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.

Reduced gluconeogenesis in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats.

The effect of a usually lethal dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 125 micrograms/kg) was studied on the conversion of 14C-alanine into 14C-glucose in male Sprague-Dawley rats by established procedures (determination of plasma alanine and blood glucose by enzymatic assays and isolation of 14C-alanine and 14C-glucose from whole blood by column chromatography). TCDD-treated rats converted significantly (p less than 0.05) less 14C-alanine into 14C-glucose than did their pair-fed or ad libitum-fed counterparts, indicating reduced gluconeogenesis as a result of TCDD treatment. This finding suggests that reduced gluconeogenesis in TCDD-treated rats contributed to the progressively developing, severe hypoglycemia observed in these animals. Corticosterone, a key hormone in gluconeogenesis, provides partial protection from TCDD-induced toxicity in hypophysectomized rats. Therefore, the conversion of 14C-alanine into 14C-glucose was also determined in hypophysectomized rats dosed with TCDD (125 micrograms/kg) and given corticosterone (25 micrograms/ml in drinking water). These rats also converted significantly (p less than 0.05) less 14C-alanine into 14C-glucose than did their pair-fed counterparts. However, in contrast to non-hypophysectomized TCDD-treated rats, these rats maintained marginal normoglycemia even at 64 days after dosing with TCDD, which suggests that the partial protective effect of corticosterone in hypophysectomized, TCDD-treated rats is unrelated to its effect on gluconeogenesis. The protection provided by corticosterone supplementation in TCDD toxicity is more likely due to reduced peripheral utilization of glucose enabling the animals to maintain marginal normoglycemia.

Mode of metabolism is altered in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats.

Male Sprague-Dawley rats were fed either a high-fat (HF) or a high-carbohydrate (HC) diet and subsequently injected with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (125 micrograms/kg) or vehicle (pair-fed controls). In all TCDD-treated animals, a reduction in caloric intake was evident as early as 1 day after dosage. Respiratory quotients (RQ) were determined at 5-day intervals. Their pattern for the HC-fed but not for the HF-fed TCDD-treated rats was different from that of the corresponding pair-fed controls. After an initial parallel decrease the RQ values remained low for TCDD-treated rats whereas they increased again for pair-fed controls. Serum total thyroxine (T4) was significantly lower in TCDD-treated animals and this reduction was not influenced by the composition of the diet. Serum triiodothyronine (T3) was neither altered by diet nor by TCDD. Thymic atrophy was as severe in pair-fed as in TCDD-treated rats fed the HC diet but not in rats fed the HF diet. Our results suggest that TCDD-treated rats are in a different mode of metabolism from pair-fed rats and that this difference is related to gluconeogenesis.

Elevated plasma corticosterone levels and histopathology of the adrenals and thymuses in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats.

The relationship between thymic atrophy and plasma corticosterone levels was examined in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated, pair-fed and ad libitum-fed male Sprague-Dawley rats given a usually lethal (125 micrograms/kg) or non-lethal (25 micrograms/kg) dose of TCDD. At both dosages, corticosterone levels in TCDD-treated animals begun to rise as early as day 4 after treatment. At later time points corticosterone levels were 5-7 times higher in rats given the non-lethal dose, and 6-10 times higher in rats administered the lethal dose than the levels observed in ad libitum-fed controls. Corticosterone levels in control rats pair-fed to the lethal dose group (as a result of the severe reduction in feed intake) were similarly elevated as in TCDD-treated rats but this was not the case in pair-fed rats of the non-lethal TCDD dosage (due to an essentially unchanged feed intake). At both dosages, relative thymus weights of TCDD-treated rats started decreasing by day 4 and continued to decline for the most part of the study. Relative thymus weights of rats pair-fed to the non-lethal TCDD dosage were not different from ad libitum-fed rats. However, the decrease in relative thymus weights of rats pair-fed to the lethal TCDD dosage paralleled that of TCDD-treated rats with an apparent 8-day lag period. Morphologically, the thymus as well as the adrenal revealed differential changes in TCDD-treated rats from those observable in pair-fed rats. These results suggest that either TCDD exerts a direct effect on the thymus and the adrenals or it causes an additional stress (e.g., a metabolic stress) over and above the starvation stress, which may be responsible for the differential morphological changes in these glands.

Corticosterone modulates acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in male Sprague-Dawley rats.

Bilateral adrenalectomy or adrenal demedullation was performed on male Sprague-Dawley rats by established surgical techniques. Subsequently, the dose-response (mortality and mean time to death) to TCDD was determined in adrenalectomized (10, 20, 40 micrograms/kg TCDD ip in 95:5 corn oil:acetone) or demedullated (15, 30, 60 micrograms/kg TCDD) rats. Adrenalectomy drastically increased mortality and greatly shortened mean time to death after dosing with TCDD. More importantly, adrenalectomized TCDD-treated rats died of hypoglycemic shock without losing much body weight. Conversely, adrenal demedullation had no effect on mortality or mean time to death caused by TCDD when compared to nondemedullated TCDD-treated controls. Thus, it was concluded that the factor(s) modulating the acute toxicity of TCDD resides in the adrenal cortex and not in the medulla. Administration of corticosterone (25 micrograms/ml in drinking water) to adrenalectomized rats returned the toxicity of TCDD to levels seen in nonadrenalectomized rats suggesting that this hormone is another key factor (in addition to the thyroid hormones) in the modulation of the acute toxicity of TCDD. Corticosterone supplementation (25, 50, or 100 micrograms/ml) to nonadrenalectomized rats, or to thyroidectomized-adrenalectomized rats (25 micrograms/ml), resulted in no additional beneficial effect indicating that a factor(s) other than thyroid hormones and corticosterone is also involved in the acute toxicity of TCDD.

Some endocrine and morphological aspects of the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Hormonal status was evaluated in TCDD-treated rats and in pair-fed and ad libitum-fed controls in order to separate hormonal changes resulting from the toxic insult of TCDD from those arising from progressive feed deprivation as it occurs in pair-fed controls. TCDD-treated rats received either a usually non-lethal (25 micrograms/kg) or a usually lethal (125 micrograms/kg) dose of TCDD whereas pair-fed and ad libitum-fed controls were given vehicle alone. Animals were terminated at predetermined time intervals and several hormones measured in serum or plasma. In addition, the morphology of the thyroid, pancreas, and pituitary was also examined. In both dosage groups, TCDD-treatment had the following effects: decreased TT4, FT4, insulin, and glucagon; mixed effects upon TT3, FT3, TSH, and GH. Pair-feeding to the non-lethal dose of TCDD had no effect on any of the hormones measured. Pair-feeding to the lethal dose of TCDD had the the following effects: slightly decreased TT4, FT4, TT3, TSH, and insulin; no effect on FT3 and glucagon. It is concluded that the endocrine status of TCDD-treated rats was different from that of pair-fed rats suggesting that some hormonal changes represent responses to an insult other than that due to starvation stress alone. A differential response between TCDD-treated and pair-fed rats was also observable morphologically in the corresponding endocrine glands indicating the importance of this additional control for morphologic observations in instances when reduced feed intake and body weight loss are prominent features of toxicity.

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.

Tissue-specific alterations of de novo fatty acid synthesis in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats.

De novo fatty acid synthesis was determined by the 3H2O method in numerous tissues and organs of TCDD-treated (125 micrograms/kg), pair-fed and free-fed male Sprague-Dawley rats to investigate if this important pathway of intermediary metabolism is altered by TCDD. Of the 12 tissues and organs examined, liver showed an increased, and interscapular brown adipose tissue (IBAT) a decreased de novo fatty acid synthesis when comparing TCDD-treated to pair-fed or free-fed control rats. De novo fatty acid synthesis was unaffected in other organs and tissues examined, with the exception that the concentration of 3H-fatty acids in plasma reflected the increased rate of synthesis seen in the liver of TCDD-treated animals. Increased de novo fatty acid synthesis in liver coincided with increased plasma triiodothyronine (T3) concentrations, whereas decreased de novo fatty acid synthesis in IBAT parallelled decreased plasma thyroxine (T4) levels. Thyroidectomy decreased de novo fatty acid synthesis, as expected, in both liver and IBAT. However, TCDD elicited no response in either of these organs in thyroidectomized rats. This finding suggests that changes observed in non-thyroidectomized rats are probably secondary effects. Indeed, known tissue-specific effects of T3 on liver and T4 on IBAT provide a likely explanation for the altered de novo fatty acid synthesis of these organs. It is suggested that increased de novo fatty acid synthesis in the liver of TCDD-treated rats might be responsible for the additional wasting away observable in these animals as compared to pair-fed controls.

Effects of vitamin A and/or thyroidectomy on liver microsomal enzymes and their induction in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats.

Vitamin A and thyroid hormone status have been shown previously to alter the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats. In the present study, we have examined the effects of a vitamin A-excess and a vitamin A-deficient diet on thyroid hormone levels, on selected drug-metabolizing enzymes in liver microsomes, and on their inducibility by TCDD in male Sprague-Dawley rats. Except for a slight increase in serum T3 levels, none of these end points was affected by feeding rats the vitamin A-deficient diet. In contrast, excess dietary vitamin A caused a decrease in serum thyroxine (T4) and triiodothyronine (T3) levels, although the levels of T3 remained in the euthyroid range (60-80 ng/dl). The concentration of liver microsomal cytochromes P-450 and b5 and the basal activity of benzo[a]pyrene hydroxylase and 7-ethoxyresorufin O-de-ethylase were unaffected by excess dietary vitamin A. This result is consistent with our previous observation that the basal activity of these enzymes is dependent more on T3 than on T4 levels. Vitamin A excess markedly suppressed the activity of liver microsomal UDP-glucuronosyl transferase toward 1-naphthol. However, no such enzyme suppression was observed in thyroidectomized rats. This suggests that the suppressive effect of vitamin A on UDP-glucuronosyl transferase activity may be dependent on T3. Neither vitamin A nor thyroid status had any major effect on the inducibility of UDP-glucuronosyl transferase and cytochrome P-450-dependent enzyme activities by TCDD. However, vitamin A and TCDD had a nearly additive effect on suppression of serum T4. It is concluded that liver microsomal enzyme induction is not associated with the modulatory effect of vitamin A and thyroid hormones on the toxicity of TCDD.

Dose-response and time course of hypothyroxinemia and hypoinsulinemia and characterization of insulin hypersensitivity in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats.

Male Sprague-Dawley rats were administered i.p. various doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in corn oil. At several time points thyroid stimulating hormone (TSH), total thyroxine (TT4), free thyroxine (FT4), total triiodothyronine (TT3), reverse triiodothyronine (rT3) and insulin were determined in serum using radioimmunoassays, and glucose was measured by the glucose oxidase method. TSH and TT3 were not affected by any dose at any time point of measurement. TT4 and FT4 were decreased in a somewhat dose-dependent manner by days 2 to 4 after dosing. Return of TT4 and FT4 to normal values by day 32 after TCDD dosage also occurred in a dose-dependent manner, except in rats that died later. rT3 was also decreased at each dose level early and returned to normal levels in a somewhat dose-dependent fashion. Rats in the 2 highest dose groups became hypoinsulinemic and in the highest dose group also hypoglycemic by day 8 after dosing. Serum insulin and glucose remained suppressed in non-survivors of TCDD until death ensued. In survivors, serum insulin returned to normal values by day 32 after dosing. The hypoinsulinemic state was further characterized by hypersensitivity towards insulin, i.e. injection of an otherwise non-toxic dose of insulin 3 days after administration of 125 micrograms/kg TCDD was lethal to 80% of the rats within 24 h. Insulin hypersensitivity preceded both hypoglycemia and hypoinsulinemia. These findings suggest that hypothyroxinemia and hypoinsulinemia may be part of an adaptive process whereby rats attempt to diminish the toxic insult of TCDD.

Composition of diet modifies toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in cold-adapted rats.

The effect of a high carbohydrate, high fat or high protein diet was studied on the acute toxicity of TCDD (125 micrograms/kg) in cold-adapted (4 +/- 1 degrees C) rats. Within 10 days after dosing, TCDD-treated rats fed a high carbohydrate or a high protein diet reduced their caloric intake by 25% whereas those fed a high fat diet consumed only 15% fewer kcal/MBS (metabolic body size). TCDD-treated rats fed a high protein diet lost body weight at the same rate as their pair-fed controls, whereas body weight loss in high fat-fed rats was significantly higher than in their pair-fed controls. In contrast, TCDD-treated rats fed a high carbohydrate diet effectively maintained their body weight in the 4 days immediately after TCDD dosage, whereas their pair-fed controls lost weight. Mortality in TCDD-treated animals was 100% irrespective of the diet; all pair-fed control rats (except one fed a high protein diet) were terminated on days corresponding to the spontaneous death of their TCDD-treated pairs. Mean time to 50% mortality and mean time to death were significantly longer in TCDD-treated rats fed a high carbohydrate diet in comparison with the other two TCDD-treated groups (p less than 0.05), although caloric intake was comparable. Serum triiodothyronine (T3) was reduced in TCDD-treated animals fed a high fat or a high carbohydrate diet but not in those fed a high protein diet; serum thyroxine (T4) was reduced in all the treated groups, irrespective of diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced serum thyroid hormone levels in hexachlorobenzene-induced porphyria.

The effect of feeding 0.1% hexachlorobenzene (HCB) for 55 days on mortality, body weight, urinary porphyrin excretion, serum thyroid hormones and induction of liver microsomal enzymes was studied in female Sprague-Dawley rats. This dosage regimen, followed by 42 days of a regular diet, resulted in 33% mortality with a mean time to death of 67 +/- 4 days. Body weight of survivors was not affected by dietary HCB, whereas non-survivors underwent a rapid weight loss (wasting) prior to death. At the end of the dosing period (day 55), rats fed the HCB diet exhibited an increase in the excretion of urinary porphyrins (4-fold) and a significant decrease in the levels of serum thyroxine (T4) and triiodothyronine (T3). When rats were returned to a regular diet the excretion of urinary porphyrins continued to rise (approx. 100 times higher than controls) and serum thyroid hormones remained suppressed. At the end of the experiment (day 97), the concentration of liver microsomal cytochrome P-450 and cytochrome bs and the activity of ethoxyresorufin-O-deethylase, pentoxyresorufin-O-dealkylase, aminopyrine-N-demethylase and UDP-glucuronosyl transferase were significantly induced, whereas the activity of NADPH-cytochrome c reductase and benzo[a]pyrene hydroxylase was not. Results demonstrate that HCB-induced lethality and porphyria occur by different mechanisms, reduced T4 and T3 serum levels accompany induction of porphyria by HCB, and induction of aryl hydrocarbon hydroxylase (with benzo[a]pyrene as substrate) is not a sensitive indicator of HCB exposure.

Organic hydroperoxide-induced lipid peroxidation and cell death in isolated hepatocytes.

Organic hydroperoxides such as tert-butyl hydroperoxide (TBHP) are cytotoxic to suspensions of isolated hepatocytes. The exact mechanism of toxicity is unknown but may involve peroxidation of cellular lipids, alkylation of cellular macromolecules, or alterations in cellular calcium homeostasis. These studies were designed to examine lipid peroxidation as a mechanism of organic hydroperoxide-induced cell death. Hepatocytes isolated from mice were more susceptible to the cytotoxic effects of TBHP than were rat hepatocytes. TBHP-induced cell death was preceded by malondialdehyde formation which was also greater in mouse than rat hepatocytes. Species differences in lipid peroxidation were due to intrinsic properties of hepatocyte membranes as lipids isolated from mouse liver and peroxidized with iron/ascorbate formed approximately eightfold more malondialdehyde than lipids isolated from rat liver. Initiation of lipid peroxidation in mouse and rat hepatocytes with iron/ascorbate caused the formation of malondialdehyde equal to that seen with TBHP and a slight depletion of cellular GSH. As with TBHP, malondialdehyde formation induced by iron/ascorbate was greater in mouse than in rat hepatocytes. However, iron/ascorbate had no effect on hepatocyte viability or morphology from either species. Furthermore, TBHP-induced malondialdehyde and ethane formation in isolated rat hepatocytes were completely blocked by promethazine whereas cell toxicity was altered only slightly. Therefore, these data do not support a role for lipid peroxidation in the acute cytotoxicity of TBHP to suspensions of isolated rat hepatocytes.

Age- and sex-dependent induction of liver microsomal benzo[a]pyrene hydroxylase activity in rats treated with pregnenolone-16 alpha-carbonitrile (PCN).

The present studies were undertaken to resolve conflicting reports on the inducibility of liver microsomal benzo[a]pyrene hydroxylase activity in rats treated with pregnenolone-16 alpha-carbonitrile (PCN). Several studies have shown that treatment of Long Evans rats with PCN causes a 5- to 10-fold induction of benzo[a]pyrene hydroxylase activity, whereas little or no induction occurs in Sprague-Dawley or Wistar rats. Studies with one-month-old-male Long Evans, Sprague-Dawley, Wistar and Holtzman rats failed to reveal an anticipated strain difference in the inducibility of benzo[a]pyrene hydroxylase activity by PCN. Studies with immature and mature male and female Long Evans rats revealed that the inducibility of benzo[a]pyrene hydroxylase activity decreases with age in male but not female rats, i.e., PCN induced benzo[a]Pyrene hydroxylase activity 5- to 8-fold in immature male, immature female and mature female rats but only 2-fold in mature male rats. The age-dependent decrease in inducibility by PCN in male rats coincided with an age-dependent increase (2.4-fold) in the basal activity of benzo[a]pyrene hydroxylase. These sex-dependent developmental changes can be explained by an age-dependent increase in the constitutive levels of the major PCN-inducible form of cytochrome P-450 (cytochrome P-450-PCN) in male but not female rats. Electrophoresis of liver microsomes and studies on the binding of metyrapone to dithionite-reduced cytochrome P-450 provided additional evidence for age- and sex-dependent differences in the levels of microsomal cytochrome P-450-PCN. In addition to reconciling the conflicting literature reports, the age- and sex-dependent differences in cytochrome P-450-PCN levels account, at least in part, for age and sex differences in certain liver microsomal enzyme activities, including benzo[a]pyrene hydroxylase activity.