Bernhard U. Stahl

Reduced activities of key enzymes of gluconeogenesis as possible cause of acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats

Male Sprague--Dawley rats (350-375 g) were injected i.p. with TCDD (25 [sublethal dose] and 125 micrograms/kg [lethal dose], respectively, in corn oil/acetone), or vehicle only; vehicle-treated animals were pair-fed to their TCDD-treated counterparts. 1, 2, 4, 8, 16, and 32 days (28 days for lethal dose) thereafter, animals were sacrificed and activities of two key enzymes of gluconeogenesis determined in livers of rats. In livers of pair-fed rats both enzyme activities were little affected. In the livers of TCDD-treated animals the activity of phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) decreased rapidly, exhibiting significant losses by the 2nd day after treatment. Time course and extent of loss of PEPCK activity (about 50%) were similar after either dose. The activity of glucose-6-phosphatase (G-6-Pase, EC 3.1.3.9) decreased more slowly as a result of TCDD treatment; statistically significant losses were observed by 4 or 8 days after the lethal and sublethal dose, respectively. These results confirm the hypothesis that reduced in vivo rates of gluconeogenesis in TCDD-treated rats are due to decreased activities of gluconeogenic enzymes. In an additional set of experiments, rats were treated with 125 micrograms/kg TCDD, 25 micrograms/kg TCDD, or with vehicle alone. The 25 micrograms/kg or vehicle-treated rats were then pair-fed to rats dosed with 125 micrograms/kg of TCDD. Mean time to death and body weight loss at the time of death were essentially identical in all groups, lending additional support to the hypothesis that reduced feed intake is the major cause of TCDD-induced death in male Sprague--Dawley rats. Both appetite suppression and reduced total PEPCK activity in whole livers occurred in the same dose-ranges of TCDD, suggesting the possibility of a cause-effect relationship.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on liver phosphoenolpyruvate carboxykinase (PEPCK) activity, glucose homeostasis and plasma amino acid concentrations in the most TCDD-susceptible and the most TCDD-resistant rat strains.

Abstract Reduced gluconeogenesis due to decreased activity of key gluconeogenic enzymes in liver, together with feed refusal, has been suggested to play an important role in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced lethality in rats. This study was carried out to further analyse the toxicological significance of reduced gluconeogenesis by comparing dose-responses and time-courses of effects of TCDD on the activity of phosphoenolpyruvate carboxykinase (PEPCK) in liver, liver glycogen concentration as well as plasma concentrations of glucose and amino acids in both genders of TCDD-sensitive Long-Evans (L-E) rats and TCDD-resistant Han/Wistar (H/W) rats. A dose-dependent decrease in PEPCK activity was observed in H/W rats, but in L-E rats the activity was not decreased. However, TCDD impaired the strong increase in liver PEPCK activity observed in pair-fed controls of the L-E strain. Liver glycogen concentrations were severely decreased in L-E rats and moderately in H/W rats. This effect seems to be secondary to reduced feed intake, since a similar decrease was seen in pair-fed controls. Decreases in plasma glucose concentrations were also more profound in L-E rats than in H/W rats, but pair-fed controls were generally less affected. Circulating concentrations of amino acids were markedly increased in TCDD-treated L-E rats, which is likely to reflect increased mobilization of amino acids and their decreased metabolism in liver. Reduction of liver PEPCK activity cannot account for the sensitivity difference of these two strains of rats in terms of mortality. Nevertheless, the response of both strains of TCDD-treated rats regarding gluconeogenesis is different from that seen in pair-fed controls and suggesting that impairment of this pathway contributes to the development of the wasting syndrome.

Reduction of hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is due to decreased mRNA levels

We have previously shown that the rate of hepatic gluconeogenesis is reduced in TCDD-treated rats and that this decrease in carbohydrate production is associated with a dose-dependent reduction of the activity of PEPCK, the rate limiting enzyme of gluconeogenesis. This derailment of glucose metabolism has been suggested to be the critical lesion in acute TCDD toxicity. To further elucidate the mechanism of decreased PEPCK activity we performed Northern blot analyses using a cDNA probe complementary to a portion of the mRNA coding for PEPCK. We have demonstrated that 4 and 8 days after TCDD treatment (125 micrograms/kg, p.o.) liver PEPCK mRNA in Sprague-Dawley rats was decreased to very low levels as compared to vehicle-treated and pair-fed control animals. This decline of PEPCK mRNA was paralleled by decreased levels of PEPCK protein, as revealed by Western blot analyses and was accompanied by a reduction in the enzymatic activity of PEPCK. These results indicate that the decrease of PEPCK activity by TCDD is most likely the result of decreased expression of the PEPCK gene. These together with previous results also suggest that many of the physiological responses occurring in TCDD-treated animals (reduced feed intake, decreased insulin, increased corticosterone, increased glucagon and cAMP levels) which would normally stimulate PEPCK gene expression, are ineffective. Furthermore tryptophan 2,3-dioxygenase (TdO) activity, which is regulated in a very similar fashion to PEPCK activity, is also reduced after TCDD treatment, suggesting a common mechanism by which TCDD alters the regulation of these enzymes. P-450 1A1 mRNA and related EROD activity were maximally induced under the conditions of these experiments and represent a positive control for TCDD-related alterations of gene expression. However, because of differences in the dose-response characteristics of TCDD-induced reduction of PEPCK activity and induction of EROD activity an involvement of the Ah receptor in the reduction of PEPCK activity cannot be postulated.

Tissue Distribution and Toxicokinetics of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in Rats after Intravenous Injection

Male Sprague-Dawley rats (240-290 g) received intravenously a nonlethal (9.25 micrograms/kg) or a lethal (72.7 micrograms/kg) dose of 14C-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) administered as an emulsion. Animals were euthanized between 5 min and 16 days (lethal dose) or 32 days (nonlethal dose) after treatment. Tissue distribution was considered complete after 24 hr, as by this time radioactivity levels in white adipose tissue had reached a maximum. The highest levels of radioactivity were found in liver (5% of dose/g tissue), followed by white fat (1% of dose/g tissue); serum was lowest at 0.01% of dose/ml serum. Relatively high levels of radioactivity were also detected in most known target organs of TCDD toxicity, e.g., brown fat, adrenals, and thyroid. The pattern of organ distribution of TCDD was essentially the same after the lethal and the nonlethal dose, but did not follow a simple lipophilicity relationship, as levels in liver were higher than those in white fat, and those in brain were extremely low. A pool of TCDD in liposomes initially trapped in lung and spleen was redistributed within 24 hr mainly to liver and adipose tissue. Affinity of TCDD to storage fat seemed to play a more important role as a driving force for redistribution than did induction of cytochrome P450 1A2. The terminal slope of elimination of TCDD from tissues indicated a half-life of 16 days after the nonlethal dose. After the lethal dose radioactivity declined in all tissues for 2 to 8 days and then increased again, reflecting shrinking tissue volumes as well as remobilization of TCDD caused by the process of body mass wasting. Distribution data for 17 tissues and serum were subjected to regression analysis and resulted in up to two uptake phases and up to three elimination phases for a given tissue. After the nonlethal dose TCDD was mainly excreted via feces; combined urinary and fecal excretions occurred with a biological half-life of 16.3 +/- 3.0 days. Much longer half-lives were detected in white fat and skin. After the lethal dose, the fecal excretion of TCDD-derived radioactivity decreased after 8 days, and urinary excretion increased starting 12 days after dosing. Radioactivity in liver and white fat and the extractable portion in feces was mainly unchanged TCDD, as determined by thin-layer chromatography. Radioactivity in urine indicated the presence of a metabolite(s) of TCDD only.

Relative potency of chlorinated dibenzo-p-dioxins (CDDs) in acute, subchronic and chronic (carcinogenicity) toxicity studies: implications for risk assessment of chemical mixtures

This paper shows that the relative toxic potency of four chlorinated dibenzo-p-dioxins (CDDs) is similar in two species with different sensitivities (guinea pig, Sprague-Dawley rat). More importantly, it also demonstrates that the relative toxic potencies of these homologues are very similar for acute, subchronic and chronic dosing in the same species (rat). Furthermore, examination of different endpoints of toxicity (mortality, porphyria, carcinogenicity) suggests that the dose-responses for these diverse end-points after acute, subchronic, and chronic administration are very similar if not identical for tetra-CDD. Based on toxicokinetic and toxicodynamic considerations, a new, possibly generalizable rule (average tissue concentration x time = toxicity) is derived for CDDs. Implicit in the relative potency arguments of CDDs is the requirement of a practical threshold dose for all endpoints of toxicity including cancer.

Comparative toxicity of four chlorinated dibenzo-p-dioxins (CDDs) and their mixture. Part II : Structure-activity relationships with inhibition of hepatic phosphoenolpyruvate carboxykinase, pyruvate carboxylase, and γ-glutamyl transpeptidase activities

Male Sprague-Dawley rats were treated with an LD20, LD50 and LD80 respectively, of tetra-, penta-, hexa-,hepta-CDD and a mixture of the four CDDs, all carrying chlorine substituents in the biologically crucial 2, 3, 7, and 8 positions. Specific activities of two key enzymes of gluconeogenesis, viz, phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC), as well as the activity of the preneoplastic marker enzyme γ-glutamyl transpeptidase (γ-GT), were determined in livers of CDD-treated and ad libitum-fed control animals. PEPCK activity showed evidence for dose-related inhibition on the second day after dosing; PC activity was slightly reduced, whereas γ-GT activity was dose-dependently inhibited. By 8 days after dosing PEPCK activities were dose-dependently decreased after administration of all four CDDs and their mixture. PC activities were significantly reduced, but no dose-response was evident. The activity of γ-GT was dosedependently inhibited, but only to a value of 25% below control activities. It is concluded that CDDs share a common mechanism of acute toxicity, viz, inhibition of glucocorticoid-dependent enzymes which results in a derailment of intermediary metabolism not compatible with survival of the animals.

Subchronic/chronic toxicity of 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD) in rats. Part II. Biochemical effects

Groups of 20 male and 20 female rats were given five different oral doses of 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD) or one dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) divided into four daily loading doses and six biweekly maintenance doses. The dosing period was 13 weeks, after which half of the rats were necropsied and the rest assigned to an off-dose period of another 13 weeks. At the end of the dosing period, liver ethoxyresorufin O-deethylase (EROD) activity was dose-dependently increased starting at the lowest dose (7- to 10-fold) with maximum induction (50- to 100-fold) at the middle or second highest dose. There was a slight reversibility of this effect in HpCDD-treated rats, particularly at lower doses, and a pronounced reversibility in TCDD-dosed rats, both in accordance with respective toxicokinetics. The activity of phosphoenolpyruvate carboxykinase in liver was dose-dependently decreased (up to 60%) at the two or three highest doses of HpCDD and also in the TCDD dosage group. Liver tryptophan 2,3-dioxygenase activity was decreased at the two highest doses of HpCDD (up to 41%), particularly in females. Serum tryptophan concentrations were elevated in rats found moribund due to wasting. There was a dose-dependent decrease in serum glucose concentrations (up to 30%) at the end of the dosing period. Serum thyroxin (T4) concentrations showed a dose-dependent decrease (78% at the highest dose) beginning in the middle dose for HpCDD and in the TCDD dosage group. Serum triiodothyronine (T3) concentrations were only slightly affected, except that they were somewhat decreased in moribund animals. The results demonstrate that similar biochemical changes occur in rats after single as after multiple dosing with HpCDD and TCDD. Based on these endpoints, the relative potency of HpCDD after subchronic exposure is in agreement with the international toxic equivalency factor (I-TEF) of 0.01 and, more specifically, with a TEF of 0.007 based on LD50 values in the same strain of rats.

Comparative toxicity of four chlorinated dibenzo-p-dioxins (CDDs) and their mixture. IV: Determination of liver concentrations

Groups of male Sprague-Dawley rats were administered orally the following chlorinated dibenzo-p-dioxins (CDDs) in corn oil/acetone (95/5; v/v): 30–60 μg/kg 2,3,7,8-tetrachlorodibenzo-dioxin (tetra-CDD), 160–270 μg/kg 1,2,3,7,8-pentachlorodibenzo-p-dioxins (penta-CDD), 630–1249 μg/kg 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (hexa-CDD) and 5000–8000 μg/kg 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (hepta-CDD) or a mixture of the four homologues such that each was present in the mixture at one quarter of its dose as a single compound. Animals were killed at 2 and 8 days after dosing. Livers were immediately removed, and aliquots frozen in liquid nitrogen. Storage occurred at −80°C until further use. About 0.2 g of each lyophilized rat liver was extracted, the extract purified by column chromatography and analyzed by GC/MS for CDD content. Results obtained suggest that the absorption of CDDs after oral administration decreases in the order of tetra-CDD≥penta-CDD>hexa-CDD>hepta-CDD, indicating that the dose was an incomplete surrogate of exposure in parts I–III of this publication series (Stahl et al. 1992; Weber et al. 1992a,b). Moreover, data also support the notion that the pharmacokinetics of CDD mixtures at high doses are somewhat different from those expected based on single compound exposures. Our findings suggest that the intrinsic relative potency in terms of toxic equivalents (TEQ) of the higher chlorinated homologues is slightly greater (about a factor of 2) than suggested by Stahl et al. (1992), because of reduced absorption, whereas the contribution to total potency of the lower chlorinated homologues in mixtures is slightly higher (about a factor of 2) because of increased relative liver concentrations. This later finding appears to be due to altered pharmacokinetics of mixtures of CDDs, probably originating in changes of partial solubility of the lower chlorinated homologues in fat under conditions of near saturation.

Toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in two substrains of male Long-Evans rats after intravenous injection.

Toxicokinetics of a nontoxic intravenous dose of 14C-labeled TCDD were studied in two substrains of Long-Evans (L-E) rats with a fivefold difference in sensitivity in terms of TCDD-induced mortality. The Turku/AB Long-Evans rat (T L-E) is the most sensitive rat strain with an oral LD50 of 17.7 microgram/kg, whereas the Charles River Long-Evans rat (CR-L-E) is a more resistant strain (oral LD50 95.2 microgram/kg). Samples of 18 tissues were collected 1, 2, 4, 8, 16, and 32 days after dosing and analyzed for radioactivity. Body weight and fecal and urinary excretion of radioactivity were monitored daily during the 32-day study period. CR L-E rats grew significantly faster than T L-E rats, increasing their body weight by 60% in 32 days compared with only 16% in T L-E rats. This difference was not caused by toxicity, because the weight gain was identical in control and TCDD-treated rats of both substrains. Tissue concentrations of [14CC]TCDD-associated radioactivity and area under the curve (AUC) values were lower in CR L-E than in T L-E rats. The most pronounced differences were found in thymus, white adipose tissue, brown adipose tissue, and adrenals. The decrease of TCDD concentration in tissues was faster in CR L-E than in T L-E rats, whereas fecal and urinary excretion was faster in T L-E than in C L-E rats. Elimination half-life was 20.2 days in T L-E rats and 28.9 days CR L-E rats. Differential toxicokinetics of TCDD in the two L-E substrains provide a likely explanation for the greater sensitivity of the T L-E strain, since observed differences in tissue concentrations and AUC values are in good agreement with the difference in susceptibility. In addition to the more efficient tissue uptake of TCDD in T L-E rats than in CR L-E rats, the major contributing factor to differences in toxicokinetics seems to be a differential growth rate (dilution by growth), which in turn appears to provide an explanation for the difference in susceptibility. More rapid excretion of TCDD in T L-E rats than in CR L-E rats is clearly a result of higher tissue concentrations in T L-E rats. However, this faster excretion rate is not sufficient to counterbalance the much slower dilution by growth in T L-E rats than in CR L-E rats. Thus, dilution by growth can be a more important factor in determining the toxicokinetics and toxicity of TCDD in rodents than is excretion.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced appetite suppression in the sprague-dawley rat is not a direct effect on feed intake regulation in the brain

The most striking sign of acute toxicity of TCDD in animals is a progressive reduction of feed intake, accompanied by loss of body weight eventually resulting in death. The mechanism(s) of this voluntary feed refusal is (are) not known but it is generally accepted that both centrally and peripherally (via feedback) acting anorectic agents exert their effect(s) in the hypothalamus. In this study direct administration into the lateral cerebral ventricle of rats resulted in much higher concentrations of TCDD in the hypothalamus and also in other regions of the brain than after a lethal intravenous (iv) injection. While rats injected iv displayed the expected cachectic syndrome, intracerebroventricularly (icv)-dosed animals ate and gained weight normally. These findings preclude the possibility of a direct effect of TCDD on appetite-regulating areas of the brain. Moreover, these results require the assumption that the appetite suppressive effect of TCDD is due to a (feedback) mechanism originating in the periphery.