaalenii PYR1. M. vanbaalenii strain PYR-1 was incubated at 28 °C with 100 μM BPA alone and with 1 mg mL
1 chloramphenicol to inhibit protein synthesis. Samples were taken over 9 nine days, extracted and analyzed by GC-MS for detection of the monomethyl and dimethyl ether metabolites of BPA. Experiments were performed in triplicate and repeated three times on separate days.

Table 2. Short Term Vs. Long Term LC50 Values and Lowest Observed Adverse Effect Levelsa 5 dpf

28 dpf

BPA

MME

DME

BPA

MME

DME

LC50

5 mg/L

0.66 mg/L

1.2 mg/L

1.8 mg/L

0.38 mg/L