Arch Toxicol (1986) 59:211-215 Archives of

Toxicology �9 Springer-Verlag 1986

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in cold-adapted rats Karl Rozman 1' 2, and Helmut Greim z

i Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66103, USA 2 Abteilung fiir Toxikologie der Gesellschaft fiir Strahlen- und Umweltforschung Miinchen mbH, D-8042 Neuherberg, Federal Republic of Germany

Abstract. The toxicity of 60 Ixg/kg 2,3,7,8-tetrachlorodi- benzo-p-dioxin (TCDD) given IP in corn oil/5% acetone was examined in male Sprague-Dawley rats adapted to 25 ~ or 4 ~ ambient temperature. Cold exposure signifi- cantly reduced mean time to death and tended to increase mortality. Body weight at the time of death was reduced at both ambient temperatures to about the same extent. Thus, the rate of body weight loss was about twice as fast in non- survivors at 4 ~ than at 25 ~ There was a continuous decrease in feed intake of the non-survivors at 25 ~ until death. However, no reduction in feed intake occurred in any of the rats at 4 ~ ambient temperature. At 14 days af- ter dosing all TCDD-dosed animals were hypothyroid in terms of T4 but essentially euthyroid in terms of T 3. Oxy- gen consumption at 10 days after dosing was reduced to the same extent in all TCDD-dosed rats without regard to survival status. By day 20 after TCDD dosage, survivors increased their oxygen consumption at both ambient tem- peratures to nearly control levels whereas non-survivors were unable to do so. Body temperature of all animals re- mained within normal range except for the non-survivors, which showed reduced rectal temperature shortly before death. It is concluded (1) that cold adaptation aggravates the toxicity of TCDD, (2) that reduced feed intake alone cannot explain TCDD-induced wasting syndrome, (3) that reduced oxygen consumption in TCDD-treated rats may be due to reduced feed intake and/or altered thyroid hor- mone status, and (4) that TCDD is likely to activate meta- bolic pathways which represent a wasteful utilization of ingested and /or stored energy.

Key words: 2,3,7,8-Tetrachlorodibenzo-p-dioxin - Toxici- ty in cold-adapted rats

Introduction

The etiology of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced "wasting syndrome" has puzzled the toxicological community for some time. Moreover, it is not clear whether wasting away and lethality are causally related or not (Gasiewicz et al. 1980; Seefeld and Peterson 1984; Kelling et al. 1985).

Recently, it has been shown that thyroid hormones play an important role in the modulation of TCDD toxici-

Offprint requests to: K. Rozman

ty (Rozman et al. 1984, 1985; Pazdernik and Rozman 1985). Furthermore, it has also been demonstrated that oxygen consumption is reduced in TCDD-exposed rats (Seefeld et al. 1984). The action of thyroid hormones on energy metabolism in conjunction with oxygen consump- tion (basal metabolic rate) has been known for a long time (Ingbar 1985). Therefore, it appeared meaningful to ex- amine the effect of altered energy requirement on the toxi- city of TCDD.

Methods

Twenty-six male Sprague-Dawley rats (Sasco, Omaha, Neb.) were used for this experiment. Rats (about 4 months old) were kept individually in wire-bottom cages with free access to Purina Rat Chow (Ralston Purina Co., St. Louis, Mo.) and water.

Prior to dosing, 13 rats were adapted for 10 days to 4+1 ~ or 25+_2~ ambient temperature, respectively. Cold (4 ~ adapted rats were housed in a cold room (La- bline Instruments Co., Melrose Park, II1., Cat. No. JM- 208) whereas 25 ~ acclimated rats were housed in regular animal quarters (12-h light/dark cycle and uncontrolled humidity) for the duration of the experiment. Rats at each ambient temperature were randomly divided into two groups. After 10 days' adaptation, ten rats were dosed IP with 60 Ixg/kg TCDD (obtained from Dow Chemical Co., Midland, Mich.) in corn oil/5% acetone (5 ml/kg), where- as three rats were given vehicle alone. The preparation of the dose and the purity of TCDD was described in more detail in an earlier publication (Rozman et al. 1984). Im- mediately prior to and 14 days after dosing, 1 ml blood was drawn from the tail vein of control and TCDD-treated rats.

Body weight and feed intake (not corrected for spil- lage) were measured daily and mortality was monitored for 60 days after dosing. Oxygen consumption was deter- mined on days 10 and 20 after TCDD dosage. The mea- surements were taken in the cold room in a suitably trans- formed desiccator (volume: 2114 ml) after 15 min adapta- tion for 10 min by an oxygen monitor (Westinghouse, Pittsburgh, Penn.). Rectal temperatures of the animals were measured by a rectal probe (VWR Scientific Inc., San Francisco, Calif.) on days 7, 17 and 27 after TCDD ad- ministration. Serum thyroxine ('1"4) and triiodothyronine (T3) levels were determined in the procured samples of blood (obtained by tail clipping) by standard radioimmu-

2t2

Table 1. Effect of 60 btg/kg TCDD on body weight, mean time to death and mortality in rats adapted to 25 ~ C or 4 ~ C ambient temperature

Ambient temperature

25~ 4~

Initial body weight(g) 432__+ 11 a (10) b 445+__15(10)

Final body b weight (g) 257 + 20 (5) 296 +__ 12 (8)

Mean time b to death (d) 33___ 5 ~ (5) 18___ 2 (8)

Mortality at 60 days (%) 50 (10) 80 (10)

a Mean _+ SE b n is number of animals c Significantly different from 4 ~ C, P< 0.05

noassay (Coming Reagent) by the Clinical Labora tory o f the Universi ty o f Kansas Medica l Center. When appropr i - ate, da ta were analyzed for statistical difference by the two-tai led t-test.

Results

It is shown in Table 1 that cold adap ta t ion significantly re- duced the mean t ime to death and tended to increase mor- tali ty in TCDD- t rea ted rats. However, total body weight loss was about the same at both ambien t temperatures.

Body weights of control and T C D D treated rats at both ambient temperatures are depicted in Fig. 1. Survi- vors and non-survivors of the T C D D dosage are shown se- parately. Body weight o f control rats did not change much at either ambient tempera ture over a per iod o f 70 days. Non-survivors o f the T C D D dosage showed the well- known wasting away at both ambien t temperatures , except that at 4 ~ this process took place twice as fast as at 25 ~ Survivors of T C D D at 25 ~ init ial ly lost some weight and then stabil ized their body weight at a lower le- vel than the controls. In contrast , body weight of survivors o f T C D D at 4 ~ was not different from controls at any t ime point.

Cold exposure increased feed intake considerably (Fig. 2). During I0 days o f cold adapta t ion , rats exposed to 4 ~ ate about 50% more feed than those animals kept at 25 ~ At 25 ~ T C D D led to reduced feed intake in both the survivors and the non-survivors. However , survivors stabil ized their feed intake and only non-survivors contin- ued to decrease their dai ly feed intake (Fig. 2). Most unex- pectedly, this was not the case with rats adapted to 4 ~ ambient temperature. These animals ate the same amount o f feed as did controls without regard to survival status, suggesting that feed intake may not be affected by T C D D in co ld-adapted rats (Fig. 2).

Thyroid hormone status of rats is depicted in Tables 2 and 3 in controls as well as in survivors and non-survivors o f the T C D D dose. Cold adapta t ion tended to decrease the concentra t ion of circulating T 4 and to increase T 3 in serum of control rats. Serum T 4 levels at 14 days after dosing (Table 2) were decreased by T C D D in both survivors and non-survivors at both ambien t temperature to about the

Ambient Temperature: 4* C 50

c

_ ~ 3o

[] �9 /" Ambient Temperature: 25 C

~400 3OO

200

IO fs

DOSING I WITH TCDD T ' ' '

�9 = * = ! ! .... I, O' io 20 30 40 50 60 70

T IME ( D A Y S )

Fig. 1. Effect of 60 Ixg/kg TCDD on body weight at 4 ~ and 25 ~ ambient temperature. Control rats are represented by open squares (El), survivors o f TCDD open circles (O), and non-survivors of TCDD by darkened circles (e ) . Number of animals is given at the last time point that it was unchanged. Each data point represents mean +SE or range

Ambient Temperature: 4 ~ C '!.! e: �9 C

~ 6

I I 2

T T

o Controls o Survivors

T �9 Non-surv vors I

I DOSING WITH TCDO

.... I ! I I,, ,o 20 30 4o 50 do ;o

TIME (DAYS)

Fig. 2. Effect of 60 Ixg/kg TCDD on feed intake at 4 ~ and 25 ~ ambient temperature. Control rats are represented by open squares (El), survivors of T C D D by open circles (O), and non-survivors of TCDD darkened circles ( e ) . Number of animals is given at the last time point that it was unchanged. Each data point represents mean +SE or range

Table 2. Effect of 60 p.g/kg TCDD on serum thyroxine (T4) levels in rats adapted to 25 ~ C or 4 ~ C ambient temperature

Day after Ambient temperature dosing with TCDD or 25 ~ C 4 ~ C vehicle

213

Controls Survivors Non-survivors Controls Survivors Non-survivors

0 5.2___0.6 a (3) b 4.9-+0.2 (5) 5.0_+0.2 (5) 3.5-+0.2 (3) 4.6-+0.5 (2) 4.0_+0.2 (8)

14 4.7+0.4(3) 2.1 • (5) 1.9_+0.1c (5) 4.2_+0.3(3) 1.7_+ 0.6~ (2) 1.9+ 0.2c (6)

a Mean (Ixg/dl T4) _+ SE b n is number of animals

Significantly different from controls, p<0.05

Talile 3. Effect of 60 Ixg/kg TCDD on serum triiodothyronine (T3) levels in Sprague-Dawley rats adapted to 25 ~ C or 4 ~ C ambient temperature

Day after Ambient temperature dosing with TCDD or 25 ~ C 4 ~ C vehicle

Controls Survivors Non-survivors Controls Survivors Non-survivors

0 56• a (3) b 54_+3 (5) 59_+5 (5) 60_-_3 (3) 66_+ 10 (2) 56+5 (8)

14 45 _+ 3 (3) 48 _+ 4 (5) 49 _+ 4 (5) 64 ___ 7 (3) 55 _+ 12 (2) 58 _+ 7 (8)

a Mean (ng/dl T3) _+ SE b n is number of animals

Table 4. Effect of 60 Ixg/kg TCDD on oxygen consumption of rats adapted to 25 ~ C or 4 ~ C ambient temperature

Day after 02 consumption (ml/min/body weight 0.75) dosing with TCDD or Ambient temperature vehicle

25~ 4oc

Controls Survivors Non-survivors Controls Survivors Non-survivors

10 11.15+0.1 la (3) b 7.82 +0.39c (5) 8.20+0.49c (5) 14.08+0.46(3) 8.78 + 0.47c (2) 9.48_ 0.65c (8)

20 10.95___0.86 (3) 10.78+0.55 (5) " 8.54+0.64c(5) 14.35+0.97(3) 12.24+1.45 (2) 9.82 (1)

a Mean + SE b n is number of animals c Significantly different from controls, p< 0.05

same degree (ca. 40% of control values). However, serum T 3 levels remained unaffected by T C D D up to 14 days af- ter dosing (Table 3).

Oxygen consumpt ion per metabolic body size of cold- adapted control rats was about 25% greater than that of rats mainta ined at 25 ~ (Table 4). T C D D reduced oxygen consumpt ion at 10 days after dosing to the same level at both ambient temperatures without regard to survival sta- tus. It is important to note that this represented a greater reduction in oxygen consumpt ion of cold-adapted rats as compared to 25 ~ main ta ined rats. More importantly, by day 20 after T C D D dosage, survivors at both ambient tem- peratures have increased their oxygen consumpt ion nearly to control levds, whereas non-survivors were unable to do so (Table 4).

Rectal temperatures of control and TCDD-treated rats are shown in Table 5. Data indicate that rats were able to main ta in their body temperature near normal without re- gard to survival status at both ambient temperatures. Body temperature of non-survivors started dropping 1 0 - 2 4 h

prior to death. However, the hearts of these rats kept beat- ing in some instances until the rectal temperature declined below 20 ~ (data not shown). The body temperature o f cold-adapted rats decreased much more rapidly (about 1.7 ~ C/h ) than that of 25 o C-exposed rats (about 0.4 o C/h).

Discussion

Reduced feed intake is thought to contribute to the devel- opment of wasting syndrome in rats lethally intoxicated with T C D D because digestibility is not impaired in these animals (Seefeld and Peterson 1984; Seefeld et al. 1984). Furthermore, recent evidence suggests that lethality is as- sociated with wasting away as mortality was similar in TCDD-treated and pair-fed Fisher F-344 rats (Kelling et al. 1985). In this experiment, cold-adapted rats wasted away much more rapidly than animals kept at room tem- perature, even though they did not seem to reduce their feed intake as compared to cold-adapted controls (Table 1,

214

Table 5. Effect of 60 Ixg/kg TCDD on body temperature of rats adapted to 25 ~ C or 4 ~ C ambient temperature

Day after Ambient temperature dosing with TCDD or 25 ~ C 4 ~ C vehicle

Controls Survivors Non-survivors Controls Survivors Non-survivors

7 37.0_0.1a (3) b 37.3___0.4(5) 37.2+0.1 (5) 37.6_+0.3(3) 37.7_+0.1(2) 37.1_+0.3(8)

17 37.0_+0.1 (3) 37.3_+0.3(5) 36.3+0.3 (5) 37.3_+0.1(3) 37.7_+0.0(2) 35.4_+1.4(3)

27 37.2-+0.2 (3) 36.7-+0.3(5) 35.8-+0.1c (3) 37.5_+0.2(3) 37.6-+0.2(2) -

a Mean (~ body temperature) _+ SE b n is number of animals c Significantly different from controls, p< 0.05

Figs. 1 and 2). This finding suggests either that digestibility is reduced in cold-adapted rats dosed with TCDD, or more likely, that energy intake is not the key factor which is responsible for the development of the wasting syn- drome. However, if the caloric intake is compatible with maintenance o f normal body function, then a metabolic disorder (wasteful utilization) is likely to be responsible for the wasting away of rats dosed with a lethal amount o f TCDD.

Serum T 4 levels (Tabel 2) at 14 days after T C D D do- sage were reduced to about 40% of control values without regard to the ambient temperature, confirming previous reports relating to the effect of T C D D on thyroid hor- mones (Potter et al. 1983; Pazdernik and Rozman 1985; Rozman et al. 1985). However, T 3 serum levels (Table 3) were not diminished at either ambient temperature, indi- cating a hormonal status that is called T3-euthyroidism in the medical literature (Ingbar 1985). In spite of T3-euthy- roid status, oxygen consumption was reduced at day 10 by approximately 30% and 40% in rats exposed to 25 ~ and 4 ~ ambient temperature, respectively. Based on data presented herein, it is not possible to resolve the question about the role o f thyroid hormones a n d / o r reduced feed intake in the decreased oxygen consumption of TCDD- treated rats at either ambient temperature.

In a cold environment rats require the expenditure of more energy for the maintenance of their body tempera- ture than at room temperature. Much of the heat required for the maintenance of homeothermia in mammals origi- nates from the respiratory chain (Ingbar 1985). Even when oxidative phosphorylat ion is tightly coupled, only about 40% of the energy gets converted into ATP while the rest dissipates and thereby serves the maintenance of body temperature (Lehninger 1981). Consequently, cold-adapt- ed control rats increase their feed intake (Fig. 2) as well as their oxygen consumption (Table 5) to produce the addi- tional heat required for the maintenance of their body temperature. However, TCDD-treated rats reduce their oxygen consumption at both 25 ~ and 4 ~ ambient tem- perature to about the same level (Table 4). These results in- dicate that cold-acclimated TCDD-treated rats maintain their body temperature by a different mechanism than control cold-adapted rats. Yet, they do maintain their body temperature within normal range until approaching death. Uncoupling of oxidative phosphorylat ion could ac- count for additional heat production. However, halogenat- ed hydrocarbons in general (Nishihara 1983) and T C D D in particular (Rozman, unpublished data) do not or do on-

ly to a very small extent uncouple oxidative phosphoryla- tion. These findings are in accordance with a report of Neal et al. (1979) that hepatic ATP levels are not reduced in TCDD-treated rats. Therefore, increased requirement for heat production in cold-adapted TCDD-dosed rats is probably met by pathways other than uncoupling of oxi- dative phosphorylation. Results o f this study do not allow conclusions about possible alternative mechanisms of heat production in TCDD-treated rats. However, much faster wasting away, in spite of normal feed intake in TCDD- dosed rats at 4 ~ as compared to 25 ~ indicates that a less efficient pathway of thermogenesis may play an im- portant role in TCDD-induced "wasting syndrome".

Acknowledgements. This research was supported by a gift of Dow Chemical Co. to the University of Kansas Endowment Associa- tion. Skilful technical assistance of Margitta Hoefler and Marilyn Ryan has greatly contributed to the conduct of these experiments.

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Received May 12, 1986/Accepted July 8, 1986