Species-Specific Endocrine Disruption: PCB- and PAH- Induced Estrogen
Effects

Project Scope

Accumulating evidence suggests that chemicals and complex mixtures are
capable of eliciting endocrine-disrupting activities that adversely
affect human health, and affect wildlife. Adverse effects of exposure to
endocrine disrupting chemicals (EDCs) have been demonstrated in numerous
species, resulting in compromised reproductive fitness. In addition,
significant increases in the incidence of hormone-dependent cancers have
been observed in humans. It has been proposed that some of these effects
may be mediated by the estrogen receptor binding activities of the EDCs.

Estrogens and the estrogen receptor (ER) play major roles in a number of
normal physiological processes such as the regulation of developmental
processes, homeostasis and fertility, as well as in pathological
conditions such as hormone-dependent diseases. Although the function and
activities of estrogen and the ER are conserved between species, the
amino acid sequences of the ligand-binding domains of the ERs are less
conserved. This suggests that species may exhibit different responses
and sensitivities to non-traditional estrogenic ligands and that a
single test species may not be sufficient to identify and to assess the
risks of environmental estrogens to human and wildlife health.

The main objective of this research was to examine, for several species,
the proposed estrogen receptor-mediated activities of a structurally
diverse set of compounds using a battery of species-based *in vitro* and
*in vivo* assays. This research project examined the estrogenic
activities of polychlorinated biphenyls (PCBs) and polyaromatic
hydrocarbons (PAHs) and their effects in a number of species including
humans, rodents, birds, fish and amphibians. The studies were performed
to define the limitations of using a single surrogate species (e.g., a
rodent) to identify and assess estrogen effects on human and wildlife
health, based on the divergence in amino acid sequences and binding
activities.

To address this objective, an integrative approach was used that
involved a series of *in vitro* and *in vivo* assays using estrogen
receptors from a number of species. Species-specific differences in
ligand preference and binding affinities initially identified using *in
vitro* assays (i.e., receptor binding, gene expression) were then
verified *in vivo* by monitoring effects on gene expression (e.g.,
vitellogenin).

Project Results and Implications

Initial species comparisons of ER binding involved 44 PCBs, 9
hydroxylated PCBs, and 8 Aroclor PCB mixtures at exposure concentrations
ranging from 1 nM to 10 uM. Further investigations were conducted with
35 structurally diverse synthetic and natural estrogenic compounds
(e.g., steroids, pharmaceuticals, phytoestrogens, mycotoxins,
organochlorine compounds, polyaromatic hydrocarbons, and other
industrial chemicals). Competitive ligand binding and reporter gene
assays identified several compounds that exhibited differential
interactions with recombinant ERs consisting of the D, E, and F domains
of the human, mouse, chicken, green anole lizard, frog (*Xenopus*), and
rainbow trout ERs (Fig. 1). Qualitatively, all receptor fusion proteins
bound the same set of compounds. However, there were substantial
differences in relative binding affinities between species. In general,
reporter gene expression data correlated with competitive binding
results. Intriguingly, the rainbow trout ER ligand binding and reporter
gene transactivation were found to be temperature dependent and
functioned more effectively closer to the physiological temperature.
Comparison of the amino acids that lined the binding pocket of the
rainbow trout and human ERs identified two conservative substitutions
(substitutions that do not change the overall electrostatic charge on
the protein), which were found to play a substantial role in this
temperature dependence.

Three compounds that exhibited differential activity *in vitro,*
β-zearalenol, genistein, and 4-tert-octylphenol, were then examined *in
vivo* to determine their ability to induce vitellogenin messenger
ribonucleic acid (mRNA) levels in juvenile trout, frog, and Japanese
quail. There was a poor correlation between the *in vitro* and *in vivo*
activities and striking differences in efficacy and potency between the
species examined. Overall, these results suggest that there general
consistency with regard to ER binding of EDCs across the species tested,
but the ability to predict the full range of *in vivo* activity of EDCs
based on *in vitro* data from a single species is questionable.

**Figure 1. Summary of the Ability of Polychlorinated Biphenyls to
Compete with \[3H\]17b-Estradiol for Binding to Recombinant Fusion
Proteins Containing the Human, Green Anole and Rainbow Trout Estrogen
Receptor D, E, and F Domains ^a,b^**

  ----------------- ----------------- ----------------- -----------------
  **PCB**           **Human**         **Anole**         **Rainbow Trout**

  Mono-*ortho*                                          

  58                non               non               Weak

  60                non               non               Weak

  68                non               non               Weak

  Di-*ortho*                                            

  18                non               non               weak

  41                non               non               \>10 μ M

  44                non               non               weak

  47                non               non               \>10 μ M

  49                non               non               weak

  87                non               non               non

  99                non               non               weak

  101               non               non               weak

  112               non               non               weak

  115               non               non               \>10 μ M

  128               non               non               weak

  138               non               non               weak

  153               non               non               weak

  Tri-*ortho*                                           

  45                non               non               \>10 μ M

  51                non               non               \>10 μ M

  84                non               non               weak

  91                non               non               \>10 μ M

  95                non               non               weak

  143               non               non               \>10 μ M

  149               non               non               weak

  151               non               non               weak

  173               non               non               \>10 μ M

  177               non               non               \>10 μ M

  178               non               non               weak

  183               non               non               weak

  187               non               non               weak

  Tetra-*ortho*                                         

  54                non               weak              \>10 μ M

  104               \>10 μ M          \>10 μ M          1.3 +/- 0.6 μM

  184               \>10 μ M          \>10 μ M          0.4 +/- 0.1μM

  188               \>10 μ M          \>10 μ M          1.3 +/- 1.2 μM

  Co-planar                                             

  126               non               non               non

  169               non               non               weak
  ----------------- ----------------- ----------------- -----------------

a\. Source: Abstracted from Table 3 of Matthews and Zachareski (2000)
(permission being sought)

b\. \"non\" = no discernable displacement of estrogen at any
concentration tested, \"weak\" = \< 10 percent estrogen displacement at
highest concentration tested, \" 10 mM\" = calculated IC50 \> 10 mM from
three experiments

Investigators

Timothy Zacharewski, Michigan State University

For More Information

Investigator Website:

<http://www.bch.msu.edu/~zacharet/>

**NCER Project Abstract and Reports:**

<http://cfpub.epa.gov/ncer_abstracts/index.cfm/fuseaction/display.abstractDetail/abstract/173/report/0>