Supplemental Information. Adverse Reproductive and Developmental Health Outcomes Following Prenatal

Transcription

1 Supplemental Information Adverse Reproductive and Developmental Health Outcomes Following Prenatal Exposure to a Hydraulic Fracturing Chemical Mixture in Female C57Bl/6 Mice Christopher D. Kassotis 1, John J. Bromfield 2,3, Kara C. Klemp 4, Chun-Xia Meng 4, Andrew Wolfe 5, R. Thomas Zoeller 6, Victoria D. Balise 4,7, Chiamaka J. Isiguzo 4, Donald E. Tillitt 8, and Susan C. Nagel 4,7 1 Nicholas School of the Environment, Duke University, Durham, NC Department of Animal Sciences, University of Florida, Gainesville, FL D. H. Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, FL Department of Obstetrics, Gynecology and Women s Health, University of Missouri, Columbia, MO Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD Department of Biology, University of Massachusetts Amherst, Amherst, MA Division of Biological Sciences, University of Missouri, Columbia, MO U.S. Geological Survey, Columbia Environmental Research Center, 4200 New Haven Road, Columbia, MO

2 Supplemental Information: Materials and Methods Heart Assessments. Whole hearts from PND21 and 85 animals were excised from one female mouse per litter and were fixed as described above. Hearts were paraffinembedded and two 5 µm midsagittal sections cut and placed on slides. For collagen assessments, one slide per heart was dewaxed and rehydrated, then stained with Picro Sirius Red (Sigma #365548) dissolved in picric acid (Sigma #P6744) at room temperature for one hour. Slides were rinsed twice in 0.5% acetic acid in distilled water and dried in a 65 C oven for 15 min, then cleared in xylene with two one-minute rinses. Slides were cover slipped and collagen was visualized as red on a pale yellow background using bright-field microscopy. A tiled image of the whole heart was created using a Zeiss Axiovert 200M microscope and MetaMorph software. A thresholding cutoff method for acceptable staining intensity was applied to assess the areas of heart and collagen using Image J. The collagen deposition was then calculated by taking the percentage of collagen area divided by the heart area. For cardiac myocyte immunostaining, one slide per heart was heated in 10mM sodium citrate buffer with 0.05% Tween 20 in a pressure cooker on full power for 3 minutes for antigen retrieval, then briefly rinsed in 1X TBS with 0.025% Triton 100 and 1% BSA. After incubation with blocking solution consisting of 0.1% cold water fish skin gelatin (Electron Microscopy Sciences, #25560), 0.1% Tween 20, and 1% BSA for 1 hour at room temperature, the sections were incubated overnight with two primary antibodies of mouse anti-troponin I visualizing cardiac myofilaments (EMD Millipore, #MAB1691, 1 µg/ml) and Alexa Fluor 594-conjugated wheat germ agglutinin visualizing cell 2

3 membranes (Invitrogen, W11262, 1 µg/ml) diluted in TBS/Triton/BSA in a humidified chamber at 4 C. The following day the sections were incubated with Alexa Fluor 488- conjugated donkey anti-mouse secondary antibody (Invitrogen, A-21202, 20 µg/ml) and DAPI DNA dye (Invitrogen, D3571, 300 nm) for 2 hours at room temperature. After rinse, the sections were cover-slipped and three images of each left ventricle at 200X were taken with an Olympus IX70 fluorescence microscope for troponin I (green), wheat germ agglutinin (red), and cell nuclei (blue). Thirty longitudinal cardiac myocytes were randomly selected and diameters across the middle of the myofiber nuclei were measured and averaged using Image J. One-way ANOVA using SPSS was used to assess significance. Thyroid-Regulated Liver Gene Expression. Whole livers from PND21 were excised from each mouse and a section of one lobe frozen with liquid nitrogen and transported to the University of Massachusetts Amherst on dry ice for gene expression analysis. Received liver samples were randomized and RNA was isolated and integrity assessed as previously described (1). Thyroid hormone responsive (Thrsp) and Malic enzyme expression, genes positively-regulated by thyroid hormone, were measured by Q-PCR and normalized to Beta-actin expression, as described previously (Supplemental Figure 4; (1). References 1. Giera S, Bansal R, Ortiz-Toro TM, Taub DG, & Zoeller RT (2011) Individual polychlorinated biphenyl (PCB) congeners produce tissue- and gene-specific effects on thyroid hormone signaling during development. Endocrinology 152(7):

4 Supplemental Table 1. Oil and Gas Operation Chemicals Used in Current Study and Their Antagonist Receptor Activities. List of the 23 oil and gas operation chemicals tested in this study and information on their uses in and outside of industry. X s denote significant receptor antagonism from in vitro reporter gene testing, published previously. ER = estrogen receptor; AR = androgen receptor; PR = progesterone receptor; TR = thyroid receptor; GR = glucocorticoid receptor. Table adapted with permission from Kassotis et al. 2015: Endocrine Disrupting Activities of Hydraulic Fracturing Chemicals and Adverse Health Outcomes After Prenatal Exposure in Male Mice. Endocrinology, 124(3); Supplemental Table 2. PND21 Organ Weight Data following Developmental Exposure to Oil and Gas Operation Chemicals. Estimated marginal means ± SEM for body and organ weights of developmentally exposed female mice at PND21. * p 0.05; # 0.05 < p 0.10 Treatments listed are as follows: VEH = 0.2% ethanol in water, MIX3/30/300/3000 = 3/30/300/3000 μg/kg/day of each of 23 chemicals listed in Table 1 (23-mix), and FLUT = flutamide at 50 mg/kg/day. N = 8, 9, 7, 8, 5, 10 litters for vehicle, Mix3, 30, 300, 3000, and flutamide, respectively. Supplemental Table 3. PND85 Organ Weight Data following Developmental Exposure to Oil and Gas Operation Chemicals. Estimated marginal means ± SEM for body and organ weights of developmentally exposed female mice at PND85. * p 0.05; # 0.05 < p 0.10 Treatments listed are as follows: VEH = 0.2% ethanol in water, MIX3/30/300/3000 = 3/30/300/3000 μg/kg/day of each of 23 chemicals listed in Table 1 (23-mix), and FLUT = flutamide at 50 mg/kg/day. N = 9, 7, 6, 8, 5, 8 litters for vehicle, Mix3, 30, 300, 3000, and flutamide, respectively. Supplemental Table 4. Litter Data following Developmental Exposure to Oil and Gas Operation Chemicals in Drinking Water. Estimated marginal means ± SEM for organ weights and pubertal metrics of developmentally exposed female mice. Data on litter size, sex ratio, and lost litters were published previously. * p 0.05; # 0.05 < p 0.10 Treatments listed are as follows: VEH = 0.2% ethanol in water, MIX3/30/300/3000 = 3/30/300/3000 μg/kg/day of each of 23 chemicals listed in Table 1 (23-mix), and FLUT = flutamide at 50 mg/kg/day. 4

5 N =11, 10, 10, 11, 10, 10 litters for vehicle, Mix3, 30, 300, 3000, and flutamide, respectively. Table adapted with permission from Kassotis et al. 2015: Endocrine Disrupting Activities of Hydraulic Fracturing Chemicals and Adverse Health Outcomes After Prenatal Exposure in Male Mice. Endocrinology, 124(3); Supplemental Figure 1: Mouse Exposure and Collection Timeline. Timeline of oil and gas operation chemical exposure to pregnant dams and assessment of developmentally exposed pups, published previously. Colored bars represent ongoing treatment or measurements and black arrows represent set collection or measurement points. GD = gestational day; PND = postnatal day Table adapted with permission from Kassotis et al. 2015: Endocrine Disrupting Activities of Hydraulic Fracturing Chemicals and Adverse Health Outcomes After Prenatal Exposure in Male Mice. Endocrinology, 124(3); Supplemental Figure 2: Developmental exposure to oil and gas chemicals does not alter thyroid-regulated gene expression in the liver. Estimated marginal means ± SEM of relative fold induction for thyroid regulated mrna expression in PND21 liver samples from developmentally exposed male mice; (A) Thyroid hormone responsive (Thrsp) mrna expression, and (B) Malic enzyme mrna expression. Treatments listed are as follows: VEH = 0.2% ethanol in water, MIX3/30/300/3000 = 3/30/300/3000 μg/kg/day of each of 23 chemicals listed in Table 1 (23-mix), and FLUT = flutamide at 50 mg/kg/day. Supplemental Figure 3: Developmental exposure to oil and gas chemicals does not alter cardiac myocyte sizes during development. Representative images of cardiac myocytes in hearts collected at PND21 and PND85 from female mice prenatally exposed to vehicle (A, G), Mix3 (B, H), Mix30 (C, I), Mix300 (D, J), Mix3000 (E, K), and flutamide (F,L) treatments. Staining consists of troponin I visualizing cardiac myofilaments (green), wheat germ agglutinin visualizing cell membranes (red), and DAPI DNA dye (blue). Scale bar: 50 µm; magnification: 200X for all samples. Supplemental Figure 4: Developmental exposure to oil and gas chemicals alters collagen deposition in the heart during adulthood. Representative images of collagen deposition in hearts collected at PND21 and PND85 from female mice prenatally exposed to vehicle (A, G), Mix3 (B, H), Mix30 (C, I), Mix300 (D, J), Mix3000 (E, K), and flutamide (F,L) treatments. Each image was extracted from the respective tiled scan of the whole heart using a Zeiss Axiovert 200M microscope with motorized stages. The collagen was visualized red on a pale yellow background. Scale bar: 200 µm; magnification: 100X for all samples. 5

6

7

8

9

10 Supplemental Table 1. Oil and Gas Operation Chemicals Used in Current Study and Their Antagonist Receptor Activities. Chemical Name CAS # Oil and Gas Operation Use Common Use Naturally occurring ER Ant AR Ant PR Ant TR Ant GR Ant 1,2,4- trimethylbenzene Surfactant Dyes, resins, perfumes, gasoline constituent X 2-(2- methoxyethoxy) ethanol Biocide, Surfactant Industrial solvent, icing inhibitor 2-ethylhexanol Defoamer, Breaker Plasticizer precursor, sunscreens X 2-methyl-4- isothiazolin-3-one Biocide Personal care products, preservative Acrylamide Scale Control, Friction Reducer Water treatment, personal care products X Benzene Paraffin Inhibitor, Surfactant Chemical precursor, resins, rubbers, plastics Bronopol Biocide Personal care products, antimicrobials X Cumene Paraffin Inhibitor Chemical precursor, paints X

11 Diethanolamine Friction Reducer, Corrosion Inhibitor Personal care products, chemical precursor Ethoxylated nonylphenol Surfactant, Corrosion Inhibitor Detergents, dust control, icing inhibitor X X Ethoxylated octylphenol Surfactant, Corrosion Inhibitor Cleaning agents, paints, coatings X X Ethylbenzene Non-emulsifier, paraffin inhibitor Styrene precursor, gasoline Ethylene glycol Crosslinker, Friction reducer Antifreeze, coolant, polyester fiber X X Ethylene glycol monobutyl ether Surfactant, Foamer Corexit 9527, personal care products Naphthalene Surfactant, Acid Inhibitor Mothballs, fumigant, chemical precursor N,ndimethylformamid e Corrosion Inhibitor Plastics, pesticides, adhesives Phenol Resin-coating for proppants Plasticizer precursor, resins X

12 Propylene glycol Gellant, Breaker Personal care products, resins X Sodium tetraborate decahydrate Crosslinker Borax, personal care products, detergents Styrene Proppant Plastics, rubber, insulation X Toluene Non-emulsifier, paraffin inhibitor Chemical precursor, paints Triethylene glycol Biocide, Dehydration Plasticizer precursor, air sanitizers, disinfectants X X Xylenes Non-emulsifier, Breaker Plasticizer precursor, polyester production X List of the 23 oil and gas operation chemicals tested in this study and information on their uses in and outside of industry. X s denote significant receptor antagonism from in vitro reporter gene testing, published previously. ER = estrogen receptor; AR = androgen receptor; PR = progesterone receptor; TR = thyroid receptor; GR = glucocorticoid receptor. Table adapted with permission from Kassotis et al. 2015: Endocrine Disrupting Activities of Hydraulic Fracturing Chemicals and Adverse Health Outcomes After Prenatal Exposure in Male Mice. Endocrinology, 124(3);

13 Supplemental Table 2. PND21 Organ Weight Data following Developmental Exposure to Oil and Gas Operation Chemicals. Treatment Body Weight (g) Thymus Heart Uterus Ovaries (paired; mg) Liver Left Kidney Spleen VEH 7.8 ± ± ± ± ± ± ± ± 3.6 MIX3 8.5 ± 0.3 * 45.7 ± ± 1.2 # 5.3 ± ± ± ± ± 5.0 MIX ± ± ± ± ± ± ± ± 2.9 MIX ± 0.2 * 47.5 ± ± 2.1 # 5.1 ± ± ± ± ± 2.9 # MIX ± ± ± ± ± ± ± ± 4.3 FLUT 8.0 ± ± ± ± ± ± ± ± 4.3 # Estimated marginal means ± SEM for body and organ weights of developmentally exposed female mice at PND21. * p 0.05; # 0.05 < p 0.10 Treatments listed are as follows: VEH = 0.2% ethanol in water, MIX3/30/300/3000 = 3/30/300/3000 μg/kg/day of each of 23 chemicals listed in Table 1 (23-mix), and FLUT = flutamide at 50 mg/kg/day. N = 8, 9, 7, 8, 5, 10 litters for vehicle, Mix3, 30, 300, 3000, and flutamide, respectively.

14 Supplemental Table 3. PND85 Organ Weight Data following Developmental Exposure to Oil and Gas Operation Chemicals. Treatment Body Weight (g) VEH 18.9 ± 0.6 MIX ± 0.5 MIX ± 0.4 MIX ± 0.5 MIX ± 0.5 FLUT 18.6 ± 0.3 Thymus 46.4 ± ± ± ± ± ± 2.4 Heart 85.6 ± ± ± ± ± ± 1.9 Gonadal Fat ± ± ± ± ± ± 8.7 Uterus Ovaries (paired; mg) 64.5 ± ± ± 6.1 ± * # 67.6 ± ± ± 4.4 ± # 68.9 ± 5.0 # 5.0 ± ± 4.3 * 5.1 ± 0.3 Liver Left Kidney ± ± ± ± ± ± ± ± ± ± ± ± 5.1 Spleen 65.5 ± ± ± ± ± ± 2.6 Renal Fat 43.2 ± ± ± ± ± ± 5.6 Estimated marginal means ± SEM for body and organ weights of developmentally exposed female mice at PND85. * p 0.05; # 0.05 < p 0.10 Treatments listed are as follows: VEH = 0.2% ethanol in water, MIX3/30/300/3000 = 3/30/300/3000 μg/kg/day of each of 23 chemicals listed in Table 1 (23-mix), and FLUT = flutamide at 50 mg/kg/day. N = 9, 7, 6, 8, 5, 8 litters for vehicle, Mix3, 30, 300, 3000, and flutamide, respectively.

15 Supplemental Table 4. Litter Data following Developmental Exposure to Oil and Gas Operation Chemicals in Drinking Water. Treatment Litters (n) Litter Size (n) Sex Ratio M/total Anogenital Distance (mm) Age of VO (d) Age of 1st Estrus (d) VO to 1E Interval (d) % Lost Litters VEH ± ± ± ± ± ± 1.6 0% MIX ± ± ± ± ± ± % MIX ± ± ± ± ± ± % MIX ± ± ± ± ± ± 1.1 0% MIX ± ± ± ± ± ± % FLUT ± ± ± ± ± ± 1.5 0% Estimated marginal means ± SEM for organ weights and pubertal metrics of developmentally exposed female mice. Data on litter size, sex ratio, and lost litters were published previously. * p 0.05; # 0.05 < p 0.10 Treatments listed are as follows: VEH = 0.2% ethanol in water, MIX3/30/300/3000 = 3/30/300/3000 μg/kg/day of each of 23 chemicals listed in Table 1 (23-mix), and FLUT = flutamide at 50 mg/kg/day. N =11, 10, 10, 11, 10, 10 litters for vehicle, Mix3, 30, 300, 3000, and flutamide, respectively. Table adapted with permission from Kassotis et al. 2015: Endocrine Disrupting Activities of Hydraulic Fracturing Chemicals and Adverse Health Outcomes After Prenatal Exposure in Male Mice. Endocrinology, 124(3);