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.

Key enzymes of gluconeogenesis are dose-dependently reduced in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats.

Male Sprague-Dawley rats (240-245 g) were dosed ip with 5, 15, 25, or 125 micrograms/kg -,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in corn oil. Ad libitum-fed and pair-fed controls received vehicle (4 ml/kg) alone. Two or 8 days after dosing five rats of each group were sacrificed, their livers removed and assayed for the activities of three gluconeogenic enzymes [phosphoenol-pyruvate carboxykinase (PEPCK; EC 4.1.1.32), pyruvate carboxylase (PC; EC 6.4.1.1), and glucose-6-phosphatase (G-6-Pase, EC 3.13.9)], and one glycolytic enzyme [pyruvate kinase (PK; EC 2.7.1.40)] by established procedures. The activity of PK was not affected by TCDD at either time point. The activity of G-6-Pase tended to be decreased in TCDD-treated animals, as compared to pair-fed controls, but the decrease was variable without an apparent dose-response. The activity of PEPCK was significantly decreased 2 days after dosing, but a clear dose-response was apparent only at the 8-day time point. Maximum loss of activity at the highest dose was 56% below pair-fed control levels. PC activity was slightly decreased 2 days after TCDD treatment and displayed statistically significant, dose-dependent reduction by 8 days after dosing with a 49% loss of enzyme activity after the highest dose. It is concluded that inhibition of gluconeogenesis by TCDD previously demonstrated in vivo is probably due to decreased activities of PEPCK and PC. The data also support the prevailing view that PEPCK and PC are rate-determining enzymes in gluconeogenesis.

Corticosterone decreases toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in hypophysectomized rats.

Male Sprague-Dawley rats were hypophysectomized by an established surgical technique. Hypophysectomy aggravated the toxicity (mortality and mean time to death) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 125 micrograms/kg ip) when compared to sham-operated rats (100% mortality with 9 +/- 1 d mean time to death vs. 90% mortality with 32 +/- 6 d mean time to death, respectively). However, administration of corticosterone (25 micrograms/ml in drinking water) to hypophysectomized rats resulted in an attenuation of the toxicity (40-60% mortality with 40-90 d mean time to death) to a range of TCDD doses (125, 250, 500 micrograms/kg) much higher than the LD50 (about 60 micrograms/kg TCDD) in nonhypophysectomized rats (about 30 d mean time to death). Furthermore, thyroid hormone supplementation in hypophysectomized rats dosed with 125 micrograms/kg TCDD restored the toxicity of TCDD to approximately "normal." Based on these data it is concluded that one or more as yet unknown key factors that are important in the modulation of the toxicity of TCDD reside in the pituitary.

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 gamma-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 gamma-glutamyl transpeptidase (gamma-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 gamma-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 gamma-GT was dose-dependently 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.

Comparative toxicity of four chlorinated dibenzo-p-dioxins (CDDs) and their mixture. Part III: Structure-activity relationship with increased plasma tryptophan levels, but no relationship to hepatic ethoxyresorufin o-deethylase activity.

Male Sprague-Dawley rats were treated with an LD20, an LD50, and an LD80 of 2,3,7,8-tetrachlorodibenzo-p-dioxin (tetra-CDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin (penta-CDD), 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (hexa-CDD), 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (hepta-CDD), respectively, and a mixture of the four homologues where each CDD was represented at one-fourth its previously established LD20, LD50, and LD80, respectively. Plasma tryptophan levels, liver ethoxyresorufin O-deethylase (EROD) activities, and liver weights were determined at 2 and 8 days after treatment. Plasma tryptophan levels were dose-dependently elevated, particularly at 8 days after treatment, by as much as 75% over control levels. EROD activity in CDD-treated animals was induced 27- to 28-fold, as compared with vehicle-treated controls, but did not show any dose-response. Liver weights were also significantly increased by the CDD treatments, but the increase was not dose related. There was no correlation between plasma tryptophan levels, a biomarker of acute toxicity of CDDs, and EROD activity, a biomarker of arylhydrocarbon (Ah) receptor-mediated enzyme induction. It is concluded that the acute toxicity of CDDs, which correlates and shows perfect structure-activity relationship with reduced activities of key enzymes of intermediary metabolism, and the induction of enzymes by much lower doses of CDDs in the liver, have different mechanisms of action.

Decreased hepatic phosphoenolpyruvate carboxykinase gene expression after 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment: implications for the acute toxicity of chlorinated dibenzo-p-dioxins in the rat.

Decreased activity of the rate limiting gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK), has been recently suggested to be the critical lesion in the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We now show that other toxicologically relevant chlorinated dibenzo-p-dioxins (CDDs), with chlorine substituents in the crucial 2-,3-,7-, and 8-positions, exert the same effect on PEPCK activity. The doses required to cause this enzyme inhibition are within the acutely toxic range for each homologue, suggesting the same mechanism of action for these compounds. To further investigate the mechanism whereby dioxins decrease PEPCK activity, Northern analysis was performed using a cDNA probe complementary to a portion of the PEPCK mRNA. We could demonstrate that after TCDD treatment hepatic PEPCK mRNA was decreased by as much as 90% compared to pair-fed control animals (day 8 after dosing). This decrease in PEPCK mRNA was paralleled by a decrease of the amount of PEPCK protein and enzymatic activity. These results indicate that the physiological changes which occur in TCDD-treated animals (decreased feed consumption, low plasma insulin and elevated plasma corticosterone levels) which under normal conditions increase PEPCK gene expression and enzyme activity, are not effective in stimulating PEPCK synthesis in TCDD-treated animals.

Correlation between toxicity and effects on intermediary metabolism in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated male C57BL/6J and DBA/2J mice.

Male mice were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by gavage. C57BL/6J (C57) mice received 0.03 to 235 micrograms/kg, DBA/2J (DBA) mice 1 to 3295 micrograms/kg. On Day 8 after dosing blood was collected, and livers and kidneys were removed. Body weights and feed intake were not much affected until Day 8 after exposure. Hepatomegaly developed at doses above 3 and 97.5 micrograms/kg in C57 and DBA mice, respectively. Ethoxyresorufin O-deethylase activity was induced in liver with an ED50 of 1.1 and 16 micrograms/kg and in kidney with an ED50 of 65 and 380 micrograms/kg in C57 and DBA mice, respectively. The activity of phosphoenolpyruvate carboxykinase (PEPCK) in livers of both mouse strains was reduced over the entire dose range, displaying a plateau in the dose response at the onset of acute toxicity of TCDD. This enzyme activity was decreased by as much as 80% at the respective lethal doses. PEPCK activity in kidney was not affected. Glucose-6-phosphatase activity (G-6-Pase) in liver was altered only in the lethal dose range with a maximum reduction of about 50%. Serum glucose concentration was reduced over the entire dose range, but the reduction was significant only at doses in which G-6-Pase activity was affected, reaching levels as low as 3 mmol/liter in DBA mice. Tryptophan 2,3-dioxygenase activity was not lowered at any dose of TCDD in either mouse strain, and no increase in serum tryptophan levels was observed. Serum levels of thyroxine (T4) and triiodothyronine (T3) were dose dependently decreased over most of the dose range administered, with T3 levels exactly paralleling T4 levels in both mouse strains. It is concluded that TCDD causes acute toxicity in male C57 and DBA mice by a severe reduction of gluconeogenesis, but, in contrast to rats, it does not affect tryptophan homeostasis. Following administration of TCDD serum T3 levels in the mouse appear to correlate with T4 levels, whereas in the rat they are independent of each other.

High sensitivity operation of discrete solid state detectors at 4 K.

Techniques are described to allow operation of discrete solid state detectors at 4 K with optimized junction field effect transistor (JFET) amplifiers. Three detector types cover the 0.6-4-mum spectral range with a noise equivalent power (NEP) of ~10(-16) Hz(-1/2) for two of the types and potential improvement to this performance for the third. Lower NEPs can be anticipated at longer IR wavelengths.