Measurement of steroids in rats after exposure to an endocrine disruptor: Mass spectrometry and radioimmunoassay demonstrate similar results

Journal of Pharmacological and Toxicological Methods 68 (2013) 314–322

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Original article

Measurement of steroids in rats after exposure to an endocrine disruptor: Mass spectrometry and radioimmunoassay demonstrate similar results☆ Brandy W. Riffle a,c, W. Matthew Henderson b, Susan C. Laws c,⁎ a

Oak Ridge Institute for Science and Education (ORISE), Research Participation Program, Oak Ridge, TN 37831, United States Ecosystems Research Division, National Exposure Research Laboratory (NERL), ORD, U.S. EPA, Athens, GA 30605, United States Endocrine Toxicology Branch, Toxicity Assessment Division, National Health and Environmental Effects Research Laboratory (NHEERL), Office of Research and Development (ORD), U.S. Environmental Protection Agency (U.S. EPA), Research Triangle Park, NC 27711, United States

b c

a r t i c l e

i n f o

Article history: Received 7 June 2013 Accepted 10 July 2013 Available online 18 July 2013 Keywords: Methods Steroids Radioimmunoassay Mass spectroscopy Quality control

a b s t r a c t Introduction: Commercially available radioimmunoassays (RIAs) are frequently used to evaluate the effects of endocrine disrupting chemicals (EDCs) on steroidogenesis in rats. Currently there are limited data comparing steroid concentrations in rats as measured by RIAs to those obtained using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). This study evaluates the concordance of serum and urine steroid concentrations as quantified by select RIA kits and LC–MS/MS following exposure to an EDC, atrazine (ATR). Methods: Adult male rats were orally dosed with ATR (200 mg/kg/day) or methylcellulose (1%, vehicle control) for 5 days. Serum was collected and separated into aliquots for analysis. Serum was assayed by RIA for androstenedione (ANDRO), corticosterone (CORT), estradiol (E2), estrone (E1), progesterone (P4), and testosterone (T). Serum was extracted prior to LC–MS/MS analysis with positive electrospray ionization in multiplereaction monitoring mode for ANDRO, CORT, P4, and T. E1 and E2 concentrations were quantified similarly by LC–MS/MS, following derivatization with dansyl chloride. To compare CORT values from urine, pregnant adult rats were orally dosed with either ATR (100 mg/kg/day) or methylcellulose for 5 days (i.e., gestational days 14–18). Urine samples were collected daily and assayed for CORT by RIA and LC–MS/MS as described above. Results: Data analyses demonstrated significant agreement between the two detection methods as assessed by Pearson product-moment correlation coefficient, Bland–Altman analysis, and the interclass correlation coefficient. No statistically significant differences were observed between RIA and LC–MS/MS means for any of the steroids assayed. Discussion: These findings indicate a significant correlation between the measurement of steroids within rat serum and urine using RIA kits and LC–MS/MS. Differences in the absolute measurements existed, but these were not statistically significant. These findings indicate that steroids may be reliably measured in rat biological media using RIAs or LC–MS/MS. Published by Elsevier Inc.

1. Introduction Abbreviations: ANDRO, androstenedione; ANOVA, analysis of variance; ATR, atrazine; AUC, area under the curve; CORT, corticosterone; CV, coefficient of variation; E1, estrone; E2, estradiol; EDC, endocrine disrupting chemical; FIFRA SAP, Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel; GLM, general linear model; GD, gestational day; HPA, hypothalamic–pituitary–adrenal; ICC, intraclass correlation coefficients; LC–MS/MS, liquid chromatography coupled with tandem mass spectroscopy; LH, luteinizing hormone; MRM, multiple reaction monitoring mode; P4, progesterone; RIA, radioimmunoassay; SD, standard deviation; SEM, standard error mean; SPE, solid phase extraction; SRM, selective reaction monitoring; T, testosterone. ☆ Disclaimer: This manuscript has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views or policy of the Agency nor does the mention of trade names or commercial products constitute endorsement or recommendation for use. ⁎ Corresponding author at: Toxicity Assessment Division (MD-72), National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, 109 T. W. Alexander Drive, Research Triangle Park, NC 27711, United States. Tel.: +1 919 541 0173; fax: +1 919 541 5138. E-mail address: [email protected] (S.C. Laws). 1056-8719/$ – see front matter. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.vascn.2013.07.003

An endocrine disrupting chemical (EDC) is defined as an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub) populations (WHO, 2002). As presented in the 2012 review by De Coster and van Larebeke, disruptions of the endocrine system have been linked to numerous pathophysiologies including the development of hormone-dependent cancers, adverse effects on sexual development and reproductive function, and metabolic disorders (De Coster & van Larebeke, 2012). Atrazine (ATR), a chlorotriazine herbicide used frequently within the United States to control grassy and broadleaf weeds for agricultural and landscape purposes, has been reported to cause adverse effects on the reproductive system in the laboratory rat (Cooper, Stoker, Goldman, Parrish, & Tyrey, 1996; Eldridge, Tennant, Wetzel, Breckenridge, & Stevens, 1994; Wetzel et al., 1994). Studies conducted by Cooper, Stoker, Tyrey, Goldman, and McElroy

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(2000) and Cooper et al. (2007) have demonstrated that ATR alters the neuroendocrine control of the hypothalamic–pituitary–ovarian axis, a mode of action that is reflected by a disruption of the pre-ovulatory surge of luteinizing hormone (LH) in female rats (Cooper et al., 2000, 2007). More recent studies have shown that ATR can also alter the activity of the hypothalamic–pituitary–adrenal (HPA) axis as indicated by the changes in circulating serum steroid concentrations. Dosedependent increases in corticosterone (CORT) and progesterone (P4) release in both male (Laws et al., 2009) and female (Fraites et al., 2009) rats have been observed following a single exposure to ATR. Increases in serum estrone (E1) and estradiol (E2) in male (Modic, 2004; Stoker, Guidici, Laws, & Cooper, 2002; Stoker, Laws, Guidici, & Cooper, 2000) and E1 in ovariectomized female (Cooper, 2010) rats following ATR administration have been previously reported as well. Thus, due to the extensive number of hormones that are changed following exposure to this dose of ATR (200 mg/kg) in laboratory rats, this chemical was chosen as the test compound for the comparison of steroid measurements using radioimmunoassays (RIAs) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) methods. One of the most commonly used methods for measuring hormones in toxicological studies is RIAs, but there are sometimes concerns regarding their validity when measuring steroids in multiple animal species and biological matrixes. For example, during a review of experimental animal and in vitro studies as part of a re-evaluation of the potential human health effects of ATR by a U.S. EPA Scientific Advisory Panel (Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA SAP)), several investigators expressed concerns about the accuracy of steroid measurements when using RIAs, in particular the levels of E1 and E2 found in male rats (Handa, 2010; USEPA, 2010). Such circumspection is based upon the fact that the majority of commercially available immunoassays for steroids have been designed and validated only for use with human serum. Thus, each laboratory is responsible for demonstrating the validity of the assay for use with other species and/or biological matrixes (e.g., urine or other target tissues). Additionally, RIAs, particularly in the lower ranges, can be subject to bias due to crossreactivity with other steroids and potential interference from proteins or the presence of the test chemical in the biological matrix being evaluated. Alternatively, commercially available RIAs are economically advantageous and can provide timely results because samples do not typically require extraction prior to analysis. Previous epidemiological and clinical studies have addressed such concerns regarding the use of RIAs in the measurement of sex hormones in serum (Hsing et al., 2007; Janse et al., 2011; Khosla et al., 2008) and urine (Falk et al., 1999; Faupel-Badger et al., 2010) from human subjects by comparing values obtained by RIA to those following LC–MS/MS. Immunoassays for the measurement of E2 have also been previously evaluated for use in mouse sera (Haisenleder, Schoenfelder, Marcinko, Geddis, & Marshall, 2011). LC–MS/MS has been recently recognized as a highly selective method for determining steroid hormone concentrations (Kushnir, Rockwood, & Bergquist, 2010; Soldin & Soldin, 2009) and is viewed by some as the “gold standard” technology for this purpose (Gust et al., 2010; Hsing et al., 2007; Rauh, Groschl, Rascher, & Dorr, 2006; Shackleton, 2010; Singh, 2008; Stanczyk, Lee, & Santen, 2007). The aim of the current study was to evaluate the concordance of steroid concentrations in serum such as P4, CORT, androstenedione (ANDRO), testosterone (T), E1, and E2 as quantified by both commercial RIA kits frequently used within our laboratory and LC–MS/MS following exposure to ATR in order to verify our previously published results (Fraites et al., 2009; Hotchkiss, Best, Cooper, & Laws, 2012; Laws, Ferrell, Stoker, & Cooper, 2003; Laws, Ferrell, Stoker, Schmid, & Cooper, 2000; Laws et al., 2009; Stoker et al., 2000; Stoker et al., 2002) and to understand whether these RIAs are “fit for purpose” to study ATR induced alterations in steroid secretion. Previous work has demonstrated that the AUC (area under the curve) for urinary CORT concentration versus time correlates well with serum values (Pruett, Lapointe, Reagan, Lawton, & Kawabata, 2008; Pruett et al., 2007), and provides a

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non-invasive method for monitoring changes in CORT homeostasis. This can be especially advantageous when evaluating potential changes in the daily circadian rhythm or during exposure regimens such as in pregnant dams to evaluate the effects of changes in maternal CORT on gestationally exposed rats. However, the CORT RIA that is typically used in our laboratory has not yet been validated for use with rat urine. Therefore, this study also compared urine CORT concentrations in pregnant dams quantified by both a commercial RIA kit and LC–MS/ MS. 2. Materials and methods 2.1. Animals All animal work was completed under the guidance and supervision of the U.S. EPA National Health and Environmental Effects Research Laboratory (NHEERL) Institutional Animal Care and Use Committee (IACUC). Adult male Wistar rats (age 73 days at start of treatment) were obtained from Charles River Laboratories (Raleigh, NC) and maintained on a 12 hour light/dark cycle (lights on at 0600 h, lights off at 1800 h) under controlled conditions [temperature (20–24 °C), humidity (40–50%)]. Animals were given access to food (Purina Laboratory Chow 5001) and water ad libitum. One week following their arrival to the facility, the animals were weight-ranked, and divided into 2 treatment groups (vehicle control, 200 mg/kg ATR). There were 10 animals per dose (total of 20 animals). An additional group of adult male Wistar rats (age 75 days at start of treatment) were obtained from Charles River Laboratories (Raleigh, NC), maintained as described above, and used exclusively for the measurement of serum E1 and E2 content as described below. As with the previous group, the animals were weight-ranked one week after their arrival to the facility and divided into 2 treatment groups (vehicle control, 200 mg/kg ATR). There were 8 animals per dose (except for ATR group; n = 7) for a total of 15 animals. Timed pregnant Sprague Dawley rats (age 73–101 days upon arrival to the facility on Gestational Day (GD) 3) were also obtained from Charles River Laboratories (Raleigh, NC) and maintained on a 14/10 hour light/dark cycle (lights on at 0600 h, lights off at 2000 h) under controlled conditions [temperature (20–24 °C), humidity (40–50%)]. Animals were given access to food (Purina Laboratory Chow 5008) and water ad libitum. One week following their arrival to the facility, the animals were weight-ranked, and divided into 3 treatment groups (cage control (no treatment), vehicle control (1% methyl cellulose), and 100 mg/kg ATR). There were 2 animals per treatment (except for ATR group; n = 3) for a total of 7 animals. 2.2. Dosing solutions and procedures ATR (CAS 1912-24-9, 97.1% purity) was a gift from the Syngenta Corporation (Greensboro, NC). All dosing solutions were prepared as a suspension of 1% methyl cellulose in distilled water (M7140, Batch # 108K0130, Sigma Aldrich, St. Louis, MO). All treatments and decapitation of the animals occurred between 0700 h and 1000 h when the circadian fluctuation of CORT was at its nadir. Animals were acclimated to the dosing procedure by pre-dosing with vehicle (1% methylcellulose by oral gavage) for 5 days prior to start of treatment. Previous work in our laboratory has demonstrated that following the pre-dosing period basal CORT levels are not affected in the control animals with subsequent handling and dosing (Fraites et al., 2009; Laws et al., 2009). At the onset of treatment, the male and pregnant rats were dosed 1 h after lights on (0700 h) with either ATR (100, 200 mg/kg) or 1% methylcellulose (vehicle control) by oral gavage delivered in a volume of 5.0 ml/kg of body weight for 5 days (pregnant rats; GD 14–18). Animals were weighed daily and the volume of solution given was adjusted for body weight. All male rats were decapitated 2 h after the final dose, and trunk blood was collected into serum collection tubes. Serum was

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isolated by centrifugation, placed in siliconized 1.7 ml centrifugation tubes, and frozen immediately at −80 °C. The serum samples were assayed for hormone content within 30 days.

2.3. Urinary corticosterone measurements Urine from pregnant rats was collected daily over 2 intervals (6 h preceding dosing and 6 h following dosing) during each of the 5 days of treatment. This was accomplished by placing animals into metabolism cages at 0100 h and returning them to their home cage at 1300 h. In order to reduce any stress associated with the dosing procedure or the use of the metabolism cages, the animals were acclimated to the cages after dosing with vehicle (1% methylcellulose by oral gavage) for 5 days prior to the start of treatment. At the end of each collection period, a 1 ml sample of the urine was collected, placed in siliconized 1.7 ml centrifugation tubes, and frozen immediately at −80 °C. The urine samples were assayed for CORT and creatinine content within 30 days. 2.4. Radioimmunoassays Serum ANDRO, P4, and T along with both serum and urinary CORT were measured using coat-a-count kits obtained from Siemens Healthcare Diagnostics Inc. (PITKAN-6, PITKPG-10, PITKTT-8, PITKRC-5, Los Angeles, CA). The reported limit of detection for each kit was as follows: ANDRO, 0.04 ng/ml; CORT, 5.7 ng/ml; P4, 0.02 ng/ml; and T, 0.4 ng/ml. Serum E1 and E2 was measured using kits obtained from Beckman Coulter (DSL8700, DSL4800, Webster, TX). The reported limits of detection for E1 and E2 were 1.2 pg/ml and 2.2 pg/ml respectively. All RIAs were performed according to manufacturers' instructions. The urinary CORT levels were standardized by the amount of creatinine in each sample (Faupel-Badger et al., 2010; Flores, Sun, Vaziri, & Miyada, 1980). ATR did not significantly alter CRT in this study, albeit the authors note that caution must be used when standardizing urinary CORT with CRT in toxicological studies given that a toxicant could affect CRT clearance in the urine (Boeniger, Lowry, & Rosenberg, 1993; Santa Maria, Vilas, Muriana, & Relimpio, 1986). Creatinine content was determined by using a colorimetric Urinary Creatinine Detection kit from Arbor Assays (K002-H5, Ann Arbor, MI) as per the manufacturer's instructions.

2.5. Steroid hormone extraction for LC–MS/MS After thawing of samples, 1 ml of serum or urine was diluted with 4 ml of 5% methanol in 13 × 100 mm disposable glass culture tubes before adding 2 μl of the internal standards (ANDRO, CORT, P4, and T, medroxyprogesterone (250 ng/ml); E1 and E2, ethinyl estradiol (150 ng/ml)). For estrogen extraction, the volume of serum used was increased to 2 ml and the diluent adjusted to 3 ml. Steroid hormones were extracted with SampliQ OPT solid phase extraction columns (30 mg, Agilent Technologies, Santa Clara, CA). Briefly, the solid phase extraction (SPE) columns were conditioned with methanol and equilibrated with water prior to loading 5 ml of the diluted sample. After washing with 30% methanol, steroid hormones were eluted sequentially with 2 ml methanol followed by 2 ml of 30% methyl tert-butyl ether in hexane. Following SPE, the samples were dried under a gentle stream of nitrogen and resuspended in 30% acetonitrile before being transferred to glass microtarget inserts in GC vials prior to analysis. For estrogens, the samples were derivatized for 10 min at 60 °C with 100 μl of dansyl chloride (2 mg/ml; Sigma Aldrich) in acetonitrile (pH adjusted with 100 μl of 10 mM sodium biocarbonate). Following derivatization, samples were filtered through Spin-X centrifuge tube filters (0.22 μM) before LC-MS/MS analysis.

2.6. Mass spectrometry Steroid hormone analysis was performed on a Varian 1200L triple quadrupole mass spectrometer with positive electrospray ionization in multiple-reaction monitoring (MRM) mode. Prior to chromatographic separation, instrumental parameters were optimized by infusing 1 μg/ml of the standards (ANDRO, CORT, E1, E2, P4, and T) into the mass spectrometer. Optimized parameters for the analysis of the steroid hormones are located in Table 1. The limits of quantification for each steroid are as follows: ANDRO, 0.68 ng/ml; CORT, 0.82 ng/ml; E1, 29 pg/ml; E2, 6 pg/ml; P4, 0.35 ng/ml; and T, 0.96 ng/ml. For quality assurance, procedural blanks and matrix spikes were included in the analytical procedure. Instrumental parameters include: drying gas temperature at 275 °C, housing temperature at 45 °C, and capillary CID at 60 V. The detector was operated at 2000 V, collision cell pressure was maintained at 2.64 mTorr and collision energies of the MRM transitions are listed in Table 1. Chromatographic separation of ANDRO, CORT, P4 and T was accomplished on a Kinetex C18 column (Phenomenex; 2.6 μm particle size, 2.1 × 150 mm) with a flow of 0.18 ml/min (method A; Table 1, Figure 1A). The initial mobile phase was 70% water with 0.1% formic acid (mobile phase 1) and 30% acetonitrile with 0.1% formic acid (mobile phase 2). Starting conditions were held for 2 min before increasing to 40% mobile phase 2 at 15 min, held for 5 min, and then increasing to 50% mobile phase 2 at 35 min. A final increase to 90% mobile phase 2 was achieved at 42 min before being returned to starting conditions. The column was allowed to equilibrate for 10 min between sample runs (total run time, 109 min). Chromatographic separation of the estrogens was achieved on an Eclipse XDB-C18 column (Agilent Technologies; 3.5 μm particle size, 3.0 × 150 mm) with a flow of 0.4 ml/min (method B; Table 1, Figure 1B). The initial mobile phase was 50% water with 0.1% formic acid (mobile phase 1) and 50% acetonitrile with 0.1% formic acid (mobile phase 2). The starting conditions were held for 1 min before increasing to 70% mobile phase 2 in 2 min, 80% mobile phase 2 in 5 min and 95% mobile phase 2 in 2 min. The column was washed with 95% mobile phase 2 for 3 min before returning to initial conditions and being reequilibrated for 12 min (total run time, 25 min). 2.7. Statistical analyses Data from treatment groups were evaluated for differences between measurement effects by analysis of variance (ANOVA) using the General Linear Model (GLM) and Tukey's Multiple Comparison post-hoc tests. Data from treatment groups were evaluated for differences between treatment effects by analysis with unpaired student's t-test. All data were tested for homogeneity of variance by Shapiro–Wilk normality test, and if heterogeneity of variance

Table 1 LC-MS/MS parameters for the analysis of steroid hormones in serum and urine. Compound

Molecular weight

Retention time (min)

Parent ion (m/z)

Fragment ions (m/z)

Collision energy (V)

P4a

314.5

40.2

315

CORT

346.4

9.6

347

ANDRO

286.4

23.0

287

T

288.4

19.8

289

E1b

270.4

14.1

504

E2

272.4

13.4

506

97 109 121 311 97 109 97 109 171 156 171 156

−14 −20 −22 −16 −16 −22 −16 −22 −34 −46 −30 −44

a b

CORT, P4, ANDRO, and T data were measured using method A. E1 and E2 data were measured using method B.

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Table 2 Serum hormone concentrations at necropsy following 5 daily doses of ATR. LC–MS/MS 0 mg/kg a

Progesterone (ng/ml) Corticosterone (ng/ml) Androstenedione (ng/ml) Testosterone (ng/ml) Estrone (pg/ml) Estradiol (pg/ml)

0.40 ± 0.07 24.50 ± 8.24 1.08 ± 0.10 2.67 ± 0.32 96.90 ±2.68b 13.25 ± 0.84b

RIA 200 mg/kg 0.64 38.99 1.13 4.24 147.77 19.56

± ± ± ± ± ±

0.04⁎ 9.56 0.17 1.01 5.57c,⁎ 1.38c,⁎

0 mg/kg 0.43 27.35 2.06 3.75 95.46 11.56

± ± ± ± ± ±

200 mg/kg 0.07 9.54 0.35 0.50 4.13b 0.3b

0.67 43.56 2.05 5.03 153.64 16.16

± ± ± ± ± ±

0.08⁎ 9.73 0.60 1.20 7.08c,⁎ 0.89c,⁎

a

Serum collected 2 h following the last of 5 daily doses of ATR. Mean ± SEM (n = 10). Serum collected 2 h following the last of 5 daily doses of ATR. Mean ± SEM (n = 8). c Serum collected 2 h following the last of 5 daily doses of ATR. Mean ± SEM (n = 7). ⁎ Significantly different from control by unpaired student's t-test, p b 0.05. b

was detected, the data were then log transformed for further statistical analyses. Method comparison analyses were completed by constructing scatter plots, calculation of Pearson product-moment correlation coefficients (Pearson's r), construction of Bland–Altman plots, and calculation of intraclass correlation coefficients (ICC). Bland–Altman plots are used to demonstrate the difference of the two measurements plotted against the mean of the two. As proposed by Bland and Altman, if 95% of the differences between values obtained by the two methods of measurement are within ± 2 standard deviations (SD), then there is good agreement between the two methods of measurement, and this is referred to as the “limits of agreement” (Bland & Altman, 1986). For all plots the mean difference between RIA and LC–MS/MS measurements (mean bias) and limits of agreement (±2 SD) were calculated (Bland & Altman, 1986). ICC values indicate the agreement between the measurements with

large ICC values (maximum of 1) signifying an excellent agreement (Hopkins, 2009; Rosner, 2005). 3. Results Serum steroid concentrations in male rats at 2 h following the last of five daily doses of ATR (200 mg/kg) or vehicle control, as determined by RIA and LC–MS/MS, are reported in Table 2. ATR significantly increased serum concentrations of P4 (1.6-fold per LC-MS/MS and RIA), E1 (1.5-fold per LC-MS/MS; 1.6-fold per RIA), and E2 (1.5-fold per LC/ MS–MS; 1.4-fold per RIA). Serum concentrations of CORT and T were similarly increased but the differences were not determined to be statistically significant (1.6-fold per LC-MS/MS and RIA for CORT; 1.6-fold per LC-MS/MS and 1.3-fold per RIA for T). Serum ANDRO was not affected by treatment with ATR.

Fig. 1. LC–MS/MS selected reaction monitoring (SRM) chromatograms for CORT, T, A and P4 (A) and E1 and E2 (B). Standards are depicted as dashed lines and serum samples are solid lines. SRM parameters are listed in Table 1.

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Fig. 2. Scatter plots (A and C) and Bland–Altman plots (B and D) of progesterone and corticosterone serum measurements. Male Wistar rats were pre-dosed with vehicle control (methyl cellulose) for 5 days prior to receiving 5 daily doses of either vehicle or ATR (200 mg/kg) by oral gavage (n = 10). Groups of animals were killed 2 h following their final dose. CORT and P4 were measured by LC–MS/MS and RIA. Plots detail the agreement of P4 (A and B) and CORT (C and D) LC–MS/MS and RIA measurements. For the Bland–Altman plots (B and D), the dashed line indicates the mean difference between the two methods (bias) and the dotted lines demonstrate 2 SD of the mean difference (limits of agreement). Filled squares represent individual measurements.

The scatter and Bland–Altman plots for serum hormone measurements are demonstrated in Figs. 2–4. The correlations between the two methods of measurement for the hormones are shown in the scatter plots and the calculated Pearson's r values. For both serum P4 and CORT (Fig. 2), the Bland–Altman plots depict the mean difference between the two measurements (bias) and the number of measurements that lie outside the limits of agreement (±2 SD of the bias) of the methods (one measurement; 5% of the total number analyzed). The mean biases of the measurements were − 0.04 ng/ml (P4) and − 3.713 ng/ml (CORT) indicating that LC–MS/MS yielded lower serum levels of both hormones than RIA. The ICC for P4 and CORT were 0.84 and 0.96, respectively, and both values suggest that there was an agreement between the two methods of measurement. Serum T, as shown in Fig. 3 (panels C and D), and serum E1, as demonstrated in Fig. 4 (panels A and B), measurements are similarly concordant with mean biases of − 0.9344 ng/ml and − 2.216 pg/ml for T and E1, respectively. There were two measurements that fell outside of the limits of agreement between the two methods for T (10% of the total number analyzed), and no measurements that fell outside the limits of agreement for E1 concentrations. RIA measurements were numerically but not statistically greater than those of LC–MS/MS for P4, CORT, T, and E1 values (average percent difference: P4, 10.6%; CORT, 13.1%; T, 12.9%; and E1, 3.2%). As depicted by the Bland–Altman plots, the differences in values obtained by RIA for E1 concentrations in samples were greater than LC–MS/MS values at higher serum concentrations (Fig. 4B) while for T measurements in serum samples RIA values were consistently greater than the values found by LC–MS/MS (Fig. 3D). Calculated ICC values indicated an agreement between LC–MS/MS and RIA measurements of T (0.97)

and E1 (0.97) serum hormone concentrations. Measurements of serum ANDRO (Fig. 3A; B) and E2 (Fig. 4C; D) content by RIA and LC–MS/MS in serum samples were found to agree as indicated by their calculated ICC values of 0.62 and 0.59 (ANDRO and E2; respectively). It is clear from the Bland–Altman plot of the ANDRO measurements (Fig. 3B) that the RIA systematically gives an elevated value when compared to those obtained by LC–MS/MS (average difference of 26.3%). The mean bias between the two methods was determined to be − 0.9153 ng/ml. LC–MS/MS evaluation of serum E2 concentrations was higher than what was determined by RIA (12.1%), and this is illustrated in the Bland–Altman plot (Fig. 4D) where the mean bias between the two methods was 2.835 pg/ml. There were no measurements that were outside of the limits of agreement between the two methods of measurement for either ANDRO or E2. Daily urinary CORT concentrations prior to and after dosing are shown in Table 3 for Sprague Dawley dams from GD 14 to 18. CORT measurements as determined by RIA and LC–MS/MS were standardized to urinary creatinine. Daily urinary CORT concentrations were elevated in post-dosing periods following daily exposures to 100 mg/kg of ATR in pregnant dams (Table 3), but the values were not analyzed for statistical significance due to a limited sample size. The correlation between LC–MS/MS and RIA for the measurement of urinary CORT is demonstrated by the scatter and Bland–Altman plot for urinary CORT measurements in Fig. 5(A and B) and the calculated ICC value of 0.94. The mean bias between the two methods of measurements was 0.039 (CORT/creatinine) indicating a slight elevation in urine LC–MS/MS CORT measurements when compared to that of RIA values. Two measurements were outside of the limits of agreement between the two methods (2.9% of the total number analyzed).

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Fig. 3. Scatter plots (A and C) and Bland–Altman plots (B and D) of androstenedione and testosterone serum measurements. Male Wistar rats were pre-dosed with vehicle control (methyl cellulose) for 5 days prior to receiving 5 daily doses of either vehicle or ATR (200 mg/kg) by oral gavage (n = 10). Groups of animals were killed 2 h following their final dose. ANDRO and T were measured by LC–MS/MS and RIA. Plots detail the agreement of ANDRO (A and B) and T (C and D) LC–MS/MS and RIA measurements. For the Bland–Altman plots (B and D), the dashed line indicates the mean difference between the two methods (bias) and the dotted lines demonstrate 2 SD of the mean difference (limits of agreement). Filled squares represent individual measurements.

4. Discussion In this study, the performances of commercially available RIAs used in our laboratory and LC–MS/MS were compared for a number of steroid hormones commonly measured in toxicological studies in serum (P4, CORT, ANDRO, T, E1, E2) and urine (CORT) following exposure to ATR. Steroid means in either serum or urine that were evaluated by RIA or LC–MS/MS analysis were found not to significantly differ within their respective treatment groups (i.e., control RIA steroid means versus control LC–MS/MS steroid means). P4, E1, and E2 concentrations were significantly higher in the sera of male rats when evaluated 2 h following the last of 5 daily doses of ATR, a finding that is consistent with previous reports of an increased P4 following a single dose of ATR (Laws et al., 2009) and elevated E1 and E2 concentrations following multiple exposures to ATR during puberty (Stoker et al., 2000, 2002). Values obtained by RIA kits and LC–MS/MS in both serum and urine were significantly correlated for each of the steroid hormones. There was excellent agreement between the two methods, as determined by the calculation of the ICC values, for the measurements of serum P4, CORT, T, E1, and urine CORT concentrations (ICC values N 0.8) and good agreement in the measurements of serum ANDRO and E2 concentrations (ICC values N 0.5). Interpretation of the ICC values can vary based on the scale used, but a conservative guideline is that an ICC value of less than 0.4 reflects poor agreement, values of 0.4–0.75 reflect fair to good agreement, and values greater than 0.75 reflect an excellent agreement (Rosner, 2005; Sampat et al., 2006). Calculated Pearson's r values, as determined by calculating the linear relationship between RIA (x-axis) and LC–MS/MS (y-values), further support the correlation between the two methods of measurement of steroid hormones (Pearson's r values N 0.81; with the exception of E2, r = 0.64). The

lower sample size used to compare LC–MS/MS and RIA for the measurement of E2 (control, n = 8; ATR, n = 7) as compared to those used for the comparison of other steroid hormones (CORT, P4, and T; n = 10) may have contributed to both the lower the ICC and Pearson's r correlation coefficient values when compared with the other steroid hormones (CORT, E1, P4, and T) despite comparable differences in the values obtained by both measurement methods, i.e., percent differences between LC–MS/MS and RIA values of less than 13% (Weir, 2005). RIA measurements of most of the steroids, with the exception of E2, were numerically but not significantly higher than the values determined by LC–MS/MS, and these findings are comparable with other published comparisons between RIA and LC–MS/MS (Chang et al., 2003; Etter, Eichhorst, & Lehotay, 2006; Faupel-Badger et al., 2010; Hsing et al., 2007; Janse et al., 2011; Rosner, Auchus, Azziz, Sluss, & Raff, 2007; Soldin & Soldin, 2009; Taylor, Machacek, & Singh, 2002) or GC–MS/MS (Dorgan et al., 2002; Haisenleder et al., 2011; Santen et al., 2007) measurements of hormones in various matrices (serum and urine). While there was no statistically significant difference in the values obtained, LC–MS/MS analysis of E2 in the serum yielded results that were, on average, 12% higher than RIA values. The elevated E2 concentrations could be a result of the hydrolysis of some of the E2 conjugates during the derivatization process. A previously reported study has demonstrated that samples that were derivatized prior to LC–MS/ MS had results 10–20% higher as compared with those that did not undergo derivatization; a finding the authors concluded was likely due to the use of a dansyl chloride solution at a high pH during the derivatization (Soldin & Soldin, 2009). Estrogens have been measured without derivatization in human serum using quantitation by MRM analysis performed in negative ion mode (Guo, Gu, Soldin, Singh, & Soldin, 2008), but this method was not evaluated here.

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Fig. 4. Scatter plots (A and C) and Bland–Altman (B and D) plots of estrone and estradiol serum measurements. Male Wistar rats were pre-dosed with vehicle control (methyl cellulose) for 5 days prior to receiving 5 daily doses of either vehicle or ATR (200 mg/kg) by oral gavage (control n = 8; ATR, n = 7). Groups of animals were killed 2 h following their final dose. E1 and E2 were measured by LC–MS/MS and RIA. Plots detail the agreement of E1 (A and B) and E2 (C and D) LC–MS/MS and RIA measurements. For the Bland–Altman plots (B and D), the dashed line indicates the mean difference between the two methods (bias) and the dotted lines demonstrate 2 SD of the mean difference (limits of agreement). Filled squares represent individual measurements.

Although not statistically significant, the greatest deviations between RIA and LC–MS/MS measurements were observed for serum ANDRO concentrations which were, on average, 26% higher by RIA than determined by LC–MS/MS. This may reflect an overestimation of the concentration due to differences in the matrix of the serum samples. As previously detailed, the RIA used in this study was designed for use with human serum and cross-reacting proteins in the matrix may lead to elevated values (Haisenleder et al., 2011; Miller & Valdes, 1991; Stenman, 2001). Samples were extracted by SPE prior to LC–MS/MS analysis and this step may have removed any interfering impurity in the serum. It is unlikely that the test chemical administered (ATR) to the rats interfered with the ANDRO RIA, because there was no significant difference between the numerical differences between RIA and LC–MS/MS measurements from control (average difference of 0.918 ±

0.35 ng/ml; 23%) or ATR treated (average difference of 1.09 ± 0.40 ng/ml; 29%) animals. Furthermore, the high degree of correlation between the two measurement methods for other steroids measured here suggests that neither ATR nor its metabolites interfered with performance of the RIA kits. There are advantages and disadvantages to both RIA and LC–MS/MS for the quantification of steroids in rat biological media. Steroid concentrations in plasma can vary in orders of magnitude depending on gender and toxicant effects, and sample volumes can be limited in toxicological studies. Therefore, there is a need for a method for measuring steroid hormones that is reliable across a wide range of values, with increased sensitivity, and that can accommodate limited sample volumes. RIAs can be beneficial in the analysis of steroid hormones in rodent samples because of the relatively rapid processing time for numerous samples,

Table 3 Daily urinary CORT concentrations as standardized to urinary creatinine content before and after dosing in pregnant dams from GD 14 to 18. Collection period

LC–MS/MS Cage control

GD 14 GD 15 GD 16 GD 17 GD 18 a b c d

PREc POSTd PRE POST PRE POST PRE POST PRE POST

0.66 0.28 0.47 0.44 0.59 0.62 0.60 0.10 0.72 0.19

± ± ± ± ± ± ± ± ± ±

0.21a,b 0.12 0.11 0.15 0.12 0.44 0.11 0.08 0.16 0.03

RIA 0 mg/kg

100 mg/kg

Cage control

0 mg/kg

100 mg/kg

0.16 ± 0.18 ± 0.32 ± 0.15 ± 0.48 ± 0.17 ± 0.54 ± 0.15 ± 0.62 ± 0.11 ±

0.24 ± 1.70 ± 0.92 ± 1.42 ± 0.79 ± 1.01 ± 0.53 ± 2.24 ± 0.65 ± 0.98 ±

0.37 ± 0.33 ± 0.48 ± 0.38 ± 0.55 ± 0.30 ± 0.53 ± 0.21 ± 0.50 ± 0.25 ±

0.20 ± 0.20 ± 0.32 ± 0.31 ± 0.47 ± 0.24 ± 0.54 ± 0.21 ± 0.52 ± 0.20 ±

0.19 ± 1.14 ± 0.78 ± 1.25 ± 0.71 ± 0.71 ± 0.54 ± 1.07 ± 0.32 ± 0.42 ±

0.02 0.06 0.05 0.02 0.10 0.03 0.07 0.03 0.05 0.00

Urinary CORT (ng/ml) was standardized to urinary creatinine content (mg/dl). Mean ± SEM (n = 2 for each control group; n = 3 for ATR). Collection period PRE is 6 h preceding dosing (0100–0700). Collection period POST is 6 h immediately following dosing (0700–1300).

0.02 0.33 0.28 0.23 0.08 0.59 0.08 0.82 0.09 0.60

0.15 0.03 0.24 0.14 0.16 0.09 0.11 0.09 0.09 0.07

0.04 0.01 0.05 0.20 0.03 0.04 0.02 0.02 0.13 0.00

0.06 0.03 0.17 0.22 0.16 0.31 0.11 0.44 0.01 0.21

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Fig. 5. Scatter plot (A) and Bland–Altman plot (B) of urinary corticosterone measurements. Timed pregnant Sprague Dawley rats were pre-dosed with vehicle control (methyl cellulose) and acclimatized to the metabolism cages daily for 5 days prior to receiving either vehicle or ATR (100 mg/kg) by oral gavage daily for 5 days (i.e., GD 14–18). Urine was collected daily during the treatment period for 2 consecutive 6 h intervals preceding (0100–0700) and following (0700–1300) dosing. Urinary CORT was measured by LC–MS/MS and RIA. Both measurements were standardized to urinary creatinine content as quantified during each respective collection period. Plots detail the agreement of urinary CORT LC–MS/MS and RIA measurements. For the Bland–Altman plot (B), the dashed line indicates the mean difference between the two methods (bias) and the dotted lines demonstrate 2 SD of the mean difference (limits of agreement). Filled squares represent individual measurements.

and they typically do not require any sample extraction. Furthermore, RIAs are technically simple to perform and relatively inexpensive per sample for all of the assays used in the current study (ANDRO, CORT, E1, E2, P4, and T). However, RIAs require the use of gamma/scintillation counters to quantify the radioactive signals, laboratory radiation material licenses, and generate radioactive waste. They also typically require larger volumes of sample in order to analyze for multiple steroids, and have the potential for cross-reactivity due to interfering proteins in the biological matrix of the sample assayed. LC–MS/MS for the analysis of steroid hormones can be advantageous in that multiple steroids can be measured from a smaller volume of sample, and it has increased sensitivity and specificity due to the high resolution of the hormones accomplished by the LC and the increase in specificity as a result of the MS/MS (Kushnir et al., 2010; Soldin & Soldin, 2009). LC-MS/MS can be expensive due to both the initial cost of the equipment and routine maintenance fees. Additionally, the use of LC–MS/MS to analyze steroid concentrations requires the use of organic solvents and is associated with longer turnaround times for results given the need for SPE before sample analysis. Furthermore, advanced technical training and skills, particularly during method development and troubleshooting, are necessary to perform mass spectroscopy. In conclusion, results from this study demonstrate a significant correlation between the measurement of steroids within rat serum and urine using commercially available RIA kits frequently employed within our laboratory and LC–MS/MS. Differences in the absolute measurements in ANDRO and E2 in the sera existed, but these differences were not statistically significant and results were proportionately comparable between control and test groups. These data confirm the accuracy of the steroid concentrations previously published by our laboratory (Fraites et al., 2009; Hotchkiss et al., 2012; Laws et al., 2009), and in particular, the E1 and E2 levels determined to be present in male rats (Stoker et al., 2000, 2002). Depending on resources and equipment available, both the commercially available RIA kits selected for comparison here and LC–MS/MS are acceptable methods for the evaluation of steroids in rat biological media following exposure to ATR. Funding This research was funded entirely by the Office of Research and Development, U.S. Environmental Protection Agency, Washington, DC 20406. Acknowledgments This work was conducted at the National Health and Environmental Effects Research Laboratory, U.S. EPA, Research Triangle Park, NC, and

supported by the U.S. EPA and the Oak Ridge Institute of Science and Education Research Participation Program. The authors gratefully acknowledge the contributions of Alvin Moore, Henry Deas, Derek Puffer, Guadalupe Moran, Priority One Services, Alexandria, VA; Faye Poythress, Shelley Bagby, Annemarie Shoffner, Patty Dillard, Marta Aguilar, and Vivian Wilson, Alpha Omega Bioservices, Baltimore, MD, for their outstanding technical support and assistance with animal care; Deborah Best, U.S. EPA, for her assistance with animal dosing; Michelle Hotchkiss and Ashley Murr, U.S. EPA, for their assistance with RIAs; and Dr. Jamie Dewitt (East Carolina University), and Dr. Jerome Goldman (U.S. EPA) for their reviews and helpful comments on earlier drafts of the manuscript.

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