##### Endocrine Disruptors: Effects on the Thyroid

# Project Scope {#project-scope .LRI-Heading-2}

Many endocrine disrupting chemicals are suspected to be thyroid tumor
promoters. It has been proposed that endocrine disruptors promote
thyroid tumors through effects on the complex hormonal control pathways
of the hypothalamus-pituitary-thyroid axis (HPTa). Specifically,
endocrine disruptors alter the HPTa by increasing thyroxin (T~4~)
glucuronidation, facilitating its elimination, and reducing serum T~4~
levels. As a compensatory feedback mechanism for this reduction in T~4~,
thyroid stimulating hormone (TSH) is released from the pituitary, which
stimulates thyroid follicular cell proliferation, ultimately leading to
hyperplasia and, potentially, neoplasia. However, preliminary research
has found that several endocrine disruptors, while they reduce serum
T~4~ levels, do not increase serum TSH. This project investigated the
mechanisms by which polychlorinated biphenyls (PCBs; known endocrine
disruptors) increase T~4~ glucuronidation and contribute to thyroid
carcinogenesis.

The main objectives of this research were to investigate:

-   The effects of PCBs on the pharmacokinetics (clearance, biliary and
    urinary excretion, and *in vitro* metabolism) of T~4~, to clarify
    the mechanism(s) by which PCBs decrease serum T~4~ levels;

-   Whether a correlation exists between the ability of specific PCB
    congeners and mixtures (Aroclors) to reduce serum T~4~ levels and
    increases in biliary excretion of T~4~ glucuronide;

-   If there are mechanisms other than glucuronidation and biliary
    excretion by which PCB congeners or mixtures of congeners decrease
    serum T~4~; and

-   Whether PCB-induced biliary excretion of T~4~ might be a consequence
    of increases in the expression of selected transport proteins.

Four sets of studies were conducted under this grant to address these
questions by investigating PCB effects on T~4~ levels and excretion
patterns in male rats.

# Project Results and Implications  {#project-results-and-implications .LRI-Heading-2}

[Effects of PCBs on circulating (plasma) T~4~ and biliary excretion of
T~4~ glucuronide:]{.underline} PCB mixtures and congeners are known to
reduce plasma T~4~ levels. This is thought to occur because of PCBs'
ability to induce the UDP-glucuronosyl transferases that conjugate T~4~,
increasing the excretion of T~4~ glucuronide into bile. PCB congeners
95, 99, 118, and 126; Aroclors 1242 and 1254; and
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were orally administered to
male rats for 7 days. Twenty-four hours after the last dose, \[^125^I\]
T~4~ was injected into the blood and common bile ducts of the rats.
Following administration of the \[^125^I\] T~4~, bile, blood, and urine
was collected over the course of 2 hours. All seven treatments were
found to decrease the plasma concentrations of T~4~. Of the pure
congeners, PCB 99 and 118 produced the largest decreases in plasma T~4~
concentration, whereas PCB 95, PCB 126, and TCDD had the least effect.
None of the seven treatments had a marked effect on the urinary
excretion of T~4~ and its metabolites. In contrast, biliary T~4~
glucuronide excretion was increased six to eight-fold after
administration of TCDD, PCB 118, or Aroclor 1254. PCB 126 administration
produced a three to five-fold increase; while PCBs 95 and 99 produced
less than a doubling of excretion. TCDD, one of the treatments that had
the least effect on decreasing the concentration of T~4~ in blood,
increased the biliary excretion the most. In contrast, PCB 99, which
caused a marked decrease on the plasma concentration of T~4~, had the
weakest effect on its biliary excretion. These results indicate that
there is not a strong correlation between the ability of PCBs and
chlorinated dioxins to decrease plasma T~4~ concentration and their
potency in increasing T~4~ biliary excretion.

[Study 2: Effects of specific PCB congeners on serum levels of T~4~ and
triiodothyronine (T~3~).]{.underline} PCB congeners are commonly divided
into three general categories. The \"TCDD type\" includes those that
have affinity for the aryl hydrocarbon receptor (AhR) and induce
cytochrome P4501A1 (CYP1A1). The \"phenobarbital type\" induce CYP2B1,
while the \"mixed type\" that induce of both CYP1A1 and CYP2B1. For this
study, one TCDD type congener (PCB 126), two phenobarbital types (PCB 95
and PCB 99), and one mixed type (PCB 118) were evaluated, along with
Aroclors 1242 and 1254 and TCDD. Each compound was orally administered
at a minimum of four dose levels to male rats for 7 days. Serum T~4~
levels were reduced to 5 to 10 percent of control values in rats
receiving Aroclor 1254 and PCBs 99 and 118, while reductions up to 50
percent occurred in rats dosed with the remaining compounds. Serum T~3~
levels were reduced to 30 percent of control values in rats administered
Aroclor 1254. The remaining compounds caused only slight or no
reductions in serum T~3~. The results of this study suggest that PCB
congeners may induce the majority of their effects on thyroid functions
via pathways that are not mediated by the AhR.

[Study 3: Other mechanisms by which PCBs might decrease serum T~4~ level
in rats.]{.underline} This study focused on the rate of disappearance of
\[^125^I\] -- labeled T~4~ from the serum of rats that received specific
PCB congeners or mixtures of PCBs. Male rats were orally given Aroclor
1254 and 1242 or individual congeners (PCBs 95 or 99, phenobarbital-like
congeners), PCB 118 (a mixed-type congener), PCB 126 (a dioxin-like
congener), or TCDD in corn oil via oral gavage daily for seven days.
Twenty-four hours after the last dose, \[^125^I\] T~4~ was administered
intravenously and blood was sampled at 15, 45, 75, and 105 minutes after
injection. At 15 minutes after the injection, serum \[^125^I\] T~4~
levels were decreased approximately 75 percent in rats administered PCB
99, PCB 118, or Aroclor 1254, and 50 percent in rats administered
Aroclor 1242, PCB 95, or PCB 126, compared to controls. There was no
significant decrease in serum T~4~ in TCDD-treated rats. Serum
concentrations of T~4~ continued to be significantly reduced in all of
the PCB-treated rats at all subsequent time points. The disappearance of
serum T~4~ from the bloodstream of PCB-treated rats appears to be too
rapid to be explained solely by increased glucuronidation and biliary
excretion. This suggests that other mechanism(s) may be involved in the
decreased serum T~4~ levels elicited by PCB exposure.

[Study 4: Effects of PCBs on T~4~ transport protein
expression.]{.underline} Transfer of T~4~ across plasma membranes is
accomplished in part via transport proteins. Two proteins thought to be
involved in this process are organic anion transporting polypeptides
Oatp2 and Oatp3. Additionally, Mrp2, a member of the superfamily of
adenosine triphosphate (ATP) binding cassette transporters, has been
implicated in the excretion of T~4~ - glucuronide into bile. The purpose
of the fourth study was to determine whether PCBs that markedly reduce
serum T~4~ might do so by enhancing the hepatic uptake of T~4~ (and
subsequent biliary excretion) through increases in the expression of
these transport proteins. Aroclors 1254 or 1242 were orally administered
to male rats for 7 consecutive days and 24 hours after the last dose,
\[^125^I\] - T~4~ was administered intravenously. Blood and liver
samples were taken immediately after injection and for up to 30 minutes
thereafter in order to measure rapid changes in blood and tissue T~4~
levels. When measured one minute after injection, animals exposed to
Aroclor 1254 showed a 56 percent decrease in serum \[^125^I\] T~4~.
Animals receiving Aroclor 1242 exhibited a 30 percent reduction within 3
minutes, compared to controls. Aroclor 1254 and Aroclor 1242
administration also produced 105 percent and 55 percent increases in the
uptake of \[^125^I\] T~4~ into liver, respectively. However, Oatp2
protein levels were reduced 87 percent by Aroclor 1254 compared to
controls, but Aroclor 1242 had no significant effect on liver Oatp2
levels. No significant changes in Oatp3 protein levels were observed in
response to any of the test agents. In contrast, administration of
Aroclors 1254 and 1242 both resulted in significant increases in Mrp2
protein levels (108 percent and 92 percent, respectively.) These
findings suggest that the large increase in hepatic uptake of T~4~ in
rats exposed to PCBs cannot be explained by increased expression of
Oatp2 and Oatp3. However, increased expression of Mrp2 may contribute to
increased biliary excretion of T~4~ glucoronide.

# Investigator {#investigator .LRI-Heading-2}

Curtis D. Klaassen - University of Kansas Medical Center, KS

# For More Information {#for-more-information .LRI-Heading-2}

<http://www.kumc.edu/pharmacology/facstaff/klaassen.html>

**NCER Project Abstract and Reports:**

<http://cfpub2.epa.gov/ncer_abstracts/index.cfm/fuseaction/display.abstractDetail/abstract/172/report/0>