Effects of perfluorodecanoic acid on thyroid status in rats

TOXlCOLOGYANDAPPLlEDPHARMACOLOGY87,430-439(1987)

Effects of Perfluorodecanoic MARC J. VAN RAFELGHEM,*

Acid on Thyroid Status in Rats’

STANLEY L. INHORN,? AND RICHARD E. PETERSON*,’

*School ofPharmacy and tDepartment QfPathology and Laboratory Medicine, University of U ‘isconsin, Madison, Wisconsin 53706

Received Juiv 8, 1986; accepted October 13, 1986 Effects of Perfluorodecanoic

Acid on Thyroid Status in Rats. VAN RAFELGHEM, M. J., INTo.xicoi. Appl. Pharmacol. 87,430-439. Treatment of rats with toxic doses of perfluorodecanoic acid (PFDA) results in reduction in feed intake, body weight. serum thyroxine (T4) and triiodothyronine (TJ concentrations. resting heart rates, and body temperatures. Some of these effects resemble changes characteristic of hypothyroidism. Therefore the effects ofPFDA on functional thyroid status were examined to relate changes in thyroid status with signs of PFDA toxicity. In the present study, the dose-related effects of PFDA on plasma thyroid hormone concentrations and a number of indices of thyroid status were investigated and compared with signs of PFDA toxicity. Young adult male Sprague-Dawfey rats were given single intraperitoneal doses of PFDA (20, 40. or 80 mg/kg), and subsequent changes were evaluated 7 days after dosing. Decreases in body weight and feed intake were used as measures of PFDA toxicity and ranged from minimal to severe. Plasma T4 concentrations and free thyroxine index were drastically reduced at all doses, and these changes were mimicked by pair feeding only at the high dose of PFDA (80 mg/kg). Plasma T3 concentrations were not affected by PFDA treatment, whereas pair feeding at the high-dose level (80 mg PFDA/kg) resulted in a significant reduction (ca. 50% from unlimited-fed control) in T?. Although PFDA caused a dose-dependent decrease in thyroid gland weight which was not completely paralleled by pair feeding, thyroid histology was unremarkable. PFDA treatment resulted in a small decrease in basal metabolic rate (8% at 80 mg PFDA/kg). A greater reduction (ca. 18%) in basal metabolic rate was observed in vehicle-treated controls pair-fed to rats of the 80 mg PFDA/kg dose group. Thermogenesis, as measured by oxygen consumption and body core temperatures, was not greatly affected by PFDA treatment, and these changes were paralleled by pair feeding. Reductions in plasma Tq concentration and free thyroxine index at a low dose of PFDA (20 mg/ kg) indicate that PFDA-induced hypothyroxinemia can be dissociated from its overtly toxic effects (i.e., severe hypophagia and body weight loss) observed at higher doses. The results obtained here suggest that despite alterations in plasma thyroid hormone levels there is no consistent pattern of effects on functional thyroid status which could explain the overt toxicity of PFDA. HORN,

S. L., AND PETERSON,

R. E. (1987).

Because of their surfactant properties, derivatives of perfluorocarboxylic and perfluorosulfonic acids have found wide use as

lubricants, surfactants, plasticizers, and aqueous film-forming foam fire extinguishants (Guenthner and Vietor, 1962; Shinoda and

’ This work was supported by the Air Force Office of Scientific Research, Air Force Systems Command, USAF, under Grant AFOSR 85-0207. and was presented at the 1986 (( 1986) Toxicologist 6, 3 15) Annual Meeting of the Society of Toxicology. The U.S. Government’s right to retain a nonexclusive royalty-free license in and

to the copyright covering this paper, for governmental purposes. is acknowledged. ’ Recipient of Research Career Development Award K-04-ES00098. and to whom reprint requests should be addressed, at 425 N. Charter Street, School of Pharmacy. University of Wisconsin, Madison, WI 53706.

0041-008X/87

$3.00

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PERFLUORODECANOIC

ACID

Nomura, 1980). Little is known about the toxicology of these perfluorinated compounds. Early reports of the presence of a nonionic fluorine fraction in human serum (Taves, 1968) and the subsequent isolation of a perfluorocarbon compound resembling perfluorooctanoic acid (PFOA, C7F15COOH) by NMR spectrometry (Guy et al., 1976) resulted in a number of studies of the toxic effects of ammonium perfluorooctanoate in rodents and primates (Griffith and Long, 1980) and the metabolism and disposition of this compound in rats (Ophaug and Singer, 1980; Hanhijarvi et al., 1982). In rodents the liver appears to be the primary target organ of perfluorooctanoate toxicity: however, while histopathological changes are more pronounced in male rats, the acute oral LD50 is lower in female (430 mg/kg) than in male rats (680 mg/kg) (Griffith and Long, 1980). Recent studies suggest that perfluorinated carboxylic acids of chain length 10 or greater may be more toxic than their shorter-chain analogs (Rogers et al., 1982; Olson and Andersen, 1983). Perfluorodecanoic acid (PFDA. CsF,&OOH) has toxic effects in the male rat similar to those seen with PFOA; however, these effects are more severe and more persistent with PFDA than with PFOA (Olson and Andersen, 1983). In addition, PFDA causes a starvation-like wasting syndrome with delayed lethalities, thymic atrophy, testicular degeneration, and a disruption of hepatic architecture in rats (Van Rafelghem et al., 1982). Reductions in serum thyroxine (T4) and triiodothyronine (TX) as early as 12 hr and for as long as 8 days after a single dose of PFDA (75 mg/kg) in rats and a concomitant reduction in body temperature and resting heart rate have led to the suggestion that some of the signs of PFDA treatment might be related to alterations in serum thyroid hormone levels (Langley and Pilcher, 1985). Most research on effects of PFDA in rats has been done following a single dose. The objective of the present study is to determine the dose-related nature of PFDA-induced

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effects on thyroid hormone levels, thyroid histology, and basal metabolic rate in rats and to determine if a correlation exists between these changes and signs of PFDA toxicity, namely reduced feed intake and body weight. Three doses of PFDA are chosen such that little or no reduction in feed intake and body weight is observed at the lowest dose while significant weight loss characteristic of the PFDA wasting syndrome is seen at the highest dose. Rats are evaluated 7 days after PFDA treatment, because overt toxicity is apparent by this time. METHODS Chemicals. Perfluorodecanoic acid (nonadecafluorodecanoic acid) was purchased from Aldrich Chemical Co.. Inc.. Milwaukee, WI (Lot No. 24 I 1 HL). This preparation was 96% pure by titration with NaOH and had a melting point of 79°C. The methyl ester of the commercial product was prepared and subjected to gas chromatography using a Varian Model 3700 gas chromatograph equipped with a 36-m vitreous silica SE-30 column and flame ionization detector. Analysis was done at 63°C with helium as carrier gas. a flow rate of 2 ml/mitt, and a split ratio of 300/l. One large peak was seen, followed immediately by a number of smaller peaks. It has been reported that analysis of these peaks by gas chromatography-mass spectroscopy indicated the large peak was the methyl ester ofPFDA. which we have confirmed by mass spectroscopy and proton NMR. and the multiple small peaks were methyl esters of fluorinated-n-decanoic acid with single or double hydrogen substitutions (George and Andersen. 1986). As a monohydrogen-substituted analog, e.g., 11 -H-eicosofluoro-n-undecanoic acid. was not available as a standard in the present study. calculated purity of the commercial product (Aldrich Product No. 17,774-l. Lot No. 2411HL) was 87.4% PFDA by gas chromatography of the methyl esters, assuming equal response of the impurities to the main peak. ~~perimentalprotoc.ol. Dosing solutions were made by dissolving PFDA in propylene glycol-water ( 1: 1. v/v) to give the following concentrations: 20, 40. and 80 mg PFDA/mI. Male Sprague-Dawley rats (300-350 g) were obtained from Harlan Sprague-Dawley (Madison, WI) and were housed individually in suspended stainless-steel cages in a room maintained at 2 1°C. A 7- to IO-day period was allowed for the animals to become accustomed to their new environment (lights on from 0500 to 1700 hr) and their feeding schedule (ground Purina Rat Chow available from 1700 to 0830 hr) before dosing was started. Rats were dosed on Day 0 by intraperitoneal in-

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jection of 20,40, or 80 mg PFDA/kg body wt. Each PFDA-dosed rat had a pair-fed control partner, which received an ip injection of 1 ml/kg of the propylene glycolwater vehicle. Control rats with access to an unlimited quantity of feed during the daily IS.5hr feeding period were also treated with vehicle. Body weights and feed intakes were measured daily for 7 days post-treatment. Thyroid histology and plasma thyroid hormones. On Day 7 after dosing, eight rats from each of the treatment groups were terminated starting at 1300 hr. Each rat was exsanguinated into a 50-ml T&Angle disposable beaker containing 100 ~1 of 15% (w/v) K-EDTA (pH 7.4) which was kept on ice. The blood was then centrifuged immediately, and plasma was stored in 0.6-ml aliquots at -70°C. Total T4 and Tr concentrations in plasma were determined by radioimmunoassay (Chopra et al.. 197 1; Pate1 and Burger, 1973). The unsaturated binding capacity of thyroid-binding proteins in the plasma was assessed by T3 uptake (Clark and Horn, 1965). Alterations in the plasma levels of these proteins are reflected in the T, uptake. These determinations were made using diagnostic kits obtained from Nuclear Medical Laboratories (Irving. Texas). Free thyroxine index, which is assumed to be proportional to the concentration of free thyroxine in the blood, was calculated as the product of the plasma T4 concentration and the T, uptake value from each rat (Rosenfeld. 1974). The thyroid gland was also excised from each animal, weighed. and fixed in 10% neutral formalin for histopathologic evaluation. The thyroids of randomly chosen rats from each PFDA-dose group (n = 4) and respective pairfed control group (n = 4) were submitted for histological examination. Thyroid glands from ad libirum-fed control rats (n = 8) were also evaluated by light microscopy. Basal metabolic rates and body core temperature. Basal metabolic rate was measured as postabsorptive resting oxygen consumption during a 4-hr period at thermoneutral temperature (27°C). an ambient temperature at which rats do not have to expend energy to maintain body core temperature (Herrington, 1940). These measurements were made by placing each rat in a herrnetitally sealed, 4-liter chamber through which atmospheric air was pumped at a constant rate of I500 ml/min. Exiting air was dried in a CaSO, (Drier&e)-filled column and passed through a paramagnetic oxygen analyzer. The volume of oxygen consumed by the rat was determined as described previously (Seefeld ef al., 1984). Oxygen consumption was measured continuously for 10 ofevery 30 min during the 4-hr period. Oxygen consumption data preceded by at least 5 min of motor inactivity were used to estimate resting oxygen consumption. Oxygen consumption data collected during periods of both motor activity and inactivity were used to estimate total oxygen consumption. Body core temperatures were measured at 0900 hr in 4-8 rats from each of the treatment groups with a rectal temperature probe connected to a digital thermometer (Model 49 TA, Yellow Springs In-

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strument Co., Inc., Yellow Springs, OH). The temperature probe was inserted 6 cm into the colon of each rat and held there for a uniform period of time (ca. 30 set). This procedure was repeated on Days 1, 3, 5, and 7. so that the animals would be= accustomed to the routine by Day 7. Sratistical analysis. The main effects of treatment (PFDA vs pair fed) and dosing (0, 20, 40, and 80 mg PFDA/kg) were analyzed by two-way ANOVA through the use of unweighted cell means (Milliken and Johnson. 1984). Significant differences between PFDA-treated and pair-fed groups were detected by pairwise comparison (Dixon et al.. 198 1). Effects of dosing within the PFDAtreated and pair-fed groups, including comparison with the vehicle-treated group of rats with unlimited access to feed. were tested by one-way ANOVA followed by Scheff& multiple comparison of means (Neter and Wasserman. 1974). Linear function was examined when a dosing effect was significant by testing for trends using orthogonal coefficients (Dixon et al.. 198 1). The computations were performed with a VAX-750 computer using BMDP (Dixon et al.. 198 1). In all cases. significance was p < 0.05.

RESULTS Body Weight and Cumulative

Feed Intake

PFDA caused a dose-dependent reduction in body weight and cumulative feed intake (Fig. 1) over a 7-day period. At 20 mg PFDA/ kg, body weight and cumulative feed intake were not significantly affected. However. at the two higher doses of PFDA (40 and 80 mg/ kg), body weights and cumulative feed intake were reduced significantly when compared with the vehicle-treated rats with unlimited access to feed. Although not statistically significant, there appeared to be a small difference in body weights between pair-fed controls and PFDA-dosed rats (at 40 and 80 mg/ kg) even though cumulative feed intake showed no such trend. Thyroid Gland tologq

Weight

and Thyroid

His-

PFDA treatment caused a significant reduction in thyroid gland weight in the highdose group (80 mg/kg) when compared with

PERFLUORODECANOIC

BODY

I

I

WEIGHT

THYROID

GLAND

*

15-

+

IO-

+

5-

+

0

PFDA

WEIGHT

20-

F

ACID

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Sections ofthyroid glands in the unlimitedfed control group, the three PFDA-treated groups, and the three pair-fed control groups showed that the glands contained uniformly small follicles lined by epithelium varying moderately in height. Approximately 4080% ofthe follicles had columnar epithelium, while 20-60s were lined by squamoid to cuboidal epithelium. These percentages did not differ significantly from one group to another. Some follicles were devoid of colloid. while others contained small quantities of clear fluid. The thyroid stroma itself contained no excessive colloid. There were no signs of inflammation in any of the thyroid glands. Plasma Concentrations mones

’

0

20 DOSE

40

00

(mg/kg)

FIG. I. Effects of PFDA treatment on body weight. cumulative feed intake, and thyroid gland weight. The data presented were obtained 7 days after ip injection of PFDA (20. 40, or 80 mg/kg) or vehicle. Vehicle-treated rats either were allowed unlimited access to feed or were pair-fed to PFDA-treated rats. On Day 7. rats were exsanguinated by decapitation. Plasma was prepared for radioimmunoassay of thyroid hormones (Table 1). and thyroid glands were removed, weighed, and fixed in 10% neutral-buffered formalin for histological evaluation. Each bar and its associated vertical line represent the mean and SE of 8-16 observations. *Significantly different from the respective pair-fed control group (p < 0.05). *Significantly different from unlimited-fed control group( p < 0.05).

that of the unlimited-fed group and with those of the two lower PFDA dosage groups (Fig. 1). However, the rats pair-fed to this high-dose group also displayed a reduction, though not as extensive, in thyroid gland weight when compared with that of the unlimited-fed group. Thus the marked hypophagia caused by 80 mg PFDA/kg partly accounted for the decrease in thyroid gland weight observed at that dose.

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of Thyroid

Hor-

Even at the lowest dose of PFDA, 20 mg/ kg, there was a marked decrease in plasma T, concentration when compared with both pair-fed control rats and vehicle-treated controls with unlimited access to feed (Table 1). Further dose-dependent decreases in this parameter were noted at the 40 and 80 mg/kg doses of PFDA. Pair feeding to animals treated with the two lower doses of PFDA (20 and 40 mg/kg) did not result in significant decreases in plasma Tq concentration, yet pair feeding to the 80 mg/kg group did lead to a reduction in this parameter which was equivalent to that seen in PFDA-treated animals. Total T3 concentrations in the plasma did not appear to be affected by PFDA treatment at any dose level when compared with those of vehicle-treated rats with unlimited access to feed (Table 1). Even at the highest dose of PFDA, where a drastic reduction in feed intake was observed (Fig. I), plasma T3 levels were similar to those in unlimited-fed controls. However, control rats pair-fed to the high-dose group (80 mg PFDA/kg) showed a 50% reduction in their plasma T3 concentration when compared with that of their PFDA-treated partners or with that of the vehi-

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INHORN. TABLE

EFFECTOF

PFDA

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PETERSON

1

TREATMENTONPLASMATHYROIDHORMONECONCENTRATIONSINRATS~

PFDA dose hx&)

Treatment group

0

Unlimited-fed

20

Total Tq (fig/W

Total T1 (ng/dl)

Ti uptake

4.41 &O.l9h

89.6 k 1 .6h

62.9 -+ 0.3”

PFDA Pair-fed

2.8 1 k 0.19*,’ 4.28f0.19h

83.2 + 4.9” 85.3 + 2Sh

64.2 i 0.5 h 63.3 +- OZh

40

PFDA Pair-fed

2.28 + 0. 17*.‘,d 4.54 I 0.2 1 h

81.1 f3.6’ 87.0 k 3.9h

63.4 f 0.3” 63. I f 0.1”

80

PFDA Pair-fed

2.00 * 0. I I <’ 2.22 ?I 0.40”

91.6f4.8*,h 42.1 f 10.5’

60.9 i OS*.’ 63.0 f 0.9”

* Significantly different from pair-fed group by pairwise comparison (p < 0.05). ’ Plasma was prepared 7 days after ip administration of PFDA or vehicle. Each value represents the mean +- SE of 8- 16 determinations. ‘.‘J Mean values in a column not followed by the same superscript are significantly different from other dose levels in the same treatment group (PFDA or pair-fed) using Scheff&‘s multiple comparison method (a < 0.05). The unlimited-fed group serves as the 0 dose level for both treatment groups.

cle-treated rats with unlimited access to feed (Table 1). The unsaturated binding capacity of thyroid-binding proteins, assessed by T3 uptake analysis, was reduced at the highest dose of PFDA (80 mg/kg) only and did not appear to be significantly affected by pair feeding (Table I). This would suggest that neither PFDA treatment nor pair feeding resulted in marked alterations in the levels of thyroid-binding proteins in the plasma. As a consequence, the treatment-related effects on free thyroxine index (data not shown) were virtually identical to the effects on total T4 concentration.

Oxygen Consumption Rate

and Basal Metabolic

Postabsorptive energy expenditure, at an ambient temperature of 27°C was quantified by measuring total and resting oxygen consumption in the whole animal. Oxygen consumption is expressed per rat as milliliters Oz/minute and per metabolic body size (MBS) as milliliters Oz/MBS/minute (MBS is

defined as body weight in kilograms raised to the 0.75 power). Total oxygen consumption per rat was decreased by PFDA in a dose-dependent fashion (Fig. 2, top panel). However, when expressed relative to metabolic body size (Fig. 2, bottom panel), total oxygen consumption was not significantly altered by the different dose levels of PFDA tested. The same pattern of changes in total oxygen consumption was observed in pair-fed control rats. Resting oxygen consumption per rat was decreased in a dose-related fashion in those rats treated with PFDA, but was also decreased in the respective pair-fed control animals (Fig. 3, top panel). Postabsorptive, resting oxygen consumption expressed as milliliters OJMBS/minute and measured at 27°C ambient temperature is an important parameter of thyroid function, because it is a measure of basal metabolic rate (BMR) (Pitt-Rivers and Tata, 1959; Himms-Hagen, 1976). The bottom panel of Fig. 3 shows that BMR was reduced significantly at the 80 mg/kg dose level when compared with that of the other two PFDA-dose levels or with that of

PERFLUORODECANOIC

ACID

=

PFDA

0

Paw-ted

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temperature, but this was not observed with pair feeding. DISCUSSION .!@ects of PFDA on Body U’eight, Feed Intake, and Circulating Thyroid Hormones

0

20 DOSE

40

80

(mg/kgl

FIG. 2. Effects of PFDA treatment on absolute and relative total oxygen consumption at thermoneutral ambient temperature (27°C). Measurements were made 7 days after ip injection of PFDA (20.40, or 80 mg/kg) or vehicle. Vehicle-treated rats either were allowed unlimited access to feed or were pair-fed to PFDA-treated rats. Total oxygen consumption, measured during periods of both motor activity and rest, was determined for each rat for 10 of every 30 min for a 4-hr period. Absolute total oxygen consumption is expressed as ml/mitt/rat (top panel) and relative total oxygen consumption as ml/ MBS/min (bottom panel) where metabolic body size (MBS) is body weight in kilograms to the 0.75 power. Each bar and its associated vertical line represent the mean and SE of 4-8 observations. *Significantly different from unlimited-fed control group (p < 0.05).

Male rats treated with a dose of PFDA (20 mg/kg) which does not exhibit overt toxic effects (i.e., body weight loss and reduced feed intake) showed significant reductions in total plasma T4 concentration and free thyroxine index. Thereby effects of PFDA on body weight and feed intake (which occurred only at higher doses of PFDA) could be dissociated from its effects on circulating thyroid hormone concentrations. Although further decreases in the total plasma Tq concentration and free thyroxine index were observed at higher doses of PFDA (40 and 80 mg/kg), the additional reductions

the unlimited-fed controls. However, in the rats pair-fed to the 80 mg/kg PFDA-treated animals an even further decrease in BMR was observed in comparison with that of their PFDA-treated partners (Fig. 3, bottom panel). Boliy Core Temperuture Two-way ANOVA indicated that there was a significant dose-related effect on body core temperature 7 days following PFDA treatment (Fig. 4). The reduction in body temperature did not appear to be significantly different when comparing PFDA-treated animals with their respective pair-fed controls. One-way ANOVA among the various PFDA or pair-fed groups indicated a significant linear trend for PFDA to decrease body core

DOSE

(mg/kg)

FIG. 3. Effects of PFDA treatment on absolute and relative resting oxygen consumption at thermoneutral ambient temperature (27°C). Oxygen consumption values preceded by at least 5 min of motor inactivity were used to estimate resting oxygen consumption. Absolute resting oxygen consumption (top panel) is expressed as ml/ min/rat and relative resting oxygen consumption (bottom panel) as ml/MBS/min. Other conditions are as in the legend to Fig. 2. Each bar and its associated vertical line represent the mean and SE of 3-8 observations. *Significantly different from the respective pair-fed control group (p < 0.05). *Significantly different from unlimited-fed control group (p i 0.05).

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RAFELGHEM,

=

PFDA

0

Pair-fed

Ra

““limited

1

*

37 u ’

INHORN.

36

i 80 DOSE

(mglkg)

FIG. 4. Effects of PFDA treatment on body core temperature. Colonic temperatures were measured 7 days after ip injection of PFDA (20.40, or 80 mg/kg) or vehicle, with a rectal temperature probe inserted 6 cm into the colon of each rat. Each bar and its associated vertical line represent the mean and SE of 4-8 observations. *Significantly different from unlimited-fed control group (p < 0.05).

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PETERSON

(8 days) reduction in circulating T4 concentrations, which was never completely matched in pair-fed control rats. More recently, Gutshall et al. ( 1986) showed that T4 supplementation (0.2 mg/kg, daily) reversed the hypophagia observed in PFDA-treated rats but not the body weight loss or the decrease in total serum T4 concentration. This also suggests that PFDA treatment reduces circulating T4 concentrations independent of its hypophagic effect. At any of the doses tested. PFDA did not result in any changes in the circulating T3 concentrations. Langley and Pilcher ( 1985) showed that a 75 mg/kg dose of PFDA resulted in significant reductions in serum T3 levels in comparison with those of ad Iibitumfed controls at 6 and 8 days post-treatment. In our studies, a nearly equivalent dose of 80 mg PFDA/kg did not affect plasma T3 concentrations 7 days following dosing. The difference in the effect of PFDA on T3 levels in these studies might be due to differences in the rat strains or in the age of the rats used. Nonetheless, it should be noted that in neither study did the circulating T3 concentrations fall to the same extent as the circulating levels of T4. Finally, both studies agree in that, approximately 1 week post-treatment. rats pair-fed to animals receiving a nearly equivalent high dose of PFDA (75 or 80 mg/ kg) showed a reduction in circulating T3 levels when compared with those of ad libiturnfed rats. This is probably due to a hypophagia-induced reduction in peripheral T4-5’monodeiodinase activity (Ingbar and Galton, 1975) which under normal conditions deiodinates T4 to T3 in peripheral tissues (Oppenheimer, 1983). It was not clear from the effects of PFDA on plasma thyroid hormones alone whether the functional thyroid status of PFDA-treated rats was altered. Therefore effects of PFDA treatment on indices of thyroid status were investigated.

were not as marked as the initial one seen at 20 mg/kg. Furthermore, vehicle-treated rats that were pair-fed to animals treated with the highest dose of PFDA (80 mg/kg) displayed reductions in both the plasma T4 concentration and the free thyroxine index that were as extensive as those observed in their PFDAtreated counterparts, supporting the contention that hypophagia causes hypothyroxinemia in rats (DeGroot et al.. 1977). The mechanism by which PFDA reduces plasma T4 concentrations is not known; however, reduced feed intake cannot fully explain this decrease in plasma T4 concentration and free thyroxine index because, at all levels of graded feed restriction, the plasma T4 and free thyroxine index results from the PFDAtreated rats and their respective pair-fed controls did not match. It was only at 80 mg/kg that these effects of PFDA could be mimicked by the paired-feed restriction. Thus, PFDA treatment results in reductions in plasma T4 concentration and free thyroxine index by a mechanism in addition to hypophagia-induced hypothyroxinemia. This interpretation is supported by the results of a timecourse study (Langley and Pilcher, 1985) Efects oj’PFDA on Basal Metabolic Rate showing that a single dose of PFDA (75 mg/ Basal metabolic rate, obtained by deterkg) resulted in a rapid ( 12 hr) and prolonged mining resting oxygen consumption in post-

PERFLUORODECANOIC

ACID

absorptive rats in a thermoneutral environment (ambient temperature of 27°C) and correcting this measurement for metabolic body size, is a classic measure of thyroid status (Pitt-Rivers and Tata, 1959; Himms-Hagen, 1976). PFDA caused a reduction in basal metabolic rate of about 8% at the highest dose tested. This was the greatest decrease in basal metabolic rate observed in PFDA-treated rats. which represents a small change relative to what would be expected in a hypothyroid animal. Decreases in basal metabolic rate of 30-45% have been reported in severely hypothyroid animals (Eberhardt et al., 1980). Vehicle-treated rats pair-fed to the animals treated with 80 mg PFDA/kg showed a significantly greater reduction in basal metabolic rate when compared with their PFDAtreated partners. This 18% reduction in basal metabolic rate (when compared with that of unlimited-fed, vehicle-treated rats) may be due to the reduction in plasma T3 concentrations observed in these animals. Thus, it appears that in the face of the marked reductions in plasma T4 concentrations, even at low doses of PFDA, the effects of PFDA on functional thyroid status cannot fully account for the overt toxicity of this compound. PFDA treatment resulted in a more pronounced effect on plasma T4 levels than did feed restriction, since T4 levels were significantly reduced in rats treated with doses of PFDA (20 and 40 mg/kg) which did not affect feed intake dramatically. These differences between PFDA treatment and pair feeding at the low doses of PFDA, however, were not translated into significant differences in basal metabolic rate between these two treatment groups. It does not appear that the changes in plasma T4 concentrations are proportional to changes in basal metabolic rate. A better correlation may exist between the plasma T3 concentrations and basal metabolic rate as evidenced by the results obtained from the vehicle-treated rats pair-fed to the animals treated with the highest dose of PFDA (80 mg/kg).

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Effects ofPFDA Treatment on Thyroid Gland Weight and Thyroid Histology Although there was a linear trend toward reduction in thyroid gland weight with increasing doses of PFDA, the decrease was significant at the 80 mg/kg dose only. A similar linear trend toward reduction in thyroid weights was observed in pair-fed control rats. At the 80 mg/kg dose level, a significantly greater reduction in thyroid weight was observed in PFDA-treated rats when compared with that in their pair-fed counterparts. The significance of the reduction in thyroid gland wet weight in either PFDA-treated or pair-fed control animals is uncertain. Since dry weights were not obtained for the thyroid glands, one cannot assume that this effect was strictly due to reduced water content. Histological examination of the thyroid glands from the PFDA-treated rats and their pair-fed controls at each of the three dose levels did not reveal any significant differences in the morphological appearance of the thyroid when comparing PFDA-treated animals with their respective pair-fed controls or with vehicle-treated controls with unlimited access to feed. There were no excessively large follicles in the thyroids of the animals from any of the treatment groups, nor was there any absorption of colloid at the periphery of follicles as would be expected in hyperactive glands. Eflects qf PFDA on Bod?l Temperature PFDA has been reported to cause decreases in body temperature in rats significantly greater than in pair-fed control rats as early as 3 days after dosing (Langley and Pilcher, 1985). This hypothermic response was not reversed by daily Tq injection (Gutshall et al., 1986) suggesting that a mechanism unrelated to PFDA-induced hypothyroxinemia is involved. The current study showed that 7 days after dosing there was a linear trend toward decreased body temperature with increasing doses of PFDA. However, body tem-

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perature was significantly reduced only in the 80 mg/kg dose group when compared with that in the unlimited-fed group and in the 20 mg/kg PFDA group. At the 80 mg/kg dose level, pair-fed controls exhibited a reduction in body temperature which was not as great as the decrease observed in their PFDA-treated partners. The difference in body temperature observed, 1 week post-treatment, between rats receiving 80 mg PFDA/kg and their pairfed controls was not as pronounced as that observed by Langley and Pilcher (1985) between rats receiving 75 mg PFDA/kg and their pair-fed partners. This again might be due to differences in age and strain of rats used in these studies. Pair-fed control rats were able to maintain normal body core temperatures at the two lower levels of the pairedfeed restriction (20 and 40 mg/kg). Thyroid Status and PFD‘4 To.xicitJ, Even though various thyroid hormonesensitive responses have been measured in PFDA-treated rats, a uniform pattern of changes consistent with hypothyroidism or hyperthyroidism was not found. A response which, in isolation. suggested that PFDAtreated animals might be slightly hypothyroid was the decrease in basal metabolic rate: however, this decrease (8%) was too small to be biologically significant and furthermore a more extensive decrease (18%) was seen in pair-fed control rats. This suggests that the reduction in basal metabolic rate of PFDAtreated animals was not due to hypothyroidism, but rather was secondary to PFDA-induced hypophagia and weight loss. Langley and Pilcher ( 1985) suggested that some of the effects of PFDA treatment (e.g., reductions in body temperature and resting heart rate) might involve changes in circulating thyroid hormone levels. However, these particular effects of PFDA are not thyroid-specific and therefore cannot be cited as evidence of PFDA treatment altering the thyroid status of rats. Other studies in our laboratory (Kelling

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PETERSON

et al.. in press) found that PFDA treatment caused an increase in the activities of L-glycerol-3-phosphate dehydrogenase and malic enzyme, two hepatic enzymes that are thyroid hormone responsive. The activities of these two enzymes are usually increased to the same extent in a hyperthyroid state, a pattern which was not observed with PFDA treatment. This suggests that a mechanism other than hyperthyroidism is responsible for the disparate increase in activities of these two hepatic enzymes following PFDA treatment. This contention is further supported by the fact that there was no microscopic evidence for hyperactive thyroid glands in PFDA-treated rats and no increases in the circulating levels of T3 were detected in these animals. Thus along with changes in circulating levels of thyroid hormones (T, decreased and Ti unchanged), certain thyroid-sensitive responses (basal metabolic rate decreased, malit enzyme and L-glycerol-3-phosphate dehydrogenase activities increased, and thyroid histology unchanged) are altered by PFDA treatment. Nevertheless, there is not enough uniformity among the functional responses of thyroid status that are altered by PFDA treatment to conclude that PFDA-treated rats are either hyperthyroid or hypothyroid. It seems more prudent to suggest that despite their hypothyroxinemia. PFDA-treated rats show no clear-cut signs of being functionally hypothyroid. ACKNOWLEDGMENTS The authors express their appreciation to Mr. Christopher Noren, Dr. Christopher Kelhng. Mr. Haakon Thorsen. Ms. Scarlett Presley, and Ms. Cindy Sowinski for technical assistance: to Ms. Joni Mitchell and Ms. Carol Gill for the typescript: and to Ms. Pamela Plantinga. statistical laboratory, for statistical analysis of the data.

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