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TABLE 1. Extract Concentrations and 17-Estradiol Equivalents (EEQs) (ng/mL) c location date NP OP NPE E2 EE2 NP/OP-EEQ a E2/EE2-EEQ b LV Wash 4/30/97 4560 172 Lake Mead 36000 10.70 1.92 0.061 10.9 LV Bay 4/30/97 3000 108 19400 8.84 2.08 0.040 9.05 9/5/97 640 ND 12710 0.752 1.01 0.008 0.86 LV Marina 9/5/97 ND ND ND 1.08 ND NA 1.08 Saddle Island 4/30/97 ND ND ND ND ND NA NA Callville Bay 9/5/97 ND ND ND Trenton Channel ND ND NA NA WWTP 8/30/97 1916 20 21600 4.26 ND 0.024 4.26 Chem. 8/30/97 3450 60 29200 3.64 ND 0.044 3.64 B. Lagoon 8/30/97 3740 264 34700 5.18 1.44 0.052 5.30 M. Creek 8/30/97 4740 324 71260 WWTPs 4.24 ND 0.066 4.25 BV-upstream 10/8/97 ND ND ND 2.50 ND NA 2.50 BV-effluent 10/8/97 148000 2350 1160000 14.6 3.04 1.90 14.9 MA-upstream 10/8/97 ND ND ND ND ND NA NA MA-effluent 10/8/97 2065 64 19400 3.62 1.43 0.027 3.77 ER-upstream 10/8/97 ND ND ND ND ND NA NA ER-effluent 10/8/97 680 ND ND 1.90 ND 0.009 1.90 a Nonylphenol and octylphenol-derived 17-estradiol equivalents. NP/OP-EEQ ) (NPrelative potency(REP) × NPconcentration) + (OPREP × OPconcentration). NPREP ) 1.25 × 10 -5 .OPREP ) 1.9 × 10 -5 . A REP estimate was not available for NPE; therefore, it was not considered when deriving EEQ estimates. b Estradiol and ethynylestradiol-derived 17-estradiol equivalents. E2/EE2-EEQ ) (E2REP × E2concentratration) + (EE2REP × EE2concentration). E2REP ) 1.0. EE2REP ) 0.10. c ND ) not detectable; NA ) not applicable FIGURE 3. Fine fractionation of LV Bay, Lake Mead (April), F3 extract using RP-HPLC with fluorescence detection followed by luciferase induction in the MVLN cell bioassay (estrogen responsive) by the corresponding fractions. Response magnitude presented as percentage of the average maximum response observed for a 1000 pM 17-estradiol standard (%-E2-max). Horizontal lines represent ( 3 SD from the mean solvent control response (set to 0%-E2-max). the samples (Tables 1 and 2). The ER agonist potency of EE2 relative to E2 for luciferase induction in the MVLN assay previously has been reported to be approximately 0.1 (25). Based on the concentrations of E2 and EE2, and their corresponding relative potencies, samples collected from LV Wash and LV Bay in April 1997 were estimated to contain 10.9 and 9.05 ng E2/EE2-derived EEQ/mL, respectively. These concentrations should have yielded doses of approximately 50 and 41 fmol EEQ/well in the MVLN bioassay. Based on regression against an E2 standard curve, such doses would be expected to yield responses of approximately 92% and 88% E2-max, respectively. Based on the range of uncertainty in the predicted responses (Table 2) and the variability of the observed bioassay responses (Figure 1c), the responses observed for the samples collected from LV Wash and LV Bay in April 1997 were not markedly different from predicted responses. Thus, the known E2 and EE2 composition of F3 of the samples collected from LV Wash and LV Bay appeared to account for all the ER agonist potency observed. Additional TABLE 2. Extract 17-Estradiol Equivalents (EEQs) (ng/mL) and Predicted MVLN Responses e location date NP/OP- EEQ a predicted response b E2/EE2- EEQ c predicted response d LV Wash 4/30/97 Lake Mead 0.061 0 10.9 92 LV Bay 4/30/97 0.040 0 9.05 88 9/5/97 0.008 0 0.86 35 LV Marina 9/5/97 NA NA 1.08 41 Saddle Island 4/30/97 NA NA NA NA Callville Bay 9/5/97 NA NA NA NA Trenton Channel WWTP 8/30/97 0.024 0 4.26 71 Chem. 8/30/97 0.044 0 3.64 68 B. Lagoon 8/30/97 0.052 0 5.30 76 M. Creek 8/30/97 0.066 WWTPs 0 4.25 71 BV-upstream 10/8/97 NA NA 2.50 59 BV-effluent 10/8/97 1.90 53 14.9 99 MA-upstream 10/8/97 NA NA NA NA MA-effluent 10/8/97 0.027 0 3.77 68 ER-upstream 10/8/97 NA NA NA NA ER-effluent 10/8/97 0.009 0 1.90 53 a Nonylphenol and octylphenol-derived 17-estradiol equivalents. NP/OP-EEQ ) (NPrelative potency (REP) × NPconcentration) + (OPREP × OPconcentration). NPREP ) 1.25 × 10 -5 .OPREP ) 1.9 × 10 -5 . A REP estimate was not available for NPE; therefore, it was not considered when deriving EEQ estimates. b MVLN bioassay response magnitudes predicted based on regression of NP/OP-derived EEQ against a 17-estradiol standard curve. Units are %E2-max. c Estradiol and ethynylestradiol-derived 17-estradiol equivalents. E2/EE2-EEQ ) (E2REP × E2concentration) + (EE2REP × EE2concentration). E2REP ) 1.0. EE2REP ) 0.10. d MVLN bioassay response magnitudes predicted based on regression of E2/EE2-derived EEQ against a 17estradiol standard curve. Units are %E2-max. e NA ) not applicable. Note: total EEQ ) NP/OP-EEQ + E2/EE2-EEQ. Predicted bioassay response magnitudes are not additive. fractionation of the F3 extracts from LV Wash and LV Bay revealed that all of the ER agonist potency was associated with the fine fractions (FFs) 3 and 4, which equate roughly to the retention times of E2 and EE2 (Figures 2 and 3). FFs 3 and 4 from the F3 extract of the LV Wash sample were collected, combined, and fractionated again by RP-HPLC using a slower flow rate and solvent gradient to separate E2 and EE2 (Figure 4). ER agonist potency was observed in fine fractions where E2 and EE2 elute, and the magnitude of VOL. 35, NO. 18, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 3623
FIGURE 4. Further fractionation of LV Wash, Lake Mead, F3 extract using RP-HPLC with fluorescence detection followed by luciferase induction in the MVLN cell bioassay (estrogen responsive) by the corresponding fractions. Response magnitude presented as percentage of the average maximum response observed for a 1000 pM 17-estradiol standard (%-E2-max). Horizontal lines represent ( 3 SD from the mean solvent control response (set to 0%-E2-max). Dashed line shows chromatography of E2 and EE2 standards with no corresponding bioassay results. induction was consistent with that predicted from EEQs calculated from the measured concentrations of E2 and EE2 and their relative ER-agonist potencies (Table 2). The greater ER agonist potency of the water extracts from LV Wash and LV Bay was most likely due to increased concentrations of E2 and EE2 as a result of WWTPs discharging into the Las Vegas Wash serving a larger population of humans. Significant ER agonist potency was also associated with F3 extracts of water from three locations on the Trenton Channel of the Detroit River (B. Lagoon, Chem., and WWTP) and BV-effluent (Figure 1). From E2 and EE2 concentrations, B. Lagoon, Chem., WWTP, and BV-effluent samples were estimated to contain 5.30, 3.65, 4.25, and 14.9 ng EEQ/mL, respectively (Table 2). Based on regression against an E2 standard curve, these concentrations of EEQ were predicted to yield responses of 76%, 67%, 71%, and 99% E2-max, respectively (Table 2). Observed MVLN cell responses for these F3 samples were, however, less than predicted (Figure 1). Further fractionation and bioanalysis of F3 extracts from BV-effluent and B. Lagoon indicated that all of the observed ER agonist potency was contained in FFs 3 and 4 (Supporting Information, Figures 4 and 5). However, the magnitude of induction of the FFs was markedly different from that of the corresponding total F3 extract. This suggests that interfering compounds and/or unidentified ER (ant)agonists present in F3 might have modulated the potency of the known ER agonists. The potential presence of interfering compounds and/or unknown ER (ant)agonists was also suggested by the lack of significant response for several samples. Based on concentrations of E2 and EE2, six additional F3 samples should have elicited significant responses in the MVLN bioassay. Concentrations of EEQs calculated from concentrations of EE2 and E2 in samples collected from LV Bay (Sept. 1997), LV Marina, M. Creek, BV-upstream, MA-effluent, and EReffluent were estimated to range from 0.85 to 4.25 ng EEQ/ mL. Regression against an E2 standard curve would result in predicted responses of 35-71% E2-max in the MVLN assay for these samples. Thus, the responses were less than predicted for these samples. The reason for this observation is unknown at this time. MVLN responses for whole extracts were similar to those for F3. In those cases where F3 elicited a significant response, the whole extract also elicited a significant response (Figure 3624 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 35, NO. 18, 2001 1). In five of the six cases, the whole extract response was slightly less than the response elicited by F3. This suggests that F1 and F2 may have contained some interfering compound(s) or ER antagonists that modulated the potency of the known ER agonists in the samples. However, no significant ER antagonist responses were observed (Figure 1). Because the decreases were slight, however, the results suggest that the bulk of potential interfering (antagonistic) compounds were present in F3. The extract of BV-effluent was the only sample for which the whole-extract response was greater than the corresponding F3 response. It was also the only sample for which the concentrations of NP and OP in F2 were predicted to yield significant ER agonist activity. NP and OP accounted for 11% of the total EEQ calculated for the BV-effluent extract. Thus, although F2 of the BV-effluent sample failed to elicit a significant response, NP and OP may have contributed to the response of the whole extract, such that the total extract response was greater than the F3 response. In general, however, NP and OP accounted for less than 1% of the total concentrations of sample EEQs present in samples. No significant ER activity was observed for blank samples, including field blanks, laboratory blanks, and solvent blanks. Water concentrations of these compounds have been described previously (26). Summary The potency balance calculations based on instrumental analyses and bioassay-directed fractionation support the conclusion that E2 and EE2 were the dominant environmental estrogens in the samples. Interfering compounds or ER antagonists present in samples (predominantly in F3) may have acted to mask or dampen the potency of the known ER agonists in the MVLN bioassay. All observed responses in the MVLN bioassay were either less than, or approximately equal to, responses predicted based on the measured concentrations and relative potencies of known ER agonists. NP and OP generally contributed less than 1% of the total EEQs. Furthermore, sample fractions containing NP and OP did not elicit significant activity. For most samples, fractions containing E2 and EE2 elicited responses slightly greater than the responses of the corresponding whole extracts. Thus, among the ER agonists detected in the samples E2 and EE2 appear to be responsible for the bulk of the activity. The fact that observed responses were generally lower than predicted suggests the presence of interfering compounds. MVLN responses for whole extracts were only slightly less than those for F3 samples. This suggests that the interfering compounds may have been present in F3. Because there were few instances where MVLN responses were greater than those predicted based on concentrations of EEQs present in the extracts, the known composition can account for the magnitude of response observed. It is unlikely that there were additional ER agonists of significant concentration that were not identified. There are insufficient data to explain differences in bioactivity among the locations investigated. It should be noted that samples from the Trenton Channel of the Detroit River in Michigan received less effluent from municipal WWTPs relative to the volume of the receiving water compared to the other sites. Also, the population served by the WWTPs varied greatly among sites. Further investigations would be necessary to determine the actual loading of bioactive compounds as a function of population density. Without knowing the available fractions and bioaccumulation potential of the various compounds and dose-response relationships for target species, it is not possible to predict the potential effects of the observed concentrations of ER agonists on biota.
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