No job name

Environ. Sci. Technol. 2001, 35, 2476-2481
Assessing the Biological Potency of
hormone biosynthesis, and/or by inducing the enzymesresponsible for steroid metabolism (1). Particular focus has Binary Mixtures of Environmental
fallen on chemicals which mimic estradiol-17 binding to the estrogen receptor(s) (ERs) to elicit agonist Estrogens using Vitellogenin
and/or antagonistic responses (2, 3). These chemicals are Induction in Juvenile Rainbow Trout
collectively described as xenoestrogens. With the exceptionof the synthetic steroids, the xenoestrogens discovered so (Oncorhynchus mykiss)
far are only weakly active when compared with endogenoussteroids. However, evidence for reproductive abnormalitiesthat are indicative of sex hormone disruption in wild fish K A R E N L . T H O R P E , * , † , § T H O M A S H .
populations, supported by in situ monitoring studies with H U T C H I N S O N , † M A L C O L M J .
caged fish, implies that some aquatic environments contain H E T H E R I D G E , † M A R T I N S C H O L Z E , # J O H N xenoestrogens at concentrations high enough to be of P . S U M P T E R , ‡ A N D C H A R L E S R . T Y L E R § concern to wildlife (4-6). These “field” observations are Brixham Environmental Laboratory, AstraZeneca UK Limited, supported by in vivo laboratory studies, where exposure to Freshwater Quarry, Brixham, Devon, TQ5 8BA, U.K., some xenoestrogens induces estrogenic effects in fish at School of Biological Sciences, The Hatherley Laboratories, environmentally relevant concentrations (7-9).
University of Exeter, The Prince of Wales Road, Exeter, The aquatic environment receives a large influx of natural Devon, EX4 4PS, U.K., Department of Biology and Chemistry, and synthetic chemicals from agricultural, industrial, and University of Bremen, Germany, and Fish Physiology Research domestic sources. In Europe, natural estrogens such as E2 Group, Department of Biological Sciences, Brunel University, and estrone, synthetic steroids including, 17R-ethynylestra- Uxbridge, Middlesex, UB8 3PH, U.K. diol, and other nonsteroidal chemicals known to haveestrogenic effects (such as alkylphenols), have been detectedin effluents that discharge into rivers (10, 11). This wide rangeof xenoestrogens in the aquatic environment highlights the Experiments were conducted to assess the in vivo importance of improving our understanding of combination potency of binary mixtures of estrogenic chemicals using effects of these chemicals in organisms as well as their plasma vitellogenin (VTG) concentrations in juvenile individual effects. In many cases xenoestrogens are present rainbow trout (Oncorhynchus mykiss) as the endpoint.
at concentrations too low to be considered of concern The estrogenic potencies of estradiol-17 (E2), 4-tert- individually, but the presence of mixtures of these chemicals nonylphenol (NP), and methoxychlor (MXC) were determined means there is a potential for additive and/or interactive following 14 day exposures to the individual chemicals effects. The existence of interactive effects implies that the and binary mixtures of these chemicals. E2, NP, and MXC estrogenic effect of a mixture somehow deviates from whatis expected, on the basis of the estrogenic effects of the single all induced concentration dependent increases in plasma agents. There are two main analytical models for defining VTG, with lowest observed effect concentrations of 4.7 and the expected effects of a mixture: the model of concentration 7.9 ng L-1 for E2, 6.1 and 6.4 µg L-1 for NP, and 4.4 and addition (CA), which assumes that the compounds act via 6.5 µg L-1 for MXC. Concentration-response curves for fixed a similar mechanism in producing an effect (12), and the ratio binary mixtures of E2 and NP (1:1000), E2 and MXC (1: model of response addition, which assumes that the com- 1000), and NP and MXC (1:1) were compared to those pounds act via independent pathways (13). If a mixture of obtained for the individual chemicals, using the model of xenoestrogens is more potent than would be expected, the concentration addition. Mixtures of E2 and NP were additive combination effect is described as more than additive at the concentrations tested, but mixtures of E2 and (synergistic), and if it is less effective, the combination effect MXC were less than additive. This suggests that while NP is described as less than additive (antagonistic). Suchdeviations from expectation should be demonstrated at more probably acts via the same mechanism as E2 in inducing than one concentration of the mixture.
VTG synthesis, MXC may be acting via a different mechanism- In this study, the estrogenic activities of three environ- (s), possibly as a result of its conversion to HPTE which mental estrogens, namely E2, 4-tert-nonylphenol [NP], and is an estrogen receptor R agonist and an estrogen receptor methoxychlor [MXC], were assessed individually and in antagonist. It was not possible to determine whether binary mixtures (to investigate possible interactive effects) mixtures of MXC and NP were additive using VTG induction, using plasma vitellogenin (VTG) concentrations in juvenile because the toxicity of MXC restricted the effect range female rainbow trout (9), as the response. VTG induction in for which the expected response curve for the binary mixture fish is specifically an estrogen-dependent process, normally could be calculated. The data presented illustrate that restricted to mature females. During reproductive develop- the model of concentration addition can accurately predict ment in female fish, the hypothalamic-pituitary-gonadalaxis stimulates the ovary to produce E2, which is released effects on VTG induction, where we know that both into the bloodstream and transported to the liver. Here it chemicals act via the same mechanism in mediating a diffuses passively into hepatocytes and binds to the ER, stimulating transcription of the VTG gene(s). The VTGsynthesized is then transported to the ovary and sequestered Introduction
A large number of natural and synthetic chemicals have been
* Corresponding author phone: (44) 1803-882882; fax: (44) 1803- labeled as endocrine active, due to their ability to mimic 882974; e-mail: karen.thorpe@brixham.astrazeneca.com.
endogenous hormones. Endocrine active chemicals mediate their effects by binding to hormone receptors as agonists or antagonists, by inhibiting the enzymes responsible for steroid 2476 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 35, NO. 12, 2001
by the developing oocytes, to be stored as yolk for the N-(tert-butyldimethylsilyl)-N-methylthrifluoroacetamide with subsequent embryo (14). Although VTG synthesis is normally 1% tert-butyldimethylchlorosilane (MTBSTFA + 1% TBD- restricted to maturing females, immature females and male MCS). After cooling, 300 µL of bis(trimethysilyl)trifluoroac- fish possess the machinery for VTG production, and exposure etamide with 1% trimethylchlorosilane (BSTFA + 1% TMCS) to estrogens (and their mimics) can trigger VTG synthesis via was added, and the vial was heated to 60 °C for 20 min. The the ER. Given that all the available evidence shows that steroid reagents were removed under nitrogen, and the extracts were estrogens and their mimics act via the ER to induce VTG resuspended in 250 µL of dichloromethane. The derivatized synthesis, the model of CA was used to assess the estrogenic samples were analyzed on a Hewlett-Packard 6890 gas activity of binary mixtures of the test chemicals. A simulation chromatograph (GC) coupled to a Hewlett-Packard 5973 mass technique termed “bootstrap” (15) was used with the model spectrometer (MS), using helium as the carrier gas at 1 mL of CA to construct a 95% confidence belt around the line of min-1. The analysis conditions were as follows; sample prediction. This provided a statistical basis to determine volume, 1 µL; GC column, HP-5MS 30 m × 0.25 mm (id) whether deviations from expectation were significant (those fused silica with 0.25 µm film thickness; injector temperature, that fall outside the 95% confidence of the predicted curve) 300 °C; column program, (1) 50 °C for 10 min, (2) increase or simply due to natural variation in the biological response to 300 °C at 8 °C min-1, (3) isothermal at 300 °C for 10 min.
(those that fall within the 95% confidence of the predicted The MS was operated in the electron impact ionization mode (70 eV) with selected ion monitoring (SIM) of the M-57 ionsfor the TBDMS/TMS derivative of E2 and its deuterated Materials and Methods
analogue, ions m/z 401 and 403, respectively.
Test Organisms. Female juvenile rainbow trout (approxi-
For measurement of NP, 1 L water samples were extracted mately three months old) were obtained from West Country under vacuum (50 mL min-1) onto preconditioned 47 mm Trout, Trafalgar Farm, Cornwall, U.K. (experiments I and II) C18 Empore disks (3M). NP was eluted from the disk using and from Houghton Springs Fish Farm, Dorset, U.K. (experi- 10 mL of methanol, and the extract was diluted 1:1 with ment III). The body weight of the fish used was 10.47 ( 0.71 HPLC grade water. Extracts were analyzed on a HPLC, using g (mean ( SEM; n ) 24) in experiment I; 6.51 ( 0.48 g (n ) a Jasco PU980 LC pump with a mobile phase composition 24) in experiment II; and 7.89 ( 0.22 g (n ) 24) in experiment of 80:20 methanol:water at 2 mL min-1. The analysis III. In all experiments, fish were maintained for 14 days under conditions were as follows; sample volume, 50 µL; HPLC flow-through conditions in de-chlorinated water at 15.0 ( column, Hypersil H5ODS 150 mm × 4.6 mm (id) (Hichrom); 1 °C, with a 16 h light:8 h dark photoperiod, with 20 min Jasco FP920 fluorescence spectrometer detection at 230 nm dawn and dusk transition periods. Prior to the start of each experiment, fish were acclimated in the same conditions for For measurement of MXC, water samples were liquid/ a minimum of 10 days. Throughout the exposures, fish were liquid extracted using hexane; extraction ratios for sample: provided with a feeding ration of 1% of body weight per day hexane were 80:1 (controls), 10:1 (2.4-7.5 µg L-1 treatments), of Keystart Hatchery 1200 fish food pellets (BOCM Pauls or 1:1 (13.5-24.0 µg L-1 treatment). Extracts were analyzed on a Varian 3400 GC, using nitrogen as the carrier gas at 35 Test Chemicals. Methoxychlor (99% purity) was pur-
mL min-1. The analysis conditions were as follows: sample chased from ChemService, Greyhound, Birkenhead, U.K. (Lot volume, 1 µL; GC column, 1 m × 2 mm (i.d.) column packed 180-80A), technical grade NP (99% purity) was purchased with 3% OV17 on 100/120 mesh (Phase Separations); injector from Acros, Fisher Scientific, Loughborough, U.K. (Lot temperature, 255 °C; column isothermal at 235 °C; electron A010020701), and E2 (98% purity) was purchased from Sigma, Experimental Design. Fish were exposed to five con-
Water Supply and Test Apparatus. The supply of dechlo-
centrations of each of the individual chemicals and three rinated water to the laboratory dosing system was monitored concentrations of each binary mixture. Each experiment daily for conductivity, hardness, and free chlorine and was included a dilution water control (DWC) and a solvent control tested for alkalinity and total ammonia twice weekly. The (SC). In all experiments, each treatment consisted of a single conductivity of the test water ranged from 204 to 238 µS replicate containing 12 fish. The test vessels had a working cm-1, the hardness from 41.3 to 47.7 mg L-1 (as CaCO volume of 45 L and were constructed of glass, with a minimum free chlorine was < 2 µg L-1. Alkalinity ranged from 21.2 to of other materials (silicon rubber tubing and adhesive) in Dissolved oxygen concentrations and pH levels were deter- Stock solutions of each chemical were prepared weekly mined in the individual tanks on days 0 and 1 and then twice in HPLC grade methanol (Fisher Scientific) and dosed to weekly throughout the exposure period. In all experiments, glass mixing vessels by means of a peristaltic pump, at a rate the dissolved oxygen concentration remained >80% of the of 0.040 mL min-1, to mix with the dilution water flowing to air saturation value throughout the exposures and pH values the mixing vessels at a rate of 400 mL min-1. From each ranged from 7.04 to 7.54. Water temperatures were monitored primary mixing vessel the test solution flowed into a second constantly throughout the exposure period and ranged from mixing vessel to produce the binary mixtures and then into 15.2 to 15.8 °C. Dilution water and test chemical flow rates the exposure tanks. The SC vessel received the same rate of were checked at least three times per week. The flow-rate addition of methanol, such that the water in all test vessels, provided a 99% replacement time of approximately 7 h.
except the DWC, contained 0.01 mL methanol per liter.
Analytical Chemistry. The actual concentrations of the
For each experiment a “fixed ratio” design was used for reference chemicals were monitored throughout all experi- the binary mixtures, in which the ratio of the two test ments. Water samples were collected from each tank into chemicals was kept constant, while the total concentration solvent-cleaned flasks on days 0, 7, and 14 of the exposures.
of the mixture was varied. The ratio used for each experiment For measurement of E2, 2.5 L water samples were spiked was selected on the basis of earlier work in which E2 was with 5 ng L-1 deuterated-E2 and then extracted under vacuum found to be approximately 1000-fold more potent than NP (50 mL min-1) onto preconditioned 47 mm C18 Envi-disks (Supelco). E2 was eluted from the disks using 30 mL of Experiment I - E2 + NP. Groups of 12 juvenile female
methanol, and the residual solvent was removed under a rainbow trout were exposed for 14 days to nominal con- stream of nitrogen. The extracts were derivatized by heating centrations of E2 at 2.4, 4.2, 7.5, 13.5, and 24.0 ng L-1 and NP to 120 °C for 20 min with 200 µL of pyridine and 300 µL of at 2.4, 4.2, 7.5, 13.5, and 24.0 µg L-1 and to binary mixtures VOL. 35, NO. 12, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2477
TABLE 1. Mean Measured Tank Concentrations of Test Chemicals, in the Individual and Binary Mixture Exposures over the 14
Daysa
Experiment I. 17 -Estradiol and 4-tert-Nonylphenol
mean measured concn estradiol (ng/L)
mean measured concn nonylphenol (µg/L)
individual
individual
Experiment II. 17 -Estradiol and Methoxychlor
mean measured concn estradiol (ng/L)
mean measured concn methoxychlor (µg/L)
individual
individual
Experiment III. 4-tert-Nonylphenol and Methoxychlor
mean measured concn nonylphenol (µg/L)
mean measured concn methoxychlor (µg/L)
individual
individual
of E2 + NP at concentrations of 4.2 ng L-1 + 4.2 µg L-1, 7.5 The model parameter θmin describes the minimal mean ng L-1 + 7.5 µg L-1, and 13.5 ng L-1 + 13.5 µg L-1, respectively.
effect (control response), θmax is the asymptotical maximal Experiment II - E2 + MXC. Groups of 12 juvenile female
effect, θ1 is termed the “location” parameter, and θ2 rainbow trout were exposed for 14 days to nominal con- characterizes the “steepness” of the concentration response centrations of E2 at 2.4, 4.2, 7.5, 13.5, and 24.0 ng L-1 and relationship. The experiments were not designed to deter- MXC at 2.4, 4.2, 7.5, 13.5, and 24.0 µg L-1 and to binary mine maximal effects, so estimation of θmax contains a high mixtures of E2 + MXC at concentrations of 4.2 ng L-1 + 4.2 degree of statistical uncertainty. Due to heterogeneous µg L-1, 7.5 ng L-1 + 7.5 µg L-1, and 13.5 ng L-1 + 13.5 µg L-1, nonrandom variabilities in the replicated data (heterosce- dasticity), each model was fitted using the estimation method Experiment III - MXC + NP. Groups of 12 juvenile female
of generalized least squares (15). To fulfill the statistical rainbow trout were exposed for 14 days to nominal con- prerequisite of symmetrically distributed effect data for this centrations of MXC at 2.4, 4.2, 7.5, 13.5, and 24.0 µg L-1 and estimation method, the plasma VTG concentrations were 4-NP at 2.4, 4.2, 7.5, 13.5, and 24.0 µg L-1 and to binary log10-transformed. LOECs were determined using a non- mixtures of MXC + NP at concentrations of 4.2 µg L-1 + 4.2 parametric Wilcoxon’s rank sum test (17).
µg L-1, 7.5 µg L-1 + 7.5 µg L-1, and 13.5 µg L-1 + 13.5 µg L-1, The expected concentration-response relationships of the binary mixtures were determined using the model of CA Fish Sampling. In all experiments, a subgroup of fish (n
(12). The model can only be used to calculate mixture effects ) 24) was sampled at the outset (day 0) of the experiment, for the same effect range observed for the individual and then all exposed fish were sampled on day 14. Fish were components of the mixture, and if these effects are quan- sacrificed in a lethal dose (200 mg L-1) of MS222 (3- titatively describable in a statistically valid way. Therefore, aminobenzoic acid ethyl ester, methanesulfonate salt) (Sigma), the expected curve of the binary mixtures can only be buffered with 1 M NaOH to pH 7.3. Blood was collected by calculated up to an effect range which is determined by the cardiac puncture, using a heparinized syringe (5000 Units minimum of the two estimated model parameter θmax of both heparin mL-1) and centrifuged (7000g; 5 min, 15 °C), and the compounds. The concentration-response curve for VTG plasma was removed and stored at -80 °C until required for induction is very steep, covering several orders of magnitude VTG analysis. Plasma samples were assayed for VTG using (ng/mL to mg/mL) and often results in a high variability in an established homologous rainbow trout RIA (16).
the vitellogenic response between individuals in a treatment.
Statistical Analyses. For the description of the concen-
Such an inherent variability can complicate any analysis on tration effect relationships for the individual test compounds exposure to individual or mixtures of chemicals. For this and for the binary mixtures, a four-parameter logit regression reason the “bootstrap” methodology (15) was employed with the model of CA, to determine the statistical accuracy of thepredicted combined effects.
Results and Discussion
Measured Concentrations of the Test Chemicals. Mean
where x ) concentration and f(x) ) mean effect.
measured tank concentrations of the individual chemicals 2478 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 35, NO. 12, 2001
FIGURE 1. Plasma vitellogenin (VTG) concentrations in female juvenile rainbow trout exposed to (a) estradiol-17 (E2) (closed circles)
and 4-tert-nonylphenol (NP) (open circles) and (b) fixed ratio binary mixtures (1:1000) of E2 and NP (open circles). In some cases the plasma
VTG concentrations were very similar between fish within a treatment; therefore, not all data points are visible. For each of these exposures
the 95% confidence belts (light gray shaded regions) of the fitted concentration-response relationships (gray lines) are shown. The
expected vitellogenic response for the binary mixture, calculated using the model of concentration addition, is shown as a black line
(b) with a 95% confidence bootstrap belt (dark gray shaded region).
and their binary mixtures are given in Table 1. All exposure estrogen and as an antiandrogen in male fathead minnows concentrations are described using means of the actual (18) and in recombinant yeast reporter gene assays (3), and measured concentrations. For most chemical exposures the it also affects some of the cytochrome p450 mixed function mean measured concentrations were between 70 and 110% oxidase enzymes (19). Some estrogenic activity of NP may of nominal. However, for the highest concentration of NP, also result from alterations it can cause in concentrations of 24 µg L-1, the mean measured concentration was between endogenous E2, rather than by effects mediated via the ER 51% and 68% of nominal. In experiment II (E2 + MXC), the directly (20). The concentration additive behavior of NP with mean measured concentration of E2 was 190% of nominal E2 in these experiments, however, together with the ability for the lowest test concentration (2.4 ng L-1), and the mean of NP to bind the ER (but not the AR) in rainbow trout liver measured concentrations of MXC were between 60% and and brain cells (21), suggests that the VTG response induced by NP is mediated via the ER alone. This may also be true Plasma Concentrations of VTG in Control Fish. The
for the related alkylphenolic chemical, octylphenol, which concentrations of VTG in the plasma of juvenile female fish has been shown to act in an additive manner with E2, in at the onset of the experiments were 500 ( 80 ng mL-1, 310 inducing VTG synthesis in rainbow trout (22).
( 50 ng mL-1, and 540 ( 80 ng mL-1 for experiments I, II, Mixtures of E2 + MXC. For fish exposed to 13.0 µg L-1
and III, respectively. There were no detectable increases in MXC there was a 58% mortality by day 14, and for fish exposed plasma VTG concentrations in either the DWC or in the SC to the mixture of 13.2 µg L-1 MXC + 12.8 ng L-1 E2 there was fish after the 14 day exposure period in any of the experiments a 25% mortality. In all other chemical exposures, 100% of the fish were alive at the end of the experiment. A previous study Mixtures of E2 and NP. There were no mortalities in fish
reported a 96 h median lethal concentration for MXC of 31.2 exposed to E2 and NP or in fish exposed to binary mixtures µg L-1 in juvenile rainbow trout (23). The toxicity of MXC has of these chemicals. Estradiol-17 (concentrations ranging been demonstrated to increase with duration of exposure, from 2.3 to 21.3 ng L-1) and NP (concentrations ranging from and this may account for the apparent greater toxicity of 1.8 to 12.2 µg L-1) produced concentration-dependent MXC observed in this study, compared with Heming et al.
increases in plasma VTG (Figure 1), with LOECs of 4.7 ng L-1 (plasma VTG concentration of 4100 ( 940 ng mL-1, p < 0.05) In experiment II, E2 (concentrations ranging from 4.2 to and 6.1 µg L-1 (plasma VTG concentration of 1450 ( 230 ng 23.0 ng L-1) and MXC (concentrations ranging from 1.4 to mL-1, p < 0.05) for E2 and NP, respectively. The mixture of 13.0 µg L-1) produced concentration-dependent increases E2 and NP, at a fixed 1:1000 ratio, also produced a in plasma VTG (Figure 2), with LOECs of 7.9 ng L-1 for E2 concentration-dependent increase in plasma VTG, with the (plasma VTG concentration of 22 460 ( 8660 ng mL-1, p < lowest mixture concentration tested (4.9 ng L-1 E2 and 3.3 0.05) and 4.4 µg L-1 for MXC (plasma VTG concentration of µg L-1 NP) inducing a 17-fold increase in VTG concentration 9340 ( 6590 ng mL-1, p < 0.05). The mixture of E2 and MXC, (8720 ( 4370 ng mL-1, p < 0.05). The observed VTG induction at a fixed 1:1000 ratio, also produced a concentration- data for the mixture of E2 and NP was close to that predicted dependent increase in plasma VTG (Figure 2), with the lowest by the model of CA (Figure 1). The 95% confidence belt of mixture concentration tested (4.3 ng L-1 E2 and 3.8 µg L-1 the fitted concentration-response relationship for the MXC) inducing a plasma VTG concentration of 18340 ( 7680 observed mixture data overlapped with the 95% confidence ng mL-1 (p < 0.05). When comparing the measured VTG bootstrap belt for the calculated mean of CA, for the whole induction that occurred for the mixture, with the expected mixture effects, calculated according to the model of For many estrogen mimics the pathways by which they concentration addition, mixtures of E2 and MXC were shown alter estrogen-sensitive pathways have yet to be fully to act in a less than additive manner (Figure 2). The characterized. NP has been shown to have multiple mech- concentration-response curve for the experimental mixture anisms of action on the endocrine system; it acts as both an of E2 and MXC is displaced to the right of the predicted VOL. 35, NO. 12, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2479
FIGURE 2. Plasma vitellogenin (VTG) concentrations in female juvenile rainbow trout exposed to (a) estradiol-17 (E2) (closed circles)
and methoxychlor (MXC) (open circles) and (b) fixed ratio binary mixtures (1:1000) of E2 and MXC (open circles). In some cases the VTG
concentrations were very similar between fish within a treatment; therefore, not all data points are visible. For each of these exposures
the 95% confidence belts (light gray shaded regions) of the fitted concentration-response relationships (gray lines) are shown. The
expected vitellogenic response for the binary mixture, calculated using the model of concentration addition, is shown as a black line
(b) with a 95% confidence bootstrap belt (dark gray shaded region).
FIGURE 3. Plasma vitellogenin (VTG) concentrations in female juvenile rainbow trout exposed to (a) methoxychlor (MXC) (closed circles)
and 4-tert-nonylphenol (NP) (open circles) and (b) fixed ratio binary mixtures (1:1) of MXC and NP (open circles). In some cases the VTG
concentrations were very similar between fish within a treatment; therefore, not all data points are visible. For each of these exposures
the 95% confidence belts (light gray shaded regions) of the fitted concentration-response relationships (gray lines) are shown. The
expected vitellogenic response for the binary mixture, calculated using the model of concentration addition, is shown as a black line
(b) with a 95% confidence bootstrap belt (dark gray shaded region).
concentration-response curve, and there is no overlap Although, ER has not yet been identified in the rainbow between the two 95% confidence belts for the whole effect trout, it has been identified in another species of teleost, the seabream (26). In the seabream, ERR and ER are coexpressed The model of CA is based on the assumption that the in the liver, although whether they are both involved in the components of a mixture mediate their effects by similar regulation of vitellogenesis has still to be investigated. If, modes of action. The small but significant deviation from however, both ERR and ER are present in the liver of rainbow additivity for the mixture of E2 and MXC suggests that MXC trout and both play a role in the regulation of vitellogenesis, mediated a vitellogenic response via a different mechanism then the mixed agonist/antagonist activity of HPTE, in the (or pathway) to E2. This complies with reports in the literature presence of E2, might well account for the observed deviation that MXC does not interact directly with the ER, in the same way as E2. The estrogenic activity of MXC is thought to result Mixtures of NP + MXC. There was 100% mortality of fish
from its primary metabolite, 2,2-bis-(p-hydroxyphenyl)-1,1,1- exposed to MXC at a concentration of 20.2 µg L-1 after 6 days trichloroethane (HPTE), which is produced in the liver (by and 50% mortality in fish exposed to 14.3 µg MXC L-1 after O-demethylation) (24). In mammals, HPTE has been found 14 days. In fish exposed to the binary mixture of 11.4 µg L-1 to act as an agonist for ERR but as an antagonist for ER (25).
MXC + 11.9 µg L-1 NP, there was a 58% mortality after 14 2480 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 35, NO. 12, 2001
days, but in all other chemical exposures all of the fish were even simple mixtures of environmental estrogens, on a clearly defined endpoint with a known mechanism of action.
NP (concentrations ranging from 1.8 to 16.3 µg L-1) and MXC (concentrations ranging from 2.0 to 20.2 µg L-1) Acknowledgments
produced concentration-dependent increases in plasma VTG This research was co-funded by the UK Environment Agency, (Figure 3), with LOECs of 6.4 µg L-1 for NP (plasma VTG National Centre for Ecotoxicology and Hazardous Substances, concentration of 128 760 ( 117 540 ng mL-1, p < 0.05) and Wallingford and the AstraZeneca Shared Research Program 6.5 µg L-1 for MXC (plasma VTG concentration of 16 390 ( (supported by AstraZeneca, Avecia, ICI and ZENECA Agro- 7160 ng mL-1, p < 0.05). The mixture of NP and MXC, at a chemicals), Brixham Environmental Laboratory, Devon. The fixed 1:1 ratio, produced a concentration-dependent increase authors wish to acknowledge the valuable technical assistance in plasma VTG, with the lowest mixture concentration tested of colleagues at Brixham Environmental Laboratory, espe- (3.0 µg L-1 NP and 3.5 µg L-1 MXC) inducing a plasma VTG cially, Rob Cumming, Nadine Pounds, and Colin Woods.
concentration of 723 055 ( 481 814 ng mL-1 (p < 0.05).
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the data from these experiments suggest that it is suitable (26) Socorro, S.; Power, D. M.; Olsson, P.-E.; Canario, A. V. M. J. for the prediction of combination effects, where the com- Endocrinol. 2000, 166, 293.
ponents of the mixture act through the same mechanism of (27) Belfroid, A. C.; Van der Horst, A.; Vethaak, A. D.; Schafer, A. J.; action on a clearly defined endpoint. The model accurately Rijs, G. B. J.; Wegener, J.; Cofino, W. P. Sci. Total Environ. 1999,
predicted the combination effects on VTG induction for the mixture of E2 and NP, where we know that both chemicals (28) Rodgers-Gray, T. P.; Jobling, S.; Morris, S.; Kelly, C.; Kirby, S.; Janbakhsh, A.; Harries, J. E.; Waldock, M. J.; Sumpter, J. P.; Tyler, act via the ER in mediating a vitellogenic response. For the C. R. Environ. Sci. Technol. 2000, 34, 1521.
mixture of E2 and MXC the model of CA overpredicted thecombination effect, but this is perhaps not surprising given Received for review October 13, 2000. Revised manuscript the complex mechanism by which MXC has been reported received March 23, 2001. Accepted March 25, 2001. to elicit an estrogenic response (24, 25). Our studies furtherhighlight the difficulties in assessing combination effects of VOL. 35, NO. 12, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2481

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