Carol M. Schiller

Dose-related effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in C57BL6J and DBA2J mice☆

The dose-related effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied in B6D2F1/J (B6D), C57BL/6J (C57), and DBA/2J (DBA) mice. A 14-fold difference in lethality was observed in C57 and DBA mice, based upon 30-day LD50 values of 182 and 2570 micrograms TCDD/kg body wt, respectively. The 30-day LD50 for B6D mice was 296 micrograms TCDD/kg body wt. A progressive loss of body weight in all strains of mice was observed during the 30-day LD50 studies, with maximal weight losses of 24.7, 34.0, and 33.4% prior to death of C57, B6D, and DBA mice, respectively. In separate experiments, it was found that decreased feed consumption did not contribute to weight loss in C57 mice exposed to lethal or sublethal doses of TCDD until the animals were moribund. Time-course studies in C57 mice treated with 200 micrograms TCDD/kg body wt indicated that decreases in serum glucose and triglyceride concentrations and increases in hepatic triglyceride content occurred within 4 to 8 days of exposure, and were maximally altered within 17 to 21 days postexposure, concomitant with a 25% body weight loss. C57 mice fasted for 24 to 96 hr lost 18% of body weight and also exhibited alterations in glucose and lipid parameters; however, these changes were substantially different than the effects of TCDD exposure. In concert, these observations demonstrate that decreased feed consumption (hypophagia) does not account for weight loss and changes in carbohydrate and lipid metabolism in TCDD-treated C57 mice. Dose-response experiments resulted in comparable changes in glucose and lipid parameters when DBA mice were exposed to 10-fold higher doses of TCDD than C57 mice. Parallel LD50 responses and parallel changes in carbohydrate and lipid metabolism, at 10- to 15-fold differences in dose range, are indicative of a common mechanism of toxicity in TCDD-treated C57 and DBA mice.

Comparative toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in four (sub)strains of adult male rats.

Four (sub)strains of adult male rats were given single oral doses of various concentrations of TCDD to establish and compare the oral 30-day LD50 values. The strains of rats were Fischer (F/334N) supplied by Harlan Industries, Frederick Cancer Research Center, and Charles River Breeding Laboratories; and CD supplied by Charles River Breeding Laboratories. The Charles River/Fischer rats were most sensitive to TCDD (LD50 = 164, 95% confidence limits 104-217 micrograms/kg), the Frederick/Fischer and Charles River/CD rats were moderately sensitive to TCDD (LD50 = 303, 250-360; and 297, 240-360 micrograms/kg, respectively), and the Harlan/Fischer rats were most resistant to TCDD (LD50 = 340, 281-409 micrograms/kg). The mean times of death were from 24.5 +/- 1.0 to 28.3 +/- 0.5 days and the percentage body weight loss at death was 37.4 +/- 1.2 to 42.7 +/- 1.3%. One week after exposure of the Charles River/Fischer animal to 45 micrograms TCDD/kg (1/4 the established 30-day LD50 dose), the same serum profile was induced as previously observed in the Harlan/Fischer rat, which includes hypoglycemia, hypertriglyceridemia, and hypercholesterolemia. These results emphasize the importance of indicating the precise dose, strain of rat, and time after dosing before termination in reporting the effects of TCDD on a particular biological response.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and fasting on body weight and lipid parameters in rats

This study compared the effect of fasting (feed deprivation) and the effect of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on body weight and on key lipid parameters. The time-course study indicated a statistically significant (p less than 0.05) body weight loss in the TCDD-exposed rats 5 days after the oral administration of corn oil with TCDD (60 micrograms/kg body wt). Animals demonstrated a 10% body weight decrease either 1 week after this TCDD administration or after 72 hr of fasting. Marked increases in serum triglyceride and cholesterol were observed only in the TCDD-exposed rats, but not in the 0- or 72-hr fasted control rats. These results indicated that some body weight loss and decreased feed consumption occurred after TCDD exposure, but that the metabolic response, with respect to serum lipid metabolism, was not that of a control rat that had lost a similar amount of body weight due solely to fasting.

Effect of phthlate esters on energy coupling and succinate oxidation in rat liver mitochondria

Abstract Isolated rat liver mitochondria were exposed to mono- and di-n-butyl phthalate (MBP and DBP) and mono- and di(2-ethylhexyl)phthalate (MEHP and DEHP) and examined for effects on mitochondrial energy-dependent processes, including oxidative phosphorylation and active K+ uptake. Additional studies on the effects of these phthalate esters on succinate oxidation and on mitochondrial membrane integrity are also included. DBP and MEHP stimulated succinate state 4 respiration, impaired K+-valinomycin induced swelling with succinate, ascorbate, or ATP as the energy sources, and inhibited succinate state 3 respiration and succinate cytochrome c reductase activity. MEHP was found to act as a non-competitive inhibitor of succinate dehydrogenase activity, with an apparent Ki = 2.4 × 10−4 M. At concentrations which uncouple energy linked reactions, MEHP and DBP produced only slight energy-independent swelling and release of soluble proteins from isolated mitochondria. MBP caused only slight stimulation of state 4 respiration and impairment of K+-valinomycin induced swelling with each of the 3 energy sources, however, of the 4 phthalate esters, it produced the greatest energy-independent swelling and led to the greatest release of soluble mitochondrial proteins. DEHP had no apparent effect on any of these processes except for slight impairment of ATP-dependent K+-valinomycin induced swelling. It is concluded that phthalate ester toxicity in liver mitochondria is due to uncoupling of energy linked reactions and/or inhibition of succinate dehydrogenase activity. Uncoupling by MBP may involve disruption of mitochondrial membrane integrity, while uncoupling by DBP and MEHP is probably due to an increase in membrane permeability to H+ and other small ions.

The differential response of isolated intestinal crypt and tip cells to the inductive actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The cell specific induction of uridine diphosphate(UDP)-glucuronyltransferase by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in intestinal epithelium was studied by administering [14C] TCDD (8 microgram/kg to adult female rats). Intact epithelial cells from the tip and crypt regions were isolated by differential vibration of rat duodenum. Cell separation was monitored by electron microscopy and marker enzymes. UDP-glucuronyltransferase and radioactivity were assayed in both cell types 0 h, 3 h, 10 h, 1 day, 3 days and 5 days after treatment. UDP-glucuronyltransferase activities were not significantly changed in either cell type isolated from TCDD-treated rats until 24 hr after treatment when a three-fold increase in crypt cell activity was evident. No significant changes in UDP-glucuronyltransferase activity were observed in the differentiated tip cells until 3 days after TCDD treatment. UDP-glucuronyltransferase was increased approximately two-fold in both cell types from 3 and 5 days following TCDD treatment. There was a negative correlation between the time-course of UDP-glucuronyltransferase induction and the [14C]TCDD concentrations measured in these cells. These studies suggest that the undifferentiated cells of the intestinal crypt region are more sensitive to TCDD inductive actions than are the absorptive tip cells.

Induction of colon tumors by a single oral dose of 1,2-dimethylhydrazine

Male Fischer rats were treated at 7 weeks of age with a single oral dose of 1,2-dimethylhydrazine (35 mg/kg). After 1.5 years, the 14 control and 28 treated animals were killed for general autopsy. The incidence of tumor formation in the treated animals was 78.6% as compared to 0% for the control animals. All tumors (1--3/rat) were located in the colon, with the exception of one in the Zymbals gland of the ear and one in the small intestine. The dosage of 1,2-dimethylhydrazine used in this study is the lowest single oral dose of this carcinogen reported to induce colon tumors.

Effects of hydrazine and its derivatives on the development of intestinal brush border enzymes.

Abstract Pregnant hamsters were exposed by intubation to a single oral dose of hydrazine hydrate (260 mg/kg) or 1,2-dimethylhydrazine dihydrochloride (150 mg/kg) as a neutralized solution on Day 12 of gestation (4 days before birth). Similarly, doses of methylazoxymethanol acetate (340 and 85 mg/kg) were given and found to be lethal to the dams within a 24- to 48-hr period. Groups of these animals ( N = 3) were sacrificed 1 or 2 days before birth and 3 or 4, 10, 17, 24 or 25, and 53 or 60 days after birth to evaluate the development of intestinal brush border enzymes. lactase, sucrase, and alkaline phosphatase, as compared to the enzyme development in control animals. The results indicate that exposure to hydrazine diminished neonatal lactase activity, elevated postnatal and young adult sucrase activity, and elevated the neonatal and postnatal activity of alkaline phosphatase. In contrast, after prenatal exposure to 1,2-dimethylhydrazine, the postnatal level of sucrase activity and all levels of alkaline phosphatase activity were elevated. There was no significant effect of 1,2-dimethylhydrazine on the development of lactase activity. A screen for teratogenic effects revealed no incidence of cleft palate formation in the offspring. While there are no apparent teratogenic events, biochemical alterations reveal more subtle effects of these environmental toxins on this developing organ system.

Pyruvate metabolism after in vivo exposure to oral arsenic

This study investigated altered pyruvate metabolism after prolonged oral arsenic exposure. Male rats were given access to deionized drinking water containing 0, 40 or 85 ppm sodium arsenate (As5+) for 3 weeks. Respiration studies with mitochondria isolated from treated animals indicated decreased state 3 respiration (with ADP) and decreased respiratory control ratios (RCR) for pyruvate/malate-mediated respiration, but not for succinate-mediated respiration, as compared to control respiration values. In addition, pyruvate dehydrogenase activity was measured, in both liver and intestine, before and after Mg-activation in vitro. After 3 weeks, the effects of arsenic at the highest dose level were pronounced on the basal pyruvate dehydrogenase activity (before activation) as well as the total pyruvate dehydrogenase (after activation). The inhibition of pyruvate dehydrogenase activity both before and after Mg-activation suggests an arsenic effect on mitochondrial pyruvate metabolism which, in part, involves inhibition of pyruvate decarboxylase. Evidence is also presented which may indicate an arsenic effect on the kinase and/or phosphatase which regulate pyruvate dehydrogenase activity.

Development of substrate cycle enzymes in the hamster small intestine.

Abstract A system of enzymes is required for the transport of reducing equivalents from reduced nicotinamide adenine dinucleotide (NADH) generated in the cytosol into the mitochondria by the substrate cycles. These substrate cycle enzymes are necessary for the flow of pyruvate derived from glucose into the mitochondria for oxidative decarboxylation and for the efficient production of adenosine 5′-triphosphate (ATP) for the unique intestinal nutrient transport functions. The enzymes of the l -glycerol 3-phosphate and the l -malate/ l -aspartate substrate cycles are present before birth and increase significantly at the 7-day postnatal period of development. The key enzymes monitored in the intestinal subcellular fractions were NAD-linked l -glycerol-3-phosphate dehydrogenase, flavoprotein-linked l -glycerol-3-phosphate dehydrogenase, l -malate dehydrogenase, and l -glutamate-oxaloacetate transaminase.

Reducing equivalent transport by substrate shuttles in rat liver and colon

Abstract 1. 1. The observed level and subcellular distribution of the α-glycerophosphate and malate-aspartate substrate shuttle enzymes in liver and colon were consistent with their proposed roles in reducing equivalent transport. 2. 2. Km value determinations of shuttle enzymes were performed. 3. 3. Substrate shuttles were reconstructed from isolated liver and colon mitochondria which displayed satisfactory respiratory control and P:O ratios. 4. 4. The results obtained suggest that while the malate-aspartate shuttle is the primary means of reducing equivalent transport in the liver, the α-glycerophosphate shuttle predominates in the colon.