Transcript Development of Assay for Measuring Avian UDP

POLYCHLORINATED BIPHENYL EFFECTS ON AVIAN HEPATIC
ENZYME INDUCTION AND THYROID FUNCTION
Objectives
• To develop and validate an assay for measuring
avian UDP-GT activity toward T4
• To compare UDP-GT induction by PCBs in birds
and mammals
•To study the effects of PCBs on avian thyroid
function
5 or 21 days
Blood, livers, thyroid glands
Body weight, thyroid gland weight, liver
weight, UDP-GT activity toward T4,
plasma T4 and T3 concentrations, thyroid
gland T4 and T3 content
UDP-GT
Activity
UDP-GT
Capacity
Liver wt
(g)
A
15
10
5
0
1500 B
1000
500
0
1.5 C
*
**
**
1.0
0.5
D
**
E
**
*
0
250
500
Dose of Aroclor 1254 (mg/kg)
Figure 1. Effects of Aroclor 1254 on Balb/c mice (Experiment 1):
a) UDP-GT activity (pmol T4G per mg liver per min); b) UDP-GT
capacity (pmol T4G per g body wt per min); c) liver weight; d)
plasma T4 concentrations; e) plasma T3 concentrations. Each
value represents the mean ± SE of 5 mice. Asterisks indicate
significant differences from controls (*p < 0.05; **p < 0.01).
Capacity
A
3
2
1
0
B
100
**
0
2
**
**
**
50
C
1
0
**
**
(g)
5 days
(ng/ml)
0 (vegetable oil),
500
Results
0.0
100
75
50
25
0
3
2
1
0
UDP-GT
12-day-old
Japanese quail
UDP-GT
12-day-old
Japanese quail,
weanling mice
0 (vegetable oil),
250, 500
8
D
4
0
(ng/ml)
Endpoints
measured
Experiment 2
Liver wt
Oral dose of
Aroclor 1254
(mg/kg)
Day of sacrifice
Tissues collected
Experiment 1
Plasma T3 Plasma T4
Animals
Plasma T4
(ng/ml)
Introduction. Polychlorinated biphenyls (PCBs) induce
the activity of the hepatic enzyme uridine
diphosphate-glucuronosyltransferase (UDP-GT) which
glucuronidates the thyroid hormone, thyroxine (T4), in
mammals. Glucuronidation increases T4 solubility in
water, which leads to its excretion. This enhanced
loss of T4 can then lead to hypothyroidism.
It is not clear how PCBs affect UDP-GT in birds.
Previous bird studies have used a substrate that
measures all UDP-GT isozymes so they may not have
accurately reflected T4 glucuronidation. I hypothesize
that PCBs will induce avian UDP-GT isozymes that
glucuronidate T4, increase thyroid gland (TG) weight,
decrease TG hormone content, and decrease plasma
T4 concentrations.
Experimental Design
Plasma T3
(ng/ml)
Abstract. Environmental exposure to polychlorinated biphenyls
(PCBs; common, persistent environmental contaminants) has been
associated with histological evidence of thyroid deficiency in wild
birds. PCBs decrease thyroid function in mammals by inducing a
liver enzyme, uridine diphosphate-glucuronosyltransferase (UDPGT). This enzyme adds carbohydrate to PCBs, thereby facilitating
their excretion from the body. UDP-GT has the same effect on the
predominant thyroid hormone, thyroxine (T4) and this increased
excretion of T4 can lead to thyroid deficiency (hypothyroidism). Our
objective was to determine if PCBs affected UDP-GT and thyroid
function in birds as is the case in mammals. We validated an assay
for measuring UDP-GT activity toward T4 in Japanese quail, and then
UDP-GT induction by Aroclor 1254 (common PCB mixture) was
evaluated in quail and mice. In Experiment 1, quail and mice were
dosed orally with solvent or Aroclor 1254 (250 or 500 mg/kg) and
sacrificed 5 days later. In Experiment 2, Japanese quail were dosed
orally with solvent or Aroclor 1254 (500 mg/kg) and sacrificed 5 or
21 days later. Total liver UDP-GT capacity increased with Aroclor
1254 exposure in all treatment groups of both species, but the effect
was smaller in quail than in mice. Enzyme induction led to a trend to
decreased plasma T4 concentrations at both doses and exposure
times in quail and significantly decreased plasma T4 concentrations
at both doses in mice. PCBs altered thyroid function in quail, but
they did not become hypothyroid. This was in contrast to mice,
which did become hypothyroid. Overall, quail showed a lesser
response than mice to equivalent doses of Aroclor 1254, so it
appears that birds may be less vulnerable to PCBs than mammals.
Supported by a Virginia Tech Graduate Research Development grant.
Activity
Catherine M. Webb and F. M. Anne McNabb
Department of Biological Sciences, Virginia Tech
4
E
*
2
0
0
500
0
500
5 days
21 days
Dose of Aroclor 1254 (mg/kg)
and Exposure Time
Figure 2. Effects of Aroclor 1254 on Japanese quail (Experiment 2):
a) UDP-GT activity (pmol T4G per mg liver per min); b) UDP-GT
capacity (pmol T4G per g body wt per min); c) liver weight; d)
plasma T4 concentrations; e) plasma T3 concentrations. Each value
represents the mean ± SE of 11-13 quail. Asterisks indicate
significant differences from controls (*p < 0.05; **p < 0.01).
Conclusions
•PCBs significantly increased UDP-GT activity in both species but less
in quail than mice
•Quail are less sensitive to Aroclor 1254 than mice
•Quail showed some thyroid effects but were not hypothyroid;
mice were hypothyroid (decreased plasma T4)
•Unclear whether the HPT axis was activated in quail; activation of
HPT axis appears to be inhibited in mice
•Higher dose of Aroclor 1254 may clarify some differences between
birds and mammals in their responses to PCBs
Significance
This is the first demonstration of a T4-specific mechanism by which
PCBs induce hypothyroidism in birds.