ToxSci Advance Access originally published online on April 2, 2008
Toxicological Sciences 2008 104(1):231-233; doi:10.1093/toxsci/kfn068
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Response to: Comments on "Evaluation of Estrogenic Activities of Aquatic Herbicides and Surfactants Using a Rainbow Trout Vitellogenin Assay"
Department of Environmental Science, University of California, Riverside, California 92521
Received March 15, 2008; accepted March 25, 2008
We have read the comments of Kramer et al. (unpublished data) on our paper, "Evaluation of estrogenic activities of aquatic herbicides and surfactants using a rainbow trout vitellogenin assay" (Xie et al., 2005a
).
We appreciate their comments and would like to respond to them.
"JUVENILE" TROUT MAY BE FEMALE
Kramer et al. (unpublished data) suggest that the fish utilized for the analyses were of "mixed sex" and likely confound the estrogenic assay because one or more of the animals utilized for evaluations of feminization could be "female." They indicated that Bon et al. (1997) showed that juvenile females possessed 423 µg/ml of vitellogenin whereas males had only 0.38 µg/ml. We wholeheartedly agree with Kramer et al. (unpublished data) that juvenile "females" do possess vitellogenin that is significantly higher than that of males. There are two key terms here that require definition: the first is "juvenile" and the second is "females." In order to determine whether a rainbow trout is male or female, an evaluation of gonadal tissue must be undertaken. Rainbow trout can vary in their lifestage development between hatcheries, but generally reach sexual maturity between 12 and 18 months posthatch. Without genotypic markers or histological verification, gender cannot be determined unless morphological presence of female gonads can be observed. This usually occurs after 6 months posthatch. Bon et al. (1997) evaluated rainbow trout at various life stages but did not provide ages. Instead they provided sizes and defined "juveniles" as fish of 360 g. They also measured vitellogenin (68.5 µg/ml) in "immature" females which were "less than 100 g."
Contrary to what was suggested by Kramer et al. (unpublished data), the fish utilized in Xie et al. (2005a) were less than 30 g and approximately 2 months of age (
11 cm was provided as a mean length from which a body mass can clearly be estimated). Gonadal material indicative of gender was not present in any of the fish. Thus, gender could not be determined. Carlson and Williams (1999) did not detect plasma vitellogenin in any rainbow trout (undetermined, male or female) under 65 g and 12 months of age. We recommend that if gonadal tissue indicative of gender cannot be observed, then the fish is an appropriate model. Regardless, one would also expect control groups to show the same variance in response, if the group was influenced by one or more precocious female.
NORMALIZATION TO PLASMA PROTEIN
We are definitely proponents of normalization of vitellogenin protein content to mass/moles of total protein. The rationale for this is that estradiol plays a significant role in fish osmoregulation. Several studies have demonstrated that estradiol downregulates NaK-ATPase in the gill of several fish species including rainbow trout (Carrera et al., 2007; McCormick, 1995; McCormick et al., 2005). Alteration of osmoregulation can dramatically alter plasma volumes and osmolality. To base measurements of a protein on volume when osmolality can be affected by the treatment can lead to uncertain results. This is borne out in the variance of total plasma protein concentrations among a cohort. We have observed concentrations ranging from 7 to 30 mg/l in fish of this age. Because a "high" value of vitellogenin is in the µg/ml range, there is no correlation between the two endpoints. We continue to provide our measurements normalized to total protein, but can provide the volume normalized values when asked by reviewers (Tilton et al., 2002).
ESTRADIOL EQUIVALENTS CALCULATIONS
As we have indicated in numerous publications (Schlenk, 1996, 1999, 2003; Schlenk and Di Guilio, 2002), it is imperative that molecular or biochemical biomarkers be calibrated before utilization. Concentration response curves with E2 are run "simultaneously" with each sample evaluation using the same hatch of animals. In fact, it is interesting that Kramer et al. indicated that our methods were based solely on Xie et al. (2005b). In the paper in question (Xie et al., 2005a), three references (Huggett et al., 2003; Thompson et al., 2000; Xie et al., 2005b) were provided on the method of estradiol equivalents (EEQ) calculation not the duration of exposure. As indicated Xie et al. 2005a, the duration of exposure was 7 days. The E2 exposure utilized to measure EEQs was also 7 days and performed simultaneously. All values of vitellogenin observed in herbicide or surfactant treated fish were within the linear range of E2 standards.
GREATER THAN ADDITIVE RESPONSES
As presented in Xie et al. (2005a), the estrogenicity of the mixtures was calculated based on the model of concentration addition, which assumes that mixtures act via a similar mode of action (i.e., estrogen receptor) in producing an effect (Altenburger et al., 2003; Loewe and Mulschnek, 1926). The model is based upon the premise that when compound A causes a 40% response and compound B causes a 30% response, when provided in a mixture, a 70% response would be predicted based upon assumed additivity. We utilized the term, "predicted" throughout the manuscript and figures. When the mixture value was significantly greater than the "predicted" value, we concluded a "greater than additive" response. In contrast to claims made by Kramer et al. (unpublished data), the vitellogenin levels after treatment with triclopyr alone are clearly shown in Figure 1 and that of the surfactants in Figure 2 of Xie et al. (2005a). The triclopyr values of EEQ in Figure 5, were significantly different (p 
0.05) from EEQ determined from triclopyr + the surfactant target prospreader activator (TPA). With regard to claims that greater than additive responses of estrogenic activity were observed in Anderson Pond, the text clearly states on p. 397; "However, caution should be used as water was not evaluated prior to pesticide application, and other compounds such as natural phytoestrogens may be present."
ESTROGENICITY OF 2,4-DICHLOROPHENOXYACETIC ACID
Table 1 provides a list of the references Kramer et al. (unpublished data) claim were omitted from Xie et al. (2005a). They claim that "all of which reported negative results for estrogenicity" of 2,4-dichloro-phenoxyacetic acid (2,4 D). Unfortunately, although we tend to shy away from literature we cannot obtain or interpret, the abstract of the Korean Journal (Hwang 2002) quite clearly states that 2,4 D "acted as xenoestrogenic contaminant in ER." Indeed, we are in agreement with all of these in vitro studies which (with the possible exception of Hwang, 2002 and Petit et al., 1997) would likely fail to have any biotransformation capacity. It is our hypothesis, which is clearly stated in the Xie et al. (2005a) publication, that 2,4 D is likely converted to an estrogenic metabolite (2,4 chlorophenol) in vivo. Thus, we are in total agreement that 2,4 D has weak estrogenic activity in vitro. However, in vivo activity potentially due to biotransformation to a more potent estrogenic metabolite was significantly greater. These data are consistent with other studies in our lab demonstrating in vitro activity significantly underestimates in vivo activity with the possible exception of domestic wastewater effluent lacking input from industrial sources.
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APMP2 VERSUS XIE ET AL. (2005a)
Lastly, Kramer et al. (unpublished data) note that a preliminary report provided in 2004a is inconsistent with our Xie et al. (2005a) publication. We concur with this observation and would also like to point out that this "preliminary" report differed from the Final report provided in Phase 3 of the study. Unfortunately, it would appear that Kramer et al. (unpublished data) failed to note the text on page 71 of the Phase 2 interim report (Siemering, 2004a) that states: "For pesticides or surfactants where increased estrogenic activity was observed a full dilution series of tests will be conducted in 2004." This subsequent study was Phase 3 (Siemering, 2004b), which is essentially the Xie et al. (2005a) publication. As stated in Xie et al. (2005a), the concentrations of the pesticides were measured by the California Department Fish and Game Water Pollution Control Laboratory. Each concentration was diluted 1:10 in serial dilution for the dose-response study. Thus, a better term might have been "calculated concentrations" although measured values were utilized for the initial dilution and field water exposures.
In conclusion, we appreciate the extensive review of our publication and the opportunity to provide additional comments. We hope we have clarified uncertainties. However, we are of the opinion that the comments of Kramer et al. (unpublished data) fail to merit any alteration in our results or subsequent interpretations. The work clearly shows that 2,4 D and triclopyr demonstrated estrogenic activity in this strain of rainbow trout, and that this activity was enhanced by two surfactants.
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