ELSEVlER

Toxicology Letters

Toxicology Letters 78 (1995) 253-262

Effects of gestational and lactational exposure to coplanar polychlorinated biphenyl (PCB) congeners or

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thyroid Ihormone concentrations in weanling rats

Byung-Woun Seoapb, Mei-Hui LibvC Larry G. Hansenbvc, Robert W. Moored, Richard E. Petersond, Susan L. Schantz*a3b

‘Neuroscience Pro;pram, University of Illinois at Urbana-Champaign, 1101 W. Peabody Drive, Urbana, IL 61801, USA

binstitute for Environmental Studies. University of Illinois at Urbana-Champaign, 1101 W. Peabody Drive, Urbana, IL 61801, USA ‘Department of Veterinary Biosciences, University of Illinois at Urbana-Champaign, 2001 S. Lincoln.

Urbana, IL 61801. USA ‘School of Pharmacy and Environmental Toxicology Center, University of Wisconsin, Madison, WI 53706, USA

Received 12 December 1994; revision received 9 February 1995; accepted 13 February 1995

AbSWlCt

Perinatal exposure to polychlorinated biphenyl (PCB) mixtures or to certain o&o-substituted PCB congeners dramatically reduces circulating thyroxine (T4) concentrations. It is not clear whether pcrinatal exposure to coplanar PCBs or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a similar effect. In this study, time-mated Sprague-Dawley rats were dosed with 2 or 8 mg/kg/day PCB 77 (3,3’,4,4’-tetrachlorobiphenyl), 0.25 or 1.00 &kg/day PCB 126 (3,3’,4,4’,5pentachlorobiphenyl), 0.025 or 0.10 &kg/day TCDD, or corn oil vehicle orally on gestation days 10-16. At weaning, plasma total T4 concentrations in PCB 77 and TCDD high-dose female pups were significantly depressed, but the changes were modest (84.4 and 79.6% of control, respectively). T4 concentrations in PCB 126 highdose females and all high-dose males were also depressed slightly, but the changes were not statistically significant. UDP- Glucuronosyl transferase (UDP-GT) activity towards 4-nitrophenol was increased in all high-dose groups. Thus, the modest decreases in T4 could he due in part to increased T4 glucuronidation by UDP-GT. Triiodothyronine (T3) and thyroid stimulating hormone (TSH) concentrations were unchanged in all groups. In contrast to the minor changes in thyroid hormone status, liver microsomal ethoxyresorufin-Odeethylase (EROD) was markedly induced in all expo- sure groups and thymus weights were depressed in the high-dose groups. Because doses of coplanar PCBs or TCDD that caused marked induction of EROD activity had only minor effects on T4, we conclude that changes in thyroid hormone status at weaning are not among the more sensitive effects of p&natal exposure to these compounds.

Keywords: PCBs; TCDD, Thyroid; Perinatai; Rat

* Corresponding author.

0378-4274/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0378-4274(95)03329-J

254 B.-W. Seo et al. / Toxicology Letters 78 (1995) 253-262

1. lntroduetion

Changes in circulating thyroid hormone concen- trations occur following exposure of adult animals to commercial polychlorinated biphenyl (PCB) mixtures. In most studies, both serum thyroxine (T4) and triiodothyronine (T3) concentrations were reduced [l-4], but in some serum T4 concen- trations were reduced with little effect on T3 [5-71. Several also reported decreased free T4 [5,6]. Adult exposure to individual coplanar PCB con- geners or to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) also affects thyroid hormone concentra- tions IS- 121. Decreased total and free T4, stable or slightly increased T3 and moderately increased thyroid stimulating hormone (TSH) concentra- tions have been reported.

Dramatic reductions of circulating T4 concen- trations have also been reported in pups following combined gestational and lactational exposure to commercial PCB mixtures [ 13- 151. Serum T3 con- centrations were only slightly reduced. Relatively little is known about which components of these mixtures are responsible for these effects. How- ever, several individual ortho-substituted PCB congeners have also been shown to reduce pup serum T4 concentrations, with little or no change in T3 [16,17]. PCB 118 (2,3’,4,4’$pentachloro- biphenyl) had the greatest effect, reducing pup T4 concentrations to about loo/o of control [ 161. Ma- ternal exposure to PCB 169 (3,3’,4,4’,5,5’- hexachlorobiphenyl), a coplanar congener, either alone or in combination with PCB 77 (3,3’,4,4’- tetrachlorobiphenyl), another coplanar congener, reduced both total T4 and free T4 concentrations in rat pups [18]. However, the changes were not as large as those reported for Aroclor 1254 and PCB 118.

Thyroid hormones play a very important role in brain development [ 191, especially during the early postnatal period [20]. The presence of adequate circulating T4 concentrations is especially critical for normal brain development, because T3 does not cross the blood brain barrier readily [21,22]. The large decreases in serum T4 seen after expo- sure to some PCB mixtures and congeners suggest that the neurobehavioral and neurochemical se- quelae that follow perinatal exposure to these

compounds may be mediated, at least in part, by reduced availability of thyroid hormone to the brain [15,16,18].

In this study, effects of gestational and lacta- tional exposure to 2 coplanar PCB congeners, PCB 77 and PCB 126 (3,3’,4,4’,5pentachloro- biphenyl), and TCDD on circulating thyroid hor- mone concentrations are reported. While these congeners are known to have CNS effects [23-251, the effects of perinatal exposure to them on thy- roid hormones have not been investigated pre- viously, except for one study in which PCB 77 was given in combination with PCB 169 [ 181. Rat dams were dosed with the PCBs or TCDD via gavage during gestation. T3, T4 and TSH were measured in serum from the pups on postnatal day 21. Be- cause induction of UDP glucuronosyl transferase (UDP-GT) is believed to be one mechanism that contributes to the decreased serum T4 concentra- tions following PCB exposure [26], the activities of 2 of the enzymes that conjugate T4 with UDP were measured. In addition, certain reproductive and developmental endpoints were assessed to char- acterize the degree of PCB or TCDD toxicity in pups and dams. Thymus weights and liver micro- somal ethoxyresorufin-O-deethylase (EROD) in- duction were measured as indicators of the degree of Ah receptor-mediated response in the pups.

2. Materials and methods

2.1. Animals and exposure One hundred and eight time-mated Sprague-

Dawley dams were obtained from Harlan Sprague Dawley (Indianapolis, IN) on day 1 of gestation (day of sperm plug was considered day 0). Upon arrival, the dams were randomly placed in 1 of 7 treatment groups. The animalswere housed singly in polypropylene cages with corn cob bedding. Food and water were available ad libitum. The animals were kept in temperature and humidity controlled rooms (21-22OC, 40-50% humidity), on a 12-h light/dark cycle. On gestational days lo- 16, .each dam was gavaged with corn oil vehicle or chemical dissolved in corn oil. The PCB 77 doses were 2 or 8 mg/kg/day; the PCB 126 doses were 0.25 or 1.00 &kg/day; and the TCDD doses were 0.025 or 0.10 &kg/day. The PCBs were ob-

B.-W. Seo et al. / Toxicology Letters 78 (1995) 253-262 255

tamed from AccuStandard (New Haven, CT; guaranteed > 99% pure); TCDD was obtained from Cambridge Isotope Laboratories (Wobum, MA; guaranteed > 98% pure). The doses for PCB 126 and TCDD were determined from preliminary dose range studies. The largest dose that produced no obvious signs of reproductive or developmental toxicity, for examplle reduced maternal weight gain, smaller litter sizes or fewer live births, was used as the high dose. The doses for PCB 77 were based on previous research with PCB 77 con- ducted in this laboraltory. The same criteria were used to select the doses.

On the date of birth (day 0), the pups were examined for gross abnormalities. Body weights and number of each sex were also recorded. On day 2, the litters were culled to 8,4 males and 4 fe- males when possible. To minimize litter size ef- fects, litters of 5 or less were excluded from the study [27]. After exclusion of dams who were not pregnant [24] or had litters of less than 5 pups [8] there were 10-l 1 litters per exposure group. The pups were kept with their natural mothers until weaning on day 21.

At weaning, 4-6 pups from each litter were kept for neurobehavioral testing. Studies on those pups are still under way and will be reported separately. The remaining pups were euthanized by decapita- tion. Final body weights, liver, brain, thymus, and thyroid weights were recorded, and blood was col- lected for thyroid hormone analysis. Data were collected from 1 male and 1 female per litter wherever possible. Because some litters contained fewer than 8 pups and/or were not sex balanced, there were 5-9 litters, per exposure group for these endpoints.

2.2. Thyroid hormone analysis Total T3 and T4 concentrations were deter-

mined using commercially available radio- immunoassay (RIA) kits (DPC, Los Angeles, CA). Intraassay coefficients of variation (%CVs) were 2.28 and 1.91% for T3 and T4, respectively. In- terassay %CVs were 8.5 and 9.6% for T3 and T4, respectively.

Serum TSH was determined by RIA with the materials and protocols of the National Institute of Diabetes and Digestive and Kidney Diseases

(NIDDK) National Hormone and Pituitary Pro- gram, using TSH RP-3 as the standard. [ ‘251]labeled TSH (about 5000 counts/mm in 50 ~1 buffer), serum, TSH antibody, and RIA buffer (0.1% BSA (w/v) in PBS, pH 7.6) were combined to make a total volume of 300 ~1. After mixing well, tubes were incubated overnight at 4°C. Sepa- ration of free and bound, labeled TSH was accom- plished with the addition of 300 ~1 0.3% bovine-y-globulin (w/v) in RIA buffer and 400 ~1 25% (w/v) polyethylene glycol (PEG-6000) in water, incubation for 20 min, and centrifugation for 30 min [28]. The sensitivity of the assay was 25 pgtube. Mean ( f S.E.M.) concentrations of TSH in 50, 75, 100, and 125 ~1 of a pooled sample were 3.45 f 0.38, 3.49 f 0.69, 3.51 f 0.58, 3.50 f 0.63 ng/ml, respectively. Differences in concentra- tions of TSH because of sample volumes were not observed (P > 0.75), indicating a parallel relation- ship to the standard curve. After adding 50, 250, and 500 pg of unlabeled TSH, 68.26 f 35.02, 239.46 f 24.57, and 566.01 f 54.53 pg were measured, respectively. Intra- and interassay %CVs were 18.63 and 15.38%, respectively.

2.3. Microsome preparation Immediately after sacrifice by decapitation,

livers were removed, weighed, and perfused with ice-cold 0.05 M Tris-HCYO. 15 M KC1 buffer (pH 7.4 at 4°C). The livers were homogenized in the same Tris-KC1 buffer. The crude homogenate was centrifuged at 10 000 x g for 15 min at 4°C. The supematant was then centrifuged at 105 000 x g for 60 min at 4°C. The pellet from the final cen- trifugation was resuspended in buffer (0.05 M Tris-HCl, 20% glycerol (v/v), 1 mM EDTA; pH 7.4 at 4°C) and stored at -80°C until assayed [ 171. Microsomal protein was determined by a moditi- cation of the Lowry method [29] using bovine serum albumin (BSA) as the standard.

2.4. EROD activity The 0-deethylation of 7-ethoxyresorutin was

determined by a modification of the method of Pohl and Fouts [30]. In brief, the l-ml reaction mixture contained 5 mM MgClz, 1 mg BSA, mi- crosomal suspension (0.08-0.4 mg microsomal protein), 2.5 PM 7-ethoxyresorutin and an

Table

1

Repro

duct

ive o

utc

om

es of

dam

s exp

ose

d to

PC

B

77, PC

B

126, o

r TC

DD

on d

ays

lo-1

6

of

gest

ati

on

Pup b

irth

wt.

Pup w

eanin

g w

t.

Tre

atm

ent

Dam

gest

a-

tional

wt.

gain

(g)

Dam

liv

er

wt.

G

est

ati

onal

(g)

peri

od (

days

) Li

tter

size

Li

ve b

irth

s

(W

Pup b

irth

wt.

Pub w

eanin

g w

t.

Male

s (g

) Fe

male

s (g

) M

ale

s (g

) Fe

male

s (g

)

Contr

ol

154.8

f 9.4

14.0

f 0.3

21.0

f

0.0

11.9

f 1.0

98.1

f

1.0

6.7

1 z

t 0.1

9

6.4

1 z

t 0.1

5

51.9

6 f

1.8

6

50.8

4 jz

1.4

2

PC

B 77 lo

w

141.3

f 6.6

13.5

f 0.4

21.2

*

0.1

10.7

f 1.0

98.1

f

1.4

6.4

9 z

t 0.1

8

6.2

2 z

tz 0.1

3

51.5

7 f

1.4

1

52.1

8 z

t 1.9

3

PC

B 77 h

igh

136.7

zt

11.3

13.8

zt

0.3

21.1

f

0.1

10.0

f 1.0

88.4

f 4.0

6.9

8 f

0.3

6

6.2

9 f

0.2

1

49.5

3 f

1.3

1

46.0

5 z

t 1.5

2

PC

B

126 lo

w

158.1

f 11.2

13.9

f 0.4

21.1

f 0.1

12.5

f 0.6

94.9

zt

1.8

6.7

0 f

0.0

8

6.3

5 f

0.0

8

54.5

5 f

1.3

9

51.7

0 f

1.4

0

PC

B

126 h

igh

141.5

*

9.7

13.5

*

0.3

21.2

*

0.1

11.3

*

1.2

97.6

f

1.6

6.6

7 z

tz 0.1

4

6.4

0 z

t 0.1

1

48.6

8 z

k 2.1

4

49.1

1 z

t 0.9

6

TC

DD

lo

w

139.5

zt 8

.7

15.1

f 2.2

21.1

*

0.1

11.8

f 1.0

96.9

zt

1.6

6.6

9 +

0.1

5

6.4

9 z

k 0.1

3

53.0

1 f

1.5

3

50.0

5 t

1.1

2

TC

DD

hig

h

151.5

f 10.6

14.3

+ 0

.6

21.0

f 0.0

11.2

f 1.0

92.3

f

4.4

6.7

8 z

t 0.2

0

6.5

0 f

0.1

3

50.8

1 *

2.5

5

47.9

0 z

t 2.7

4

Data

are

means f

S.E

.M.

N =

lo-1

4

exp

osu

re g

roup. M

ate

rnal dose

s were

2 o

r 8 m

&g/d

ay

for

PC

B 7

7.0

.25 o

r 1.0

&kg

/day

for

PC

B

126, a

nd 0

.025 o

r 0.1

&kg

/day

for

TC

DD

.

B.-W. Seo et al. /Toxicology Letters 78 (1995) 253-262 251

NADPH-generating system (0.8 mg NADP+, 1.5 mg glucose-6-phosphate, 1 U glucosed-phosphate dehydrogenase) in 0.05 M Tris-HCl buffer (pH 7.4). The reaction was initiated by adding the NADPH-generating system and terminated with the addition of 2 ml methanol [17]. The reaction was conducted for 2 min at 37°C. The incubation conditions were selected such that the formation of resort& was linear with respect to time and pro- tein concentration. T:he formation of resorufin was determined by measuring sample fluorescence re- lative to a known amount of resort& with excita- tion at 550 nm and emission at 585 nm in a Perkin-Elmer 203 Fluorescence Spectrophoto- meter. Each sample was measured in duplicate.

measured in duplicate, Blanks were obtained from control incubations in which the UDP-glucuronic acid was omitted. To develop the color 3 ml 0.2 M glycine-NaQH buffer (pH 10.7) was added to the reaction medium. UDP-GT activity toward phenolphthalein and Cnitrophenol was quantified spectrophotometrically by measuring the disap- pearance of the substrate at 555 and 405 nm for phenolphthalein and 4-nitrophenol, respectively.

2.6. Statistical analysis

2.5. UDP-GT activity UDP-GT activity was measured using a moditi-

cation of the method of Watanabe et al. [31]. Phenolphthalein and 4nitrophenol were used as substrates for ‘the phenobarbital and 3-methyl- cholanthrene (3-MC) inducible forms, respective- ly. The incubation medium contained microsomal fraction (0.1-0.25 mg protein), 0.1 M Tris-HCl buffer (pH 7.4), 5 mM MgCIB, 0.5 mg BSA, and either 0.25 mM phenolphthalein or 0.5 mM 4- nitrophenol in a total volume of 0.5 ml. The in- cubation was performed for 6 min for 4-nitro- phenol or 10 min for lphenolphthalein at 37°C. The reaction was started by adding 0.05 ml 30 mM UDP-glucuronic acid and stopped with 1 ml 5% trichloroacetic acid (v/v). Each sample was

The data were analyzed using ANOVA. The lit- ter was the experimental unit and sex was treated as a within litter variable. Significant overall ef- fects were further analyzed via planned com- parisons of each experimental group to the control group [32]. Significant interactions were further analyzed using tests for simple effects followed by planned comparisons of the experimental groups to the control group [32]. Statistical significance was ascribed at P < 0.05.

3. Results

3.1. Reproductive and developmental outcomes There were no signs of toxicity in the dams from

any of the treatment groups. All groups displayed gestational weight gains, liver weights, litter sizes, and percent of live births similar to controls (Table 1). Birth weights and weaning body weights of the pups also were not affected by the treatment

Table 2

Absolute thymus, thyroid, liver, and brain weights at weaning (day 21) following combined gestational and lactational exposure to

PCB 77, PCB 126. or TCDD

Treatment Thymus wt. (mg) Thyroid wt. (mg) Liver wt. (g) Brain wt. (g)

Control 238.9 f I I .6 6.4 f 0.4 1.79 f 0.06 1.33 f 0.01

PCB 77 low 234.3 zt 10.2 6.8 f 0.3 1.82 zt 0.07 1.32 * 0.02

PCB 77 high 202.5 f 10.2’ 5.9 f 0.2 1.69 f 0.06 1.32 f 0.04

PCB 126 low 237.5 f Il.4 7.8 f 0.6 2.03 f 0.07* I.31 l 0.01

PCB 126 high 211.0 f 13.0 6.6 f 0.3 2.09 f 0.03* 1.27 + 0.02

TCDD low 230.5 f 5.86 7.5 f 0.3 1.95 * 0.07 1.29 f 0.02

TCDD high 183.3 zt 20.7* 6.7 f 0.2 1.97 l 0.17 1.29 f 0.04

Data are means f S.E.M. N = 5-9/exposure group. Maternal doses were 2 or 8 mg/kg/day for PCB 77. 0.25 or I .O &kg/day for

PCB 126 and 0.025 or 0.10 &kg/day for TCDD. Exposure was on gestation days 10-16. *Significant difference from the controls.

P < 0.05.

258 B.-W. Seo et al. / Toxicology Letters 78 (1995) 253-262

(Table l), nor were brain weights at weaning (Table 2).

A significant main effect of treatment on pup liver weights was observed (P < 0.01). Further

4.0 _I I

3.5

3.0 5 ‘J

E 5 2.5

s = - 2.0

2 1.5

1 .o

0.5

0.0 Control Low High Low High Low High

3.0

2.5

2.0

1.5

1.0

0.5

0.0 Control Low High Low High Low High

PCB 77 PCB 126 TCDD

Fig. I. Serum T4 concentrations at weaning on day 21 for fe- male (A) and male (B) rats born to dams dosed with PCB 77 (2 or 8 mg/kg/day), PCB 126 (0.25 or I.0 pglkglday), TCDD (0.025 or 0. IO pglkglday) or corn oil vehicle on gestation days 10-16. Values are group means * S.E.M. N = 5-9/exposure group. Statistical significance was set at P < 0.05. *Significant difference from the controls.

analyses comparing each treated group to the con- trol group revealed significant increases in only the PCB 12dtreated groups, while the TCDD groups were marginally significant (P = 0.085). In the PCB 126 low-dose group, the liver weight was in- creased by 13.7%, and in the high-dose group the liver weight was increased by 17.1% (Table 2).

3.2. Thyroid hormone concentrations There was not a significant main effect of PCB

or TCDD treatment on serum total T4 concentra- tions in the pups at weaning (P > 0.05), but the treatment by sex interaction was significant (P < 0.05), indicating that the males and females were affected differently. Tests for simple main effects in each sex indicated a significant effect in females (P < 0.05), but not in males. Comparisons of the exposed groups to the control group showed that the PCB 77 and the TCDD high doses significantly lowered total circulating T4 levels in the female

_l #

I + . . T 150

100

50

0 Control Low High Low High Low High

PCB 77 PCB 126 TCDD

Fig. 2. Liver microsomal UDP-CT activity toward 4- nitrophenol at weaning on day 21 in rats born to dams dosed with PCB 77 (2 or 8 mg/kg/day). PCB 126 (0.25 or I.0 pglkglday), TCDD (0.025 or 0.10 &kg/day) or corn oil vehicle on gestation days 10-16. Values are group means f S.E.M. N = 5-9Iexposure group. Statistical significance was set at P < 0.05. *Significant difference from the controls; +significant dif- ference between PCB 126 low and high doses; “significant dif- ference between TCDD low and high doses.

B.-W. Seo et al. / Toxicology Letters 78 (1995) 253-262 259

pups. However, the decreases were moderate, 15.6 and 20.4%, respectively (Fig. 1). Although the males showed a similar trend toward depression in T4 levels, the effeclt was not significant.

Total T3 and TSH concentrations were not affected by the treatment (data not shown) and thyroid weights did not differ from controls (Table 2). Similarly, the dams’ hormone levels at wean- ing were unchanged by the treatment (data not shown).

3.3. UDP-GT activity There was a significant increase in UDP-GT ac-

tivity toward 4nitrophenol (P < 0.0001; Fig. 2). However, unlike the findings for T4, there was not evidence of a sex by treatment interaction. Further analysis showed that all 3 high-dose groups and the PCB 126 low-dose group were significantly in-

::, l-f 2500

1500

1000

500

0 ! Control Low High Low High Low High

IPCB 77 PCB 126 TCDD

Fig. 3. Liver microsomal EROD activity at weaning on day 21 in rats born to dams dosed with PCB 77 (2 or 8 mg/kg/day). PCB 126 (0.25 or I.0 fig/kg/day). TCDD (0.025 or 0.10 @kg/day) or corn oil vehicle on gestation days 10-16. Values arc group means * S.E.M. N = 5-9/exposure group. Statist- ical significance was set at P < 0.05. *Significant difference from the controls; ‘signilicant difference between PCB 126 low and high doses; ‘significant difference between PCB 77 low

and high doses.

duced. The PCB 77 and TCDD low-dose groups did not differ significantly from controls. The greatest induction was seen in the PCB 126 and TCDD high-dose groups, 2.8- and 3. l-fold induc- tion, respectively. No increase in UDP-GT activity toward phenolphthalein was observed.

3.4. Ah receptor markers Because the animals were treated with coplanar

PCBs or TCDD, 2 common Ah receptor markers were measured, thymus weight and EROD activ- ity. Significant EROD induction was observed (P < 0.0001; Fig. 3), with no treatment by sex effect (P > 0.05). Further analysis showed that all treat- ment groups were significantly induced. Also, the PCB 77 and PCB 126 groups showed a significant dose-dependent increase in the low- and high-dose groups. The difference between the TCDD low- and high-dose groups was marginally significant (P = 0.087). The greatest EROD induction was seen in the PCB 126 and TCDD high-dose groups, 25.6- and 23.6-fold induction, respectively (Fig. 3).

Significant thymic atrophy was also observed following treatment (P < 0.05; Table 2). The treat- ment by sex interaction was not significant, in- dicating the treatment did not affect one sex differentially. Comparisons of the exposed groups to the control group showed that the PCB 77 and TCDD high-dose groups had thymus weights significantly lower than those of the controls. Thy- mus weights were decreased by 15.2 and 23.3%, respectively (Table 2). Although thymus weights were also lower in the PCB 126 high-dose group, the decrease was not statistically significant.

4. Discussion

Gestational and lactational exposure to co- planar PCBs or TCDD resulted in small, but signi- ficant depressions in plasma T4 concentrations in female rat pups. Male pups also showed a slight depression in T4, but the effect was not significant. Plasma T3 and TSH concentrations were not changed in any of the groups. The decreases in plasma T4 ranged from about 15-20%. This small degree of T4 depression is unlikely to be of any clinical significance to the animal, particularly given the normal plasma T3 and TSH concentra-

260 B.-W. Seo er al. / Toxicology Letters 78 (1995) 253-262

tions. Coplanar PCBs and TCDD also depress factors must also be involved. Complex mixtures plasma T4 levels in adult animals, but doses in the of PCBs induce both PB and 3-MC forms of UDP- overtly toxic range are required to achieve a GT [34], which may explain the greater effects of sizable effect [8,10- 121. Aroclor 1254 on serum T4 concentrations.

Exposure to coplanar PCBs or TCDD resulted in significant increases in UDP-GT activity toward 4-nitrophenol. UDP-GT catalyzes the formation of T4-glucuronide, a deactivation reaction that precedes excretion of T4 in the bile. Early studies with PCBs [33] and TCDD [S] found increases in the biliary excretion of T4glucuronide following exposure. More recently, it has been demonstrated that serum T4 levels following exposure to a num- ber of UDP-GT inducers, including PCBs, cor- relate well with the degree of increase in UDP-GT activity toward T4 [26]. Thus, induction of UDP- GT appears to play an important role in mediating the decreased serum T4 concentrations observed following PCB exposure.

The UDP-GTs consist of a family of related en- zymes that differ in their substrate specificity and in their ability to be induced by various chemicals [5,26]. Phenobarbital (PB) induces a form of UDP-GT with activity towards chloramphenicol and phenolphthalein, whereas 3-MC induces a form with activity towards napthol and 4- nitrophenol. All types show activity towards T4 [26]. In the current study, no increase in UDP-GT activity towards phenolphthalein was observed in any of the groups. As would be expected with coplanar PCBs and TCDD, UDP-GT activity towards 4-nitrophenol was 2-3 times higher than control in the PCB 77, PCB 126, and TCDD high- dose groups. However, there was not a good cor- respondence between induction of UDP-GT activ- ity and depression in serum T4. Pups in the PCB 77 and TCDD high-dose groups had similar reduc- tions in T4, but the TCDD-exposed pups showed a much larger induction of UDP-GT. In contrast, pups in the PCB 126 high-dose group were similar to the TCDD high-dose pups in terms of UDP-GT induction, but did not show a significant decrease in T4. Furthermore, the depression in plasma T4 was more evident in female pups, but no male- female differences in UDP-GT activity were observed. Although induction of UDP-GT may play a role in mediating the decreased serum T4 concentrations, these findings suggest that other

A variety of reproductive and developmental outcome measures were also assessed to char- acterize the degree of PCB or TCDD-related toxic- ity present in the animals. Maternal weight gain during gestation, maternal liver weight, gestation length, litter size, percent live births, birth weights and postnatal growth rates were all unaffected by the exposures. However, thymus weights were decreased and liver weights were increased in pups from the high-dose groups and microsomal EROD was markedly induced in pups from all treatment groups. Thus the doses were clearly sufficient to evoke various Ah receptor-mediated responses. Other studies have shown that similar doses are also sufficient to cause alterations in various as- pects of neurobehavioral functioning including spatial learning and memory [35]. Thus changes in thyroid hormone status do not appear to be one of the more sensitive effects of these Ah receptor agonists. In contrast, some orrho-substituted PCB congeners and some PCB mixtures have very dramatic effects on plasma T4 concentrations. Doses of either PCB 118 or Aroclor 1254 that were not associated with any overt toxicity to dams or their pups reduced plasma T4 concentrations to less than 10% of control levels [ 15,161.

Thyroid hormones are known to play a very im- portant role in brain development [19]. The presence of adequate circulating T4 concentra- tions during the early postnatal period is especially critical for normal brain development. Although T3 is the more biologically active form of the hor- mone, it does not cross the blood brain barrier readily [21,22]. Instead, T4 enters the brain and is converted to T3. Neonatal rats respond to decreas- ed plasma T4 concentrations with an increase in brain type II-5 ‘-deiodinase, the major enzyme in the brain responsible for the conversion of T4 to T3 [18]. The fact that low doses of some ortho- substituted PCB congeners and PCB mixtures are capable of dramatically reducing plasma T4 con- centrations suggests that some of the neurobehav- ioral and neurochemical sequelae observed follow- ing perinatal exposure to PCBs may be mediated

B.-W. Seo et al. /Toxicology Letters 78 (1995) 253-262 261

by reduced availability of thyroid hormone to the brain. This study has demonstrated that maternal doses of coplanar PCBs or TCDD that are just below the threshold for overt fetotoxicity reduce thymus weights and markedly induce liver micro- somal EROD activity, but have only a small effect on plasma T4 concentrations at weaning. These findings suggest that reduced availability of thy- roid hormones may not be involved in mediating the neurobehavioral and neurochemical changes that have been documented following perinatal ex- posure to similar levels of these Ah receptor agon- ists. Because we sampled only at weaning, we cannot rule out the possibility that more signifi- cant decreases in T4 occurred earlier in develop- ment. However, given the importance of T4 to overall growth and development, the lack of ef- fects on birth weights or postnatal growth rates argues against a large decrease in T4 earlier in de- velopment.

Acknowledgements

The authors thank Carol Davis and Patricia Weston for their assistance in conducting the RIAs. We are also grateful to NIDDK and the Na- tional Hormone and Pituitary Program for the gift of the TSH RIA kit. This work was supported by Health Canada Project #4028 to SLS and NIH Grant #ES01332 to REP.

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