N. Voulvoulis and M.D. Scrimshaw
3.1 IN VITRO ASSAYS
3.1.1 C OMPETITIVE L IGAND B INDING
These assays are based on the primary mode of action of estrogens (and xenoestro- gens), which is binding to the ER. The implication is that binding to the receptor results in a subsequent effect on biological activity. These assays have the potential for high-throughput screening and are under consideration for assisting with the prioritization of chemicals by the U.S. EPA.16
Measurements utilize the appropriate radiolabeled hormones, predominantly [3H]17β-estradiol in the case of the ER, which is used as a control to confirm the presence of the receptor and to evaluate the effects of competitive binding to that receptor through addition of the compounds being tested. Assays for estrogenic compounds involve the extraction of the ER from a cell line such as MCF-717 and subsequent incubation with [3H]17β-estradiol, either with or without the synthetic estrogen diethylstilbestrol (DES) as a control, or the test compounds in increasing concentrations. The bound, labeled [3H]17β-estradiol is isolated with hydroxyapatite, then freed by incubation with charcoal before extraction with ethanol for scintillation counting.6 If less of the [3H]17β-estradiol is bound to the ER, this is because the test compounds have demonstrated an ability to compete for the binding sites. The greatest limitation of these assays, however, is that although compounds may bind to the receptor, the tests do not distinguish between subsequent agonistic or antag- onistic effects.7,10 Most binding assays rely on the isolation of the receptors from natural cell lines; however, bacterially expressed receptors for high-throughput test- ing have been developed for the human hERα, a reptilian receptor from the liver of the green anole (Anolis carolinensis), aER and from rainbow trout (Oncorhynchus mykiss), rtER.16 The binding of a range of PCB, hydroxylated PCB, and arochlors was also evaluated; however, compounds tested generally exhibited greater affinity for the rtER than either of the other receptors.
The range of available ligand binding assays and the lack of recognized standards for reporting data make intercomparison difficult; however, a number of tests and compounds evaluated are summarized in Table 3.1. In addition to the ER, binding assays for other receptors, such as the androgen receptor (AR), have also been developed,18 with p,p′-DDE being identified as a potent AR antagonist.19 Such assays usually require overnight incubation at around 4°C with AR isolated from rat repro- ductive tissues, such as the epididymis and prostate.14
Ligand binding assays have been used to identify estrogenic activity in a range of compounds, and some workers have compared the effectiveness of tests, for example the binding of PAH to the α and β isoforms of the human estrogen receptor (hERα and hERβ).20 This work indicated that, although the tested compounds
62 Endocrine Disrupters in Wastewater and Sludge Treatment Processes
TABLE 3.1
Compounds that Have Been Tested Using Competitive Ligand Binding with IC50 Values
Receptor Compounds Tested
hERα p-nonylphenol (7.2 µM)21
PAH (nb-28 nM)20 Organochlorines
o,p′-DDT (485 µM),21 dieldrin (>50 mM),22 endosulphan (631 µM),21 (>50 mM),22 toxaphene (470 µM),21 (>50 mM)22 PCB
Tetra-ortho PCB104, 184, 188 (>10 µM),16 2′,3′,4′,5′-tetrachloro–4-biphenylol (0.1 µM),16 2,6,2′,6′-tetrachloro–4-biphenylol (0.5 µM)16
hERβ PAH (nb-29 nM)20
Fish ER Alkylphenols
octylphenol (0.1 µM)23 nonylphenol (0.5 µM)23 NP1EC (200 µM)23 Phthalates
benzylbutylphthalate (2–10 nM)24 di-n-butylphthalate (2–10 nM)24 bis(2-ethylhexyl)phthalate (2–10 nM)24 benzophenone (2–10 nM)24
n-butylbenzene (2–10 nM)24 4-nitrotoluene (2–10 nM)24
butylated hydroxy anisole (2–10 nM)24 Chlorophenols
2,4-dichlorophenol (2–10 nM)24 PCB
Tetra-ortho PCB54 (>10 µM)16 Tetra-ortho PCB104, 188 (1.3 µM)16 Tetra-ortho PCB184 (0.4 µM)16
2′,3′,4′,5′-tetrachloro–4-biphenylol (0.27 µM)16 2,6,2′,6′-tetrachloro–4-biphenylol (0.3 µM)16 Mouse ER (B6C3F1) Whiskey, red and white wine*25
Organochlorine mixture (DDT; DDE;
methoxychlor; endosulphan; toxaphene)*25 Reptilian, aER (green anole) PCB
Tetra-ortho PCB104, 184, 188 (>10 µM)16 2′,3′,4′,5′-tetrachloro–4-biphenylol (0.25 µM)16 2,6,2′,6′-tetrachloro–4-biphenylol (0.5 µM)16 nb, non-binder; BBP, butylbenzyl phthalate; DBP, di-n-butyl phthalate; DEHP, bis(2-ethyl- hexyl)phthalate; NP1EO4-nonylphenoxycarboxylic acid
* Reported as 100% displacement of labeled estradiol for red and white wine; no effect for whiskey and OC mix
Methods for the Determination of Endocrine Disrupters 63
exhibited similar affinity for binding to both forms of the receptor, additional use of a recombinant receptor–reporter assay using MCF-7 cells indicated that the capacity for transcriptional activation was isoform specific. Parent PAH did not exhibit any binding to either the α or β forms of the receptor; however, monohy- droxylated PAH bound to different degrees.
3.1.2 CELL PROLIFERATION TECHNIQUES
These approaches are predominantly based on human-derived cell lines and utilize a number of end points to measure the cell proliferation induced through exposure to estrogenic compounds. Commonly used strains of cells are estrogen-responsive MCF-7 or T47-D human breast cancer cells.7 The E-screen assay, developed for this purpose, is based on the increased growth of MCF-7 cells in the presence of estrogens.21 When a range of concentrations are tested, the method can differen- tiate between agonists, partial agonists, and inactive compounds.8 The E-screen assay developed by Soto et al.26 compares cell yields in both positive and negative controls with those from samples exposed to test compounds. A large range of chemicals were evaluated using the E-screen by Soto et al.21,with relative pro- liferative effects of up to100% observed for a number of compounds at concen- trations of around 10 µM (Table 3.2). Polychlorinated biphenyl congeners and hydroxylated PCB were also evaluated, with the 2′,4′,6′-trichloro-4-hydroxybi- phenyl demonstrating the greatest effect of 99.8% at a concentration of 100 nM, in comparison to reduced effects for other congeners at concentrations two orders of magnitude greater. The end point of the E-screen has been modified by Körner et al.,27 who, rather than counting cells or nuclei, utilized a colorimetric end point which was claimed to be faster and easier to perform. A range of other means of quantifying cell growth has also been reported, with the alamar blue (AB) and [3H]thymidine incorporation assays exhibiting greater sensitivity than cell count- ing, DNA, and MTT assays.28 The AB assay was also described as quicker and less expensive. This test has been applied to the evaluation of effluents from several STWs in Germany and in evaluating the effect of mixtures of xenoestro- gens which included bisphenol A, octyl and nonylphenol, butylphthalate, and 4- hydroxybiphenyl.2
However, a range of cell lines for MCF-7 exists, and these display different responses that could cause problems with reproducibility. This was highlighted for response to estradiol, p-nonylphenol, and bisphenol A, using four cell lines, BUS, ATCC, BB, and BB104, with BUS exhibiting the greatest sensitivity.30 Another cell line, the E3, has also been compared to wild-type (WT) MCF-7 cells and demon- strated a more proliferative (7 compared to 6 reported for the BUS line) and less variable response to 17β-estradiol than the wild-type.28 Experimental protocols for proliferation of the MCF-7 (and other estrogen responsive lines) require that media used for growth be stripped of steroids with dextran charcoal. Some workers have found that cell lines exhibit proliferation in estrogen-free media.31,32 Issues such as this present difficulties with regard to evaluation of the technique and in setting up validated and standard methods for evaluating the estrogenicity of compounds;
however, they are not unique to the MCF-7 cell line, nor to cell proliferation assays.
64 Endocrine Disrupters in Wastewater and Sludge Treatment Processes
TABLE 3.2
Compounds and Materials Tested for Estrogenic Activity Utilizing Cell Proliferation Assays
Cell Line Compounds Tested
MCF-7 (E-screen) Alkylphenols and alkylphenol ethoxylates
nonylphenol (103%),30 (70%),23 (100%),*31 (100%),21 (105%)27
octylphenol (+ive),28 (~80% at 1.0 µM23 (100%),21 (97%)27 butylphenol (71–76%),21 (78%)27
NP2EO (> 100% at 10 µM)23, NP1EC (~ 80% at 1.0 µM)23 Bisphenols38
bisphenol A,38 (97%),30 (~ 80%),34 (82%),39 (97%)27 bisphenol A dimethacrylate (~ 80%)**34 tetrabromo-bisphenol A (52%)27 Chlorophenols
Tris-(4-chlorophenyl) methanol,28 4-chloro-3-methylphenol (44%),27 4-chloro-2-methylphenol (25%)27
Organochlorine compounds
o,p′-DDT (86%),21 p,p′-DDT (71%),21 o,p′-DDE (26%), (n/e),28 o,p′-DDD (79%),39 dieldrin (55%),21 endosulphan (81%)21
HCB (62%),39 methoxychlor (57%),21 toxaphene (52%)21 isopropyl benzene28
naphthalene (halowax 1041) PCB, OH-PCB
4-hydroxybiphenyl (87%, 10 µM),21 (71%)27 4,4′-dihydroxybiphenyl (84%, 10 µM)21 2-monochlorobiphenyl (4%) (87%, 10 µM)21
2,2′,4,5-tetrachlorobiphenyl (62%, 10 µM) (87%, 10 µM)21 4-phenylphenol (99%)39
2-phenylphenol (30%)39 Phthalates
di-n-butylphthalate (62%)27
benzylbutylphthalate (90%),21,39 (80%)27 sewage effluent Germany (33–90%)29 dental sealant (100% 5 µg ml–1)34 ZR-75 (human breast cancer) Phthalates
butylbenzylphthalate (10–5 M),24 di-n-butylphthalate (>10–4 M)24 bis(2-ethylhexyl)phthalate (10–5 M)24
butylated hydroxy anisole (10–5 M)24 Alkylphenols and alkylphenol ethoxylates p-nonylphenol (+ive),23 octylphenol (10–6 M)23 NP2EO (+ive),23 NP1EC (+ive)23
(n/e) no effect
* Control cells in dextran charcoal stripped medium also exhibited growth.
** Response had not plateaued at highest concentration tested (10–6 M)
Values are reported as relative proliferative effect (RPE) (%) for simplicity, or the lowest concen- tration at which effects were observed.
Methods for the Determination of Endocrine Disrupters 65
Other proliferative cell lines that have been utilized in testing for estrogenic compounds include the ZR-75 human breast cancer line, with a range of compounds evaluated by Jobling et al.24 These included a range of phthalates, two antioxidants (butylated hydroxyanisole and butylated hydroxytoluene), caffeine, benzoic acids, methylphenols, and benzophenone. Most of these compounds exhibited only weak activity at concentrations below 10–4 M, with the exceptions reported in Table 3.2.
Both MCF-7 and T47D cell lines were used to determine the estrogenic effect of wines and spirits in comparison to a mixture of organochlorine compounds.25 In addition, the estrogenicity of a range of more commonly used products, ranging from sun screens33 to resin-based composites and sealants used in dentistry, has been evaluated by this technique.34 Such applications demonstrate that the tests are poten- tially versatile and may well find future use in evaluation of effluents and in longer- term monitoring schemes.
There is however, a range of disadvantages associated with cell proliferation techniques, in addition to the experimental procedures that are relate to reproduc- ibility. Mammalian cells, which are best for understanding specific mechanism-based details,35 exhibit tissue-specific expression of receptor subtypes. In addition to their use in cell proliferation techniques, the MCF-7 strain has been developed, and either stable or transient transfections of the cells have been obtained with recombinant estrogen receptor/reporter genes. The use of a recombinant yeast cell bioassay (RCBA) has been compared to the MCF-7 E-screen test and was found to be twice as sensitive to estradiol.36