Influence of the Gastrointestinal Environment on the Bioavailability of

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Influence of the gastrointestinal environment on the bioavailability of ethinyl estradiol sorbed to single-walled carbon nanotubes Joseph H. Bisesi, Sarah E. Robinson, Candice M. Lavelle, Thuy N. Ngo, Blake Castillo, Hayleigh M. Crosby, Keira Liu, Dipesh Das, Jaime G. Plazas-Tuttle, Navid B. Saleh, Patrick Lee Ferguson, Nancy D. Denslow, and Tara Sabo-Attwood Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 15 Dec 2016 Downloaded from http://pubs.acs.org on December 15, 2016

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Environmental Science & Technology

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Influence of the gastrointestinal environment on

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the bioavailability of ethinyl estradiol sorbed to

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single-walled carbon nanotubes

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Joseph H. Bisesi Jr †‡, Sarah E. Robinson †‡, Candice M. Lavelle †‡, Thuy Ngo †‡,

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Blake Castillo †‡, Hayleigh Crosby†‡, Keira Liu §#∥, Dipesh Das¶, Jamie G. Plazas-

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Tuttle¶, Navid Saleh ¶, P. Lee Ferguson §#∥, Nancy D. Denslow ∇‡, Tara Sabo-Attwood

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†‡*

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† Department of Environmental and Global Health, University of Florida, 101 S. Newell

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Dr., Box 100188, Gainesville, FL 32610, United States. ‡ Center for Environmental and

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Human Toxicology, University of Florida, 2187 Mowry Road, Box 110885, Gainesville,

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FL 32611, United States. § Nicholas School of the Environment, Duke University, Box

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90328, Durham, NC 27708, United States. # Department of Civil and Environmental

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Engineering, Duke University, 121 Hudson Hall, Box 90287, Durham, NC 27708, United

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States. ∥ Center for the Environmental Implications of Nanotechnologies (CEINT), Duke

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University, 121 Hudson Hall, Box 90287, Durham, NC 27708, United States. ¶

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Department of Civil, Architectural, and Environmental Engineering, University of Texas

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at Austin, 301 E. Dean Keeton St., Austin, Texas 78712, United States. ∇ Department of

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Physiological Sciences, 2187 Mowry Road, Box 110885, Gainesville, FL 32611, United

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States. 1 ACS Paragon Plus Environment


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Corresponding Author

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*Tara Sabo-Attwood, PhD Department of Environmental and Global Health Center for Environmental and Human Toxicology University of Florida Box 110885 2187 Mowry Road Gainesville, FL 32611 Email: [email protected] Phone: (352)294-5293

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ABSTRACT

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Recent evidence suggests that if single-walled carbon nanotubes (SWCNTs) make their

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way into aquatic environments, they may reduce the toxicity of other waterborne

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contaminants due to their sorptive nature. However, few studies have examined

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whether contaminants remain adsorbed following ingestion by aquatic organisms. The

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objective of this study was to examine the bioavailability and bioactivity of ethinyl

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estradiol (EE2) sorbed onto SWCNTs in a fish gastrointestinal (GI) tract. Sorption

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experiments indicated that SWCNTs effectively adsorbed EE2, but, the chemical was

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still able to bind and activate soluble estrogen receptors (ER) in vitro. However,

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centrifugation to remove SWCNTs and adsorbed EE2 significantly reduced ER activity

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compared to EE2 alone. Additionally, the presence of SWCNTs did not reduce the EE2

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driven induction of vitellogenin 1 in vivo compared to organisms exposed to EE2 alone.

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These results suggest that while SWCNTs adsorb EE2 from aqueous solutions, under

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biological conditions EE2 can desorb and retain bioactivity. Additional results indicate

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that interactions with gastrointestinal proteins may decrease estrogen adsorption to

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SWCNTs by 5%. The current study presents valuable data to elucidate how SWCNTs

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interact with chemicals that are already present in our aquatic environments which is

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essential for determining their potential health risk.



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INTRODUCTION Single-walled carbon nanotubes (SWCNTs) have many unique properties, such

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as high tensile strength and electrical conductivity, that have driven increased

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production of these materials for use in consumer and industrial products over the past

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decade [1]. Accompanying this increase in production are concerns over the potential

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for environmental release of SWCNTs, whether accidental or intentional, and

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associated health impacts [2, 3] on ecosystems. Studies examining the environmental

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fate of SWCNTs suggest that if these materials are introduced to aquatic systems, they

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will partition to organic material and sediments [4]. As a result, SWCNTs are likely to

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enter aquatic food chains through dietary routes [2, 5, 6].

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The majority of research studies exploring the toxicity of SWCNTs in aquatic

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systems have been heavily focused on waterborne exposures; however, a few studies

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have examined the impacts of SWCNTs through feeding exposure routes and report

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that these materials exhibit little overt toxicity and are not readily absorbed through the

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gastrointestinal system [2, 6, 7]. Bisesi et al. 2015 [5] has demonstrated that these

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materials can impart more subtle effects that include modulation of nutrient transporter

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expression levels in the gastrointestinal tract from exposure to SWCNTs in the low µg

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range. Such effects have not been extensively explored but imply that energy and

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fitness outcomes could be impacted.

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It is well established that sorption to activated carbon can reduce the

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bioavailability of hydrophobic organic contaminants (HOCs), including estrogens to fish

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and benthic invertebrates [8]. However the influence of carbon nanomaterials on

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bioavailability, toxicity, and bioaccumulation of HOCs is variable. In several cases, 4 ACS Paragon Plus Environment

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SWCNTs have been shown to sorb both polar and non-polar environmental chemicals,

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effectively sequestering such compounds as the plasticizer bisphenol-A (BPA) and the

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synthetic hormone, 17α-ethinyl estradiol (EE2) [9-11]. Studies by Ferguson et al., 2008

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[12] and Parks et al., 2014 [13] found that carbon nanotubes effectively bind HOCs like

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polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs),

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decreasing bioaccumulation and toxicity in sediment dwelling organisms. Linard et al.

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2015 [14] similarly demonstrated that multi-walled carbon nanotubes (MWCNTs)

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effectively decrease waterborne exposure to fluoranthene in fish. These studies imply

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that carbon nanotubes strongly sorb such chemicals in the water column, reducing their

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bioavailability. Conversely, Schwab et al.[15], showed that MWCNT sorption to the

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herbicide diuron was partially reversible and Ferguson et al,[12] showed SWCNTs

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increased the bioavailability and bioaccumulation of PAHs in benthic copepods.

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Investigations that examine the sorption behavior of chemicals to carbon nanotubes as

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they transit the gastrointestinal system have not been performed.

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The synthetic estrogenic hormone EE2 is commonly found in oral contraceptives

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that are prescribed worldwide [16, 17]. As a result, EE2 is continuously introduced into

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our waste streams through normal excretion, where it reaches wastewater treatment

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plants (WWTPs) which are moderately effective at removing this compound with

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reported efficiencies of approximately 80% [18]. The remaining EE2 that is not

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removed during wastewater treatment can be discharged to surface water where it has

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been measured at low ng/L levels [19]. EE2 has been reported to be a potent

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endocrine disruptor in fish causing a number of reproductive abnormalities at low ng/L

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levels including male vitellogenin production [20], intersex [21], reduced fecundity [22], 5 ACS Paragon Plus Environment


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reduction in male secondary sex characteristics, impacts on reproduction behavior [23],

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and even collapse of entire populations [24]. EE2 is moderately hydrophobic (Kow: 3.7)

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and as a result has been shown to adsorb to SWCNTs under environmental conditions

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[9-11]. Its presence in aquatic environments, its associated hazards, and the potential

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for adsorption to SWCNTs, which are also likely to be present in the same aquatic

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reservoirs, make EE2 an ideal candidate for studying the adsorption behavior of

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chemicals to SWCNTs in the gastrointestinal system.

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The fish gastrointestinal system is a complex milieu of pH gradients, proteins,

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lipids, and surfactants that act in concert to aid in the digestion and absorption of key

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nutrients. The gut also serves as an important barrier that protects fish from harmful

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constituents in their environment [25]. As materials and chemicals, both together and

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separately, move through the gastrointestinal system, fluctuations in parameters such

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as pH, ionic strength, and surfactant properties of biomolecules may influence the

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sorption capacity of SWCNTs and thus change the bioavailability of sorbed compounds.

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The objective of the current study was to use in vitro assays and in vivo

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biomarkers of estrogenic exposure to determine how the gastrointestinal system of fish

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impacts the bioavailability of EE2 adsorbed to SWCNTs. Sorption assays were

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performed to determine the adsorption capacity of EE2 to SWCNTs. These mixtures

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were then tested in estrogen receptor (ER) binding and activity assays to determine the

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bioavailability of EE2 adsorbed to SWCNTs under in vitro conditions. Mixtures were

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also administered to two freshwater fish species (fathead minnow and largemouth bass)

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that are likely to contact SWCNT-EE2 in aquatic environments, by oral gavage to

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determine if movement through the gastrointestinal system influences the bioavailability 6 ACS Paragon Plus Environment

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of EE2 adsorbed to SWCNTs. Finally, the impact of gastrointestinal proteins on EE2

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adsorption to SWCNTs was tested to determine if proteins may drive desorption of EE2.

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EXPERIMENTAL

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Chemicals. Pristine, non-functionalized, semi-conducting SWCNTs (SG65) were

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generously donated by Southwest Nanotechnologies (Norman, OK, USA) in dry form.

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EE2, 17β-Estradiol (E2), Tris-HCl, triethylene glycol (TEG), CHAPS, glycerol, KCl,

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NaN3, and bovine gamma globulin were purchased from Sigma-Aldrich (St. Louis, MO,

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USA). Gum arabic, molecular grade isopropanol, chloroform, RNAsecure, RNase

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away, Scintiverse liquid scintillation counter cocktail, and molecular biology grade

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ethanol were purchased from Fisher Scientific (Pittsburg, PA, USA). RNA Stat-60 was

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purchased from Tel-test (Friendswood, TX, USA). Tritium labeled E2 was purchased

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from Perkin-Elmer (Waltham, MA, USA).

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Suspension Preparation and Characterization. Suspensions were prepared

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according to methods described in previous studies [2, 5]. Briefly, 5 mg of SWCNTs

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were weighed in a glass test tube and 5 mL of 0.5% gum arabic was added to the tube.

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The mixture was then sonicated and centrifuged to remove unsuspended aggregates

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and the concentration of the resulting solution was measured by near infrared

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fluorescence (NIRF) spectroscopy using previously described methods [26]. Using this

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method we are able to produce suspensions with consistent characteristics including an

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average SWCNT cluster size of 132 nm (DLS), that moderately compact with a fractal

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dimension of 2.2-2.3. The catalyst metals leaching from the SWCNTs are less than 5%

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w/w. Data from the current and previous studies indicate that these suspensions are



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stable with the particle characteristics described above being maintained for at least a

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week [2, 5].

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EE2 and SWCNT Adsorption. EE2 was bound to SWCNTs by first diluting the

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measured SWCNT stocks described above in 0.5% gum arabic to reach a final

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concentration 25,000 µg/L in glass test tubes. Calculated volumes of concentrated EE2

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stocks, dissolved in 70:30 TEG:ethanol v/v, were added to each test tube to reach

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desired EE2 concentrations. Tubes were then capped and placed at a 45° angle on an

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orbital shaker for one hour to achieve maximum adsorption. The resulting suspensions

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with EE2 adsorbed to SWCNTs were either used directly in in vitro and in vivo assays or

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ultracentrifuged at 250,000 × g to remove SWCNTs and adsorbed EE2, followed by the

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use of the supernatant in the assays. All EE2 only controls were also ultracentrifuged at

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250,000 × g to account for any loss of EE2 during centrifugation by adsorption to the

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centrifuge tubes.

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Quantitation of Adsorption Affinity. Adsorption experiments were performed

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in triplicate by mixing EE2 (2 µg/L-4000 µg/L) and SWCNTs (25,000 µg/L) as described

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above. Following mixing, tubes containing EE2, with and without SWCNTs, were

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ultracentrifuged at 250,000 x g to remove SWCNTs and adsorbed EE2 from suspension

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and the remaining EE2 dissolved in the supernatant was measured using an EE2

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ELISA (Abraxis, Warminster, PA) following the manufacturers protocol. The resulting

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measured concentrations of dissolved EE2 (Cw) were used to calculate corresponding

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adsorbed EE2 loadings on the SWCNT (Q) by assuming a complete mass balance.

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Isotherms were plotted and a Freundlich model was used to describe adsorption

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behavior of SWCNTs and EE2. 8 ACS Paragon Plus Environment

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Estrogen Receptor Binding Assays. To determine the impact of EE2

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adsorption to SWCNTs on estrogen receptor (ER) binding by EE2, a fluorescence

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polarization (FP) based ER binding assay was utilized. The advantages of this assay

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over traditional radioligand binding assays and detailed methods have been described

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previously [27]. Briefly, recombinant human estrogen receptor 1 ligand binding domain

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(ESR1-LBD) was produced in bacterial cells and purified for use in the assays.

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Additionally, a fluorescein labeled estrone (Fl-E1) probe was produced and purified by

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HPLC to serve as the native ligand for the assays. Predetermined concentrations of

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ESR1-LBD and Fl-E1 (110 nM and 5.5 nM, respectively) were mixed in binding buffer

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for one hour to allow for binding to form a complex. Serial dilutions of E2 and EE2 (395

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– 0.002 µg/L) was prepared in binding buffer in triplicate and placed in a black 96 well

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plate to generate a standard curve of ESR1 binding. The E2 standard curve is run as a

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QA/QC measure to ensure consistency between runs. Six treatments were tested in

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triplicate: EE2 alone (3 and 0.3 µg/L nM) which was centrifuged at 250,000 × g for 1

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hour before being diluted in binding buffer, EE2 (3 and 0.3 µg/L ) with SWCNTs (25,000

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µg/L) mixed for one hour and diluted in binding buffer, and EE2 (3 and 0.3 µg/L) and

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SWCNTs (25,000 µg/L) mixed for one hour followed by centrifugation at 250,000 × g to

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remove SWCNTs and adsorbed EE2 and the supernatant diluted in binding buffer. The

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standard curve and treatments were prepared in binding buffer in a 96 well plate (100

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µL final volume), 10 µL of the ESR1-LBD and Fl-E1 complex was added to each well

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and the mixture allowed to incubate while shaking in the dark for one hour. Following

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the incubation, fluorescence polarization was read on a Biotek Synergy H1 (Biotek,

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Winooski, VT, USA) and the percentage of the Fl-E1 displaced by the competitors was



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calculated for each concentration of EE2, E2, and the treatments described above.

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Percent inhibition of the EE2 and E2 were plotted against concentration and used to

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calculate the concentration of EE2 present in the mixtures with and without SWCNTs.

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Estrogen Receptor Activation. HEK293 (human embryonic kidney) cells were

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acquired from American Type Culture Collection (ATCC®) and cultured following the

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protocol previously described [28, 29]. Cell culture media consisted of Dulbecco’s

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modified Eagle Media supplemented with 2 mM L-Glutamine, 100 µg/mL, and 10% fetal

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bovine serum (FBS). During transfection and exposure, media with charcoal stripped

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FBS was used to reduce receptor activation by any hormones present. Each assay was

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performed in triplicate. Cells were seeded at a density of 200,000 cells/well in 24-well

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plates, and transfected with 500 ng of either the full length largemouth bass (LMB) ERα

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in the pCMV4 mammalian expression vector [29] or empty pCMV4 vector, 200 ng of

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constitutively active pRL-TK renilla (Promega, Fitchburg, WI, USA), and 1000 ng of a

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2x-ERE-Luciferase vector [29] using the Fugene 6 reagent (Promega, Fitchburg, WI,

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USA) in antibiotic free media. After 18 hours, media in the wells was changed to the

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complete media described above with 10% charcoal stripped FBS and exposed to one

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of the following treatment conditions: mixtures of SWCNTs (25,000 µg/L) and EE2 (3,

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0.3, 0.03, 0.003 µg/L) following a one hour incubation, supernatant from the same

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mixtures that were centrifuged at 250,000 × g to remove SWCNTs and sorbed EE2 from

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suspension, or EE2 (3, 0.3, 0.03, 0.003 µg/L ) without SWCNTs that was centrifuged at

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250,000 × g before use. After 24 hours cells were harvested in 100 µL of passive lysis

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buffer (Promega, Fitchburg, WI, USA), followed by gentle rocking for 20 min at room

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temperature, centrifuged for 3 min at 12,000 rpm at 4°C, and then assayed for 10 ACS Paragon Plus Environment

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transcriptional activation using a Dual Luciferase Reporter Assay System (Promega,

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Fitchburg, WI, USA) on a Synergy H1 microplate reader (Biotek Instruments, Winooski,

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VT). Relative luciferase activity was plotted as fold change from control (no SWCNTs or

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EE2 exposure).

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Determining Oral EE2 Dose for vtg1 Expression. All animal research was

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performed under University of Florida IACUC approved protocols (IACUC Study

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#201507459). Largemouth bass (LMB, Micropterus salmoides) were purchased from

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the American Sportfish Hatchery (Montgomery, AL) and fathead minnows (FHM,

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Pimephales promelas) were obtained from in house breeding cultures at the University

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of Florida Aquatic Toxicology Core. These species were chosen based on their different

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life histories and distinct gastrointestinal morphology. To determine the dose-response

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relationship between oral EE2 exposure and vitellogenin 1 (vtg1) expression for each

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fish species, male largemouth bass (LMB, n = 5 to 9 fish /treatment) and fathead

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minnows (FHM, n = 6 fish/treatment) were gavaged 0.01, 0.001, 0.0001, 0.00001 µg

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EE2/g body weight. EE2 was dissolved in 70:30 TEG:ethanol (v/v) then diluted in 0.5%

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gum arabic for gavage. The final concentration of TEG and ethanol in the gavage

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solution was less than 0.03%. The solvent/gum arabic mixture was also gavaged into

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control fish. Following gavage, LMB were held in 37 L flow through tanks for 48 hours.

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At the end of the 48 hours, fish were euthanized using Tricane-S, weighed, measured,

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and their livers removed. Livers were flash frozen in liquid nitrogen and stored at -80°C

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until processed for total RNA extraction and vtg1 expression analysis. This dose

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response relationship was used to determine the final EE2 concentration used in

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subsequent experiments with SWCNTs. 11 ACS Paragon Plus Environment


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Fish SWCNT and EE2 Mixture Exposures. Mixtures of EE2 and SWCNTs were

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prepared as described above at a final concentration of 200µg EE2/L (LMB) or 4000 µg

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EE2/L (FHM) and 25,000 µg SWCNT/L to achieve a final concentration of 0.001 µg

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EE2/g body weight for LMB or 0.01 µg EE2/g body weight for FHM. These mixtures

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were placed on an orbital shaker for 60 minutes to allow time for EE2 and SWNT

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sorption. Fish were gavaged with one of the following treatments: carrier control, EE2

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alone that was centrifuged at 250,000 × g, SWCNTs alone, EE2+SWCNTs or the

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supernatant of the EE2+SWCNTs that were centrifuged at 250,000 × g to remove EE2

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adsorbed to SWCNTs. Four to eight male LMB replicates and 8 male FHM replicates

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were used for each treatment and the same procedure was followed after the gavage as

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for the dose response study described above.

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Vitellogenin mRNA Expression. Total RNA was extracted from liver samples

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using a modified method previously described by Bisesi et al. 2015 [5]. In brief, tissues

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were homogenized in RNA Stat-60 (Tel-Test, Friendswood, TX) using a handheld rotary

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homogenizer followed by addition of chloroform. Aqueous RNA was precipitated with

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isopropanol, washed with ethanol, re-dissolved in RNAsecure (Life Technologies, Grand

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Island, NY, USA), and measured using a Synergy H1 plate reader (BioTek Instruments,

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Winooski, VT, USA). Samples were then DNase treated (Quanta PerfeCTa DNase I;

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Quantabio, Beverly, MA, USA) and reverse transcribed to cDNA (Quanta qScript cDNA

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synthesis kit; Quantabio, Beverly, MA). Expression of vtg1 in liver tissue was measured

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using quantitative real-time PCR and normalized using 18s rRNA and rpl8 as reference

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genes for LMB and FHM, respectively. Reactions for quantitative PCR were prepared

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as follows: 10 µL of SYBR® Select Master Mix (Life Technologies, Grand Island, NY, 12 ACS Paragon Plus Environment

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USA), 1 µL forward primer (18 µM), 1 µL reverse primer (18 µM), 1 µL template, 7 µL

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RNase/Dnase free water. Primers used for each of the genes can be found in table 1

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below. Quantitative PCR reactions were performed using a MyiQ™ Real-Time PCR

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Detection System (Bio-Rad, Hercules, CA, USA) with the following parameters: 1 cycle,

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95 °C for 3 min; 40 cycles, 95 °C for 10 s, 58 °C for 1 min. Using the iQ™5 optical

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system software (Bio-Rad), threshold cycles were used to determine relative fold

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change in mRNA expression from controls using the 2-∆∆Ct method.

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Protein Impacts on Adsorption of EE2 to SWCNTs. To determine whether

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proteins commonly present in the fish gastrointestinal system may influence the

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adsorption behavior of estrogenic hormones and SWCNTs, adsorption experiments

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were performed with and without the addition of trypsin and pepsin. Mixtures of

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SWCNTs (25,000 µg/L) and tritium labeled E2 (3 µg/L, specific activity: 120 Ci/mmol)

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were prepared with and without trypsin (100,000 and 500,000 µg/L) or pepsin (100,000

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and 500,000 µg/mL). Additionally, the order of the protein addition was tested, where

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protein was either added before or after the addition of E2. The total volume of the

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mixtures was 1 mL. SWCNTs were first diluted in 0.5% gum arabic and either E2 or

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protein was added and allowed to mix on an orbital shaker table for one hour. Following

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the first hour of mixing, the final addition of E2 or protein was added and allowed to mix

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for another hour. At the end of the second hour all mixtures were centrifuged at

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250,000 × g for one hour to remove SWCNTs and adsorbed E2. To measure the E2

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remaining in the supernatant 50 µL was diluted in 5 mL scintiverse LSC cocktail and

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counts per minute (CPM) were measured on a Beckman Coulter LS6500 scintillation



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counter. CPM from mixture supernatants were quantified against a standard curve of

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E2 for comparison between treatments.

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Statistical Analysis. All graphs and statistical analyses were performed in

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GraphPad Prism 5 (GraphPad Software Inc, La Jolla, CA). All data were tested for

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normality using the Kolmogorov-Smirnov test and homogeneity of variances using the F

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tests of equality of variances. For ERα transactivation assays, unpaired t-tests with

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Holm-Sidak corrections for multiple comparison testing were used to determine

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statistical significance (p < 0.05). For gene expression and protein impact on adsorption

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experiments, a one way ANOVA followed by Tukey’s HSD test was performed to

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determine statistical significance and multiple comparisons testing, If data were

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normally distributed. For non-normally distributed data, the Kruskal-Wallis

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nonparametric test was performed followed by Dunn’s multiple comparison test. P

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values

Influence of the Gastrointestinal Environment on the Bioavailability of

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