Subchronic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and their reversibility in male Sprague-Dawley rats

Subchronic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and their reversibility in male Sprague-Dawley rats

ELSEVIER Toxicology 97 (1995) 133-140 Subchronic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and their reversibility in male Sprague-Dawle...

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Toxicology 97 (1995) 133-140

Subchronic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and their reversibility in male Sprague-Dawley rats Xuelin

Lia, Karl

K. Rozman*a’b

‘Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160-7417, USA bSection of Environmental Toxicology, GSF-lnstitut ftir Toxikologie, 85758 Neuherberg, Germany

Received 19 May 1994; accepted 9 August 1994

Abstract

The hypothesis tested in this experiment is that effects of 2,3,7,8-tetrachlorodibenzoq-dioxin (TCDD) show identical dose-responses after subchronic as after acute exposure when the dose is corrected for toxicokinetics. Groups of male Sprague-Dawley (S-D) rats were administered orally a total dose of 0, 0.2, 2.3, 11.5, 35, 70 or 115 &kg of TCDD over a period of 10 weeks at 4 ml/kg of vehicle. Body weight was recorded weekly. One week after the last dose of TCDD one half of the rats was killed and tryptophan 2,3dioxygenase (TdO), 7_ethoxyresorutin-Odeethylase (EROD) and phosphoenolpyruvate carboxykinase (PEPCK) activities were measured in livers, whereas tryptophan and total T4 (lT4) were determined in serum. The results show that the dose-responses for decreased TdO and PEPCK activity and elevated serum tryptophan levels are similar if not the same as the dose-response for subchronic retardation of body weight increase. They also demonstrate that the dose-responses for the induction of EROD activity and the reduction of serum TT4 occurred at much lower doses than those for decreased TdO and PEPCK activities or elevated tryptophan levels and mortality. After a 6-week recovery period, PEPCK and TdO activities in liver as well as tryptophan in serum returned to near control values, whereas EROD activity and serum lT4 still displayed a dosedependent induction and reduction, respectively, albeit both shifted to the right in accordance with toxicokinetics. These data support the notion that subchronic dose-responses of TCDD are similar to acute dose-responses when corrected for toxicokinetics and that at least some TCDD-induced effects are reversible also in accordance with toxicokinetics. Keywords: TCDD; Subchronic toxicity; PEPCK, TdO; EROD; Tryptophan;

Serum TT4

1. lntroductlon

contaminant produced during the manufacture of chlorophenols, hexachlorophene, and phenoxy

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most toxic chemicals in some mammalian species (Vos, 1978). TCDD was an unwanted

herbicides (Lilienfeld and Gallo, 1989). More recently the major source of TCDD reaching the environment is municipal waste incineration (Alexandrou and Pawliszyn, 1989) and metal industries in many countries. TCDD exerts

* Corresponding author. 0300-483X195/309.50 0 1995 Elsevier Science Ireland SSDI 0300-483X(94)02938-Q

Ltd. All rights reserved

134

X. Li, K.K. Rozman / Toxicology 97 (1995) 133-140

characteristic effects in laboratory animals (Skene et al., 1989). In a somewhat species-dependent fashion, these include a wasting syndrome, delayed lethality, hepatotoxicity, thymic atrophy, chloracne, porphyria, carcinogenicity, reproductive and developmental toxicity, etc. Some progress has been made in recent years to elucidate the mechanism of acute toxicity of TCDD in rats by showing that TCDD-induced death is probably due to a combination of appetite suppression (Christian et al., 1986; Stahl et al., 1991; Weber et al., 1991a) and reduction of gluconeogenesis (Weber et al., 1991a,b,c) leading to an eventually lethal hypoglycemia. Reduced gluconeogenesis was shown to be the result of a dose-dependent decrease in the activity of the key enzyme of this biochemical pathway, viz. phosphoenolpyruvate carboxykinase (PEPCK). Appetite suppression, on the other hand, was assumed to be the result of a dosedependent decrease in the activity of the key regulatory enzyme of the kynurenine pathway, viz. tryptophan 2,3-dioxygenase (TdO), which in turn leads to a dose-dependent increase in serum and tissue concentrations of tryptophan (Rozman et al., 1991; Weber et al., 1992b; Fan and Rozman, 1994). Tryptophan, in turn, is known to modulate the dose-response to endogenous appetite suppressants such as cholecystokinin (Debas et al. 1975). In this experiment the hypothesis tested was that some of the biochemical and toxicological doseresponses after either acute or subchronic exposure to TCDD are identical when the dose is corrected for pharmacokinetics. A confirmation of this hypothesis was expected to provide further support for the notion that dose x time = constant toxicity for TCDD as was suggested by Rozman et al. (1993).

2. Materials and methods 2. I. Animals Male Sprague-Dawley rats (Sasco, Omaha, NE, 200-225 g) were distributed randomly into seven groups (six to seven rats for each dosage and one control group). Rats were housed in suspended stainless steel wire-bottom cages individually with free access to feed (Ralston Purina Co., St. Louis,

MO) and water (room temperature: 25 f 2°C; 12 h light-dark cycle and uncontrolled humidity). After one week of adaptation, all animals were dosed orally once a week for 10 weeks with different doses of TCDD or vehicle alone. Body weight was measured weekly and mortality recorded. TCDD (Cambridge Isotope Laboratories, Wobum, MA, Purity > 99%) was dissolved in 95:5 (v:v) corn oil:acetone (Fisher Allied Scientific/Sigma Chem. Co., St. Louis, MO) and administered at 4 ml/kg. The dosing was conducted by gastric intubation at 0.00,0.02,0.23, 1.15, 3.50, 7.00, 11.50 &kg per week, respectively. This dosage regimen resulted in cumulative doses of 0, 0.2, 2.3, 11.5, 35, 70 or 115 &kg. One half of the rats in each group was sacrificed at week 10 (one week after the last dose), whereas the others were terminated at week 16. Livers were removed and stored at -80°C for later biochemical analyses. Trunk blood was also collected and serum stored frozen for later tryptophan and TT4 determinations. All animals were deprived of feed for 24 h prior to termination. 2.2. Subcellular fractionation Frozen liver samples were homogenized with a Teflon-pestled Potter-Elvehjem homogenizer in three volumes of 0.25 M sucrose or in 10 volumes of potassium phosphate buffer (20 mM, pH 7.0) at 0-4°C. Sucrose homogenates were centrifuged for 30 min at 10 000 x g (L5-65 ultracentrifuge, Beckman Instnmrents Inc., Palo Alto, CA), the pellets discarded and the supematants centrifuged for 1 h at 100 000 x g. The resulting supematants were employed as cytosolic fraction for measuring PEPCK activity, whereas the resuspended pellets (0.25 M sucrose) were used for determination of EROD activity. Protein concentration in raw homogenates was determined by the Biuret method (Technical Bulletin #514, Sigma Chemical Co., St Louis, MO) after solubilization with 5.3% cholic acid and ultrasonication (10 min, bath ultrasonifier B52OOR-1;Fisher Scientific, St Louis, MO). Protein concentration in the cytosol was measured by the Bradford method (Bradford, 1976) using a bovine serum albumin standard. Spectrophotometric measurements were performed by a Shimadzu UV16OU (Delta Instrument Co., Waterloo, IL).

X. Li. K.K. Rozman / Toxicology 97 (1995) 133-140

2.3. PEPCK activity

according to Yamuda et al. (Yamuda et al., 1983). Serum was prepared from trunk blood, and deproteinized by addition of 1 volume of 5% trichloroacetic acid. After dilution with 1 volume of 0.01 M acetate buffer (PH 4.3), lipids were extracted with 2 volumes of chloroform, and the supematant diluted 140 with mobile phase (0.01 M acetate buffer, pH 4.3, with 30% methanol). Twenty ~1 of this solution were injected into a Zorbax C8 reverse phase column of a Shimadzu SCL 6-A HPLC equipped with a RF-535 fluorometric detector (Delta Instrument Co., Waterloo, IL). The flow rate was 1.2 ml/mm at 30°C. A 50 @ml tryptophan solution (Sigma Chemical Co., St Louis, MO) served as standard.

Liver PEPCK activity was determined as described by Petrescu et al. (Petrescu et al., 1979) using deoxyguanosine 5’-diphosphate as the nucleotide substrate. A 50-~1aliquot of supematants, adjusted to a protein content of 400 pg, was used per assay. No rotenone was added. Oxaloacetate formed during the (reverse) enzyme reaction was determined by reduction with malate dehydrogenase in the presence of NADH; the change in absorbance was measured spectrophotometrically at 340 nm. Blanks contained neither bicarbonate nor CO*. The reaction was allowed to proceed for 5 min at 23°C. 2.4. TdO activity TdO activity in liver was measured according to Metzler et al. (Metzler et al., 1982). Frozen liver samples were homogenized in 10 volumes of icecold potassium phosphate buffer (20 mM, pH 7.0) containing 2.5 mM tryptophan and 1.36 mg methemoglobin per 10 ml. The reaction was allowed to run at 37’C for either 40 or 80 min and was terminated by addition of perchloric acid/ethanol/ water (1: 1:1). The standard sample containing 200 ~1 of 0.15 mM L-kynurenine was assayed parallel to the 80 min run. The formation of an azo-dye derivate of kynurenine was measured at 560 nm. The enzyme activity was calculated based on the difference in the absorbance between the two time points. 2.5. Tryptophan Serum tryptophan

was determined by HPLC

Table 1 Body weight and mortality

of rats exposed

Total dose (N&g) 0.0 0.2 2.3

11.5 35.0 70.0 115.0 ‘Mean f SE. (n = 6-7). %I = 2.

subchronically

2.6. TT4 TT4 in serum was measured by radioimmunoassay (Diagnostic Products Corp., Los Angeles, CA). 2.7. EROD activity EROD activity in liver was determined fluorometrically as described by Dutton and Parkinson (Dutton and Parkinson, 1989). The protein concentration of the microsomes was adjusted to 700 &ml. Ethoxyresorufin was added to the samples as substrate. The reaction was started by addition of 50 ~1 of a NADPH regenerating system, and was incubated at 37°C for 60 min. The reaction was terminated by addition of 2 ml icecold acetone. Blanks were prepared by addition of acetone prior to the NADPH regenerating system. Standard samples which had been incubated

to TCDD

Body weight (g) Initial 244 225 241 242 237 245

f * f f f f

21a 7 14 14 12 16

242 f 17

Mortality

Mean time to death (days)

l

016 O/6 O/6 O/6 O/6 O/6

-

322 f 46b

411

53.5 f 8.8

Week 10

Week 16

400 384 392 340 325 298

445 458 399 341 333 306

f f f * zt f

51 42 21 29 39 39

245 A 53

135

5-l f 46 zt 56 + 26 l 38 ct 80

X. Li, K. K. Roman / Toxicology 97 (1995) 133-140

136

without ethoxyresorutin were prepared by addition of 5 and 20 ~1, respectively, of a 500 PM resorufin solution in ethylene glycol. Alter centrifugation at 2500 rpm for 5 min, fluorescence in the supernatants was measured at 535 nm (excitation) and 585 nm (emission) in a Shimadxu RF-594 fluorometer (Delta Instrument Co., Waterloo, IL).

0.25

2.8. Statistical analysis Data from controls were compared to TCDDtreated animals by a two-tailed Student’s t-test with P < 0.05 deemed significant. 3. Results

60 3

50

5 C 5

30

E &I z 40

Subchronic exposure of rats to high doses of TCDD for 10 weeks resulted in the expected wasting syndrome and mortality (Table 1). There was a dose-dependent retardation of body weight increase in the four higher groups, but no effect in the two lowest dosage groups (Fig. 1). Hepatic TdO activity was decreased at the two highest dosages with a concurrent increase in serum tryptophan levels (Fig. 2). The dose-responses for TdO activity and serum tryptophan levels displayed the expected inverse relationship. Hepatic PEPCK activity was also dose-dependently reduced (Fig. 3). Moreover, the dose responses of reduced TdO and PEPCK activities and elevated serum tryptophan concentrations were very similar if not identical to the dose-responses of subchronic retardation of

% E 20 5 uY 10 0 Controls

1

0.1

10

Fig. 2. Hepatic TdO activity (upper panel) and serum tryptophan levels (lower panel) after 10 weeks of subchronic exposure to TCDD. Values are mean f S.E. of three rats. *Significantly different from controls (P < 0.05).

* “1 $

L.3..._ i

t

*

*

*

Controls

0.1

1

10

100

1000

Total dose @g/kg)

Fig. I. Body weight change of rats after 10 weeks of subchronic exposure to TCDD. Values are mean l S.E. of three rats. *Significantly different from controls (P < 0.05).

1000

Total dose @g/kg)

+

-50

100

Ccmtrds

0.1

1

10

*

100

1000

Total dose @g/kg)

Fig. 3. Hepatic PEPCK activity after 10 weeks of subchronic exposure to TCDD. Values are mean f S.E. of three rats. *Significantly different from controls (P < 0.05).

X. Li, K.K. Roman/Toxicology 97 (1995) 133-140

137

8000,

E

5 6000-

E” z 5 g

4000-

.G .s H n 0 2000. f.5

Fig. 4. Hepatic EROD activity after 10 weeks of subchronic exposure to TCDD (B) and after an additional 6 week recovery period ( 0). Values are mean f SE. of three rata, except in the 115-@kg dosage group where the values are mean f range of two rats. *Significantly different from controls (P < 0.05).

reduced, but the slope and ED, of this doseresponse appeared different from both induction of EROD activity and subchronic toxicity with its associated biochemical effects (Fig. 5). After a 6-week recovery period, PEPCK and

body weight increase (see Table 1 and Figs. l-3). EROD activity was induced even at the lowest dose of TCDD and induction was at maximum before any signs of subchronic toxicity occurred (Fig. 4). Serum TT4 was also dose-dependently 7-

0

--.Y Controls

0.1

1

10

100

1000

Total dose @g/kg)

Fig. 5. Serum ‘IT4 concentrations aRer 10 weeks of subchronic exposure to TCDD. Values are mean f S.E. of three rats. lSignitTcantly different from controls (P < 0.05).

X. Li, K.K. Rozman / Toxicology 97 (1995)

133-140

(Fig. 6), respectively, albeit both shifted to the right in accordance with toxicokinetics. 4. Discussion

b

d

II.3

2.3

Tdel

Dxe

35

70

115

(JQ’@)

Fig.

6. Hepatic PEPCK (panel a) and TdO activities (panel b) as well as serum tryptophan (panel c) and lT4 (panel d) levels after the dweek recovery period. Values are mean f SE. of three rats, except in the 115&kg dosage group where the values are mean * range of two rats. *Significantly different from controls (P < 0.05).

TdO activities as well as serum tryptophan levels returned to near control values (Fig. 6), whereas EROD activity and serum TT4 still displayed a dose-dependent induction (Fig. 4) and reduction

This subchronic toxicity study provides additional support for the hypothesis that subchronic (multiple dose) toxicity of TCDD is in many ways identical to its acute (single dose) toxicity when the dose is corrected for pharmacokinetics. Expressed differently, the cumulative dose minus the portion of dose already eliminated (= dose remaining to be eliminated = body burden) alone determines toxicity (Table 1). and associated biochemical effects of TCDD as suggested by Rozman et al. (1993). For example, retardation of body weight increase did not occur until a total dose of about 5-10 &kg was reached (Fig. l), which is about the single dose of TCDD causing signiticant body weight effect (Seefeld et al., 1984; Stahl et al., 1992). Similarly, reduced PEPCK (Fig. 3) and TdO (Fig. 2) activities as well as elevated serum tryptophan levels became manifest at doses virtually identical to those causing these effects after single doses of TCDD (Weber et al., 1991a,b,c, 1992a; Rozman et al., 1991). Induction of EROD activity was as expected based on single dose experiments (Roth et al., 1988) at or near maximum already at a total dose of about 5- 10 &kg of TCDD after 10 weeks (Fig. 4). During the 6 weeks of recovery, corresponding to about 2 half lives (tllz = 20 days; Weber et al., 1993) during which 75% of TCDD’s near steadystate body burden after the IO-week dosing period had already been eliminated, the induction of EROD activity became partially reversible manifested as a shift of the dose-response curve to the right (Fig. 4). Serum IT4 levels were clearly reduced by all but the lowest dose of TCDD (Fig. 5). The dose selection for establishing a dose response for this TCDD effect was not ideal. It is apparent that the three highest doses caused already maximum reduction in serum ‘IT4 levels. Therefore, the dose-response for this effect must be assumed to be between 0.1 and 10 @g/kg of total TCDD dose. In fact the lack of complete reversibility of this effect after 6 weeks of recovery (Fig. 6) suggests that the EDso for this dose-response is either closer to the

X. Li. K.K. Rozman/

1 &kg cumulative dose or that this effect is less reversible than induction of EROD activity. Both PEPCK and TdO activity displayed trends towards reversibility after the 6-week recovery period (Fig. 6), although complete reversibility did not occur at the highest cumulative dose (Fig. 6), which was 115 hg/kg of TCDD. This is in agreement with pharmacokinetic considerations that, assuming a constant half-life, the remaining body burden from this dosing/recovery regimen corresponds to a single dose of about 12 @g/kg of TCDD, which is a dose at the lower end of the dose-response curve for reductions of these enzyme activities (Weber et al., 199la,b,c, 1992a,b). In accordance with the reversibility of decreased TdO activity after subchronic dosing with TCDD, serum tryptophan levels returned to near normal values (Fig. 6) at the end of the 6-week recovery period. In conclusion, this experiment supports the claim by Rozman et al. (1993) that subchronit/chronic toxicity of TCDD appears to follow Haber’s (Haber, 1924) and Druckrey’s (Druckrey and Kiipfmiiller, 1948) law for special cases in toxicology when dose x time = constant toxicity, provided that appropriate pharmacokinetic considerations are taken into account. References Alexandrou, N. and Pawlisxyn, J. (1989) Supercritical fluid extraction for the rapid determination of polychlorinated dibenzo-pdioxins and dibenxofurans in municipal incinerator fly ash. Anal. Chem. 61, 2770-2776. Bradford, M.M. (1976) A rapid and sensitive method for the quantization of microgram quantities of protein using the principle of protein-dye binding. Anal. B&hem. 72, 248-254. Christian, B.J., Inhom, S.R. and Peterson, R.E. (1986) Relationship of the wasting syndrome to lethality in rats treated with 2,3,7,8-tetrachlorodibenxo-p-dioxin. Toxicol. Appl. Phannacol. 82, 239-255. Debas, H.T., Farooq, O., Grossman, MI., (1975) Inhibition of gastric emptying is a physiologic action of cholecystokinin. Gastroenterology 68, 1211-1217. Druckrey, V.H. und Kiipfmiiller, K. (1948) Quantitative Analyse der Krebsentstehung. Z. Naturforsch. 3b, 254-266. Dutton, D.R. and Parkinson, A. (1989) Reduction of 7alkoxyresorufins by NADPH-cytochrome P450 reductase and its differential effects on their 0-dealkylation by rat

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