TCDD-mediated oxidative stress in male rat pups following perinatal exposure.

2,3,7,8-tetrachlorododibenzo-p-dioxin (TCDD) is a highly persistent trace environmental contaminant and is one of the most potent toxicants known. Exposure to TCDD has been shown to cause oxidative stress in a variety of animal models. In this study, pregnant Long Evans rats were dosed with 1 microg TCDD/kg on gestational day (GD) 15 so as to investigate oxidative stress in the liver of male pups following gestational exposure to TCDD. Lipid peroxidation (TBARS), production of reactive oxygen species (ROS), and total glutathione (GSH) were assayed to identify changes in oxidative stress parameters in the pup liver at GD 21 and postnatal days (PND) 4, 25, 32, 49, and 63. Mean ROS levels in pups were elevated at all time points tested with a significant elevation at PND 4 and PND 25. However, pup hepatic lipid peroxidation was unchanged throughout the time course. In addition, hepatic total GSH levels were not significantly changed although the means for the TCDD-treated groups were less than those of the controls at all time points except PND 49. The results indicate that although the levels of ROS are increased following gestational/lactational exposure, this increase does not translate to direct oxidative damage or significant changes to endogenous antioxidant defense mechanisms. Further investigation into the effect of gestational/lactational exposure in pups should include additional endpoints for further characterization of the time course of the response, the effect upon extrahepatic tissues, and investigation of differences between male and female offspring.

Tissue disposition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in maternal and developing long-evans rats following subchronic exposure.

Prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces alterations in the reproductive system of the developing pups. The objective of this study was to determine the disposition of TCDD in maternal and fetal Long-Evans (LE) rats following subchronic exposure, since the adverse reproductive and developmental effects have been extensively characterized in this strain of rat. LE rats were dosed by gavage with 1, 10, or 30 ng [(3)H]TCDD/kg in corn oil, 5 days/week for 13 weeks. At the end of 13 weeks, females were mated and dosing continued every day throughout gestation. Dams were sacrificed on gestation day (GD) 9, GD16, GD21, and post-natal day 4 and analyzed for [(3)H]TCDD-derived activity in maternal and fetal tissues. Maternal body burdens were equivalent at different time points, indicating that the dams were at steady state. Maternal body burdens were approximately 19, 120, and 300 ng TCDD/kg following doses of 1, 10, and 30 ng TCDD/kg, respectively. Individual embryo concentrations on GD9 were 1.6, 7, and 16 pg TCDD/g after maternal exposure of 1, 10, and 30 ng/kg/d, respectively. On GD 16, fetal liver, urogenital tract, head, and body concentrations were similar and averaged 1.4, 7.8, and 16.4 pg TCDD/g after administration of 1, 10, or 30 ng TCDD/kg/d, respectively, indicating no preferential sequestration within the different fetal tissues. These concentrations of TCDD within fetal tissues after subchronic exposure are comparable to those seen after a single dose of 50, 200, or 1000 ng TCDD/kg administered on GD15, a critical period of gestation.

Acute administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in pregnant Long Evans rats: association of measured tissue concentrations with developmental effects.

Prenatal exposure to TCDD interferes with fetal development at doses lower than those causing overt toxicity in adult animals. Exposure to TCDD during development produces alterations in the reproductive system of the developing pups- delayed puberty and reduced sperm counts in males and malformations in the external genitalia of females. The objectives of this study were to determine maternal and fetal tissue concentrations of TCDD after acute exposure and whether these tissue concentrations can be used to estimate the intensity of the developmental abnormalities reported by other laboratories. Pregnant Long Evans rats received a single, oral dose of 0.05, 0.20, 0.80, or 1.0 microg [3H]-TCDD/kg on gestation day (GD) 15, and maternal and fetal tissue concentrations of TCDD were measured on GD16 and GD21. On GD16, maternal liver contained the greatest amount of TCDD (30-47% administered dose). One day after administration of 0.20 microg TCDD/kg on GD15, there were 13.2 pg TCDD/g present in an individual fetus. This concentration is associated with delayed puberty and decreased epididymal sperm counts in male pups as well as malformations in the external genitalia of females. For the responses studied, tissue concentration measured during a critical period of gestation adequately predicts the intensity of the response. In addition, there was a strong correlation between fetal body burden and maternal body burden on GD16. A dose of 0.05 microg TCDD/kg resulted in maternal body burdens of 30.6+/-3.1 and 26.6+/-3.1 ng TCDD/kg on GD16 and GD21, respectively. In conclusion, low-level TCDD exposure during the perinatal stage of life can produce adverse effects within the developing pups and that tissue concentration measured during a critical period is the appropriate dose metric to predict adverse reproductive and developmental effects.

2,3,7,8-Tetrachlorodibenzo-p-dioxin in pregnant Long Evans rats: disposition to maternal and embryo/fetal tissues.

Prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) interferes with fetal development at doses lower than those causing overt toxicity in adult animals. In a multigeneration study (Murray et al., 1979), female rats that were administered 0.01 microgram TCDD/kg/day in their diet did not experience reduced fertility; however, reduced fertility was seen in the F1 and F2 generations. Exposure to TCDD during development produces alterations in the reproductive system of the developing pups, such as delayed puberty and reduced sperm counts in males (Mably et al., 1992a; Gray et al., 1995) and malformations in the external genitalia of females (Gray and Ostby, 1995). Therefore, the objectives of this study were to determine maternal and fetal tissue concentrations of TCDD that are associated with the adverse reproductive effects seen by Gray and co-workers. Pregnant Long Evans rats received a single oral dose of 1.15 micrograms [3H]TCDD/kg on Gestation Day (GD) 8 and maternal as well as fetal tissue concentrations of TCDD were measured on GD9, GD16, and GD21. On GD9, the highest level of TCDD localized in the maternal liver (25.1% dose). In addition, the amount reaching all the embryos on GD9 was 0.01% of the administered dose, which resulted in a concentration of 0.02% dose/g. The amount of TCDD reaching the fetal compartment (fetuses + placentas) increased to 0.12% dose/tissue on GD16 and 0.71% by GD21. The concentration of TCDD within the fetal compartment (0.01% dose/g) on GD16 was comparable to that found in the maternal blood and spleen. Concentrations of TCDD in a single embryo/fetus were 39.6, 18.1, and 22.1 pg/g on GD9, GD16, and GD21, respectively. Estimates of hepatic half-life of elimination in pregnant rats suggested that TCDD may be eliminated faster in pregnant LE rats. Therefore, measurements of biliary elimination were made in pregnant and nonpregnant LE rats to compare rates of metabolism; however, biliary elimination of TCDD is not affected by pregnancy. In conclusion, this dose administered during a critical period of organogenesis causes adverse effects on the developing reproductive system of rodents. This dose produced a body burden of 22.1 pg TCDD/g within a single fetus on GD21. This indicates that low-level TCDD exposure during the perinatal stage of life can produce adverse effects within the developing pups.

Pathways of hepatic oxalate synthesis and their regulation.

Important features of hepatic oxalate synthesis remain uncertain despite its clinical significance. To clarify the terminal steps of the biosynthetic pathway and their modulation, we have examined oxalate and glyoxylate synthesis in vitro using isolated guinea pig peroxisomes and purified lactate dehydrogenase (LDH). Glycolate was rapidly oxidized to glyoxylate by isolated peroxisomes followed by a slower conversion of glyoxylate to oxalate. The glycolate oxidase (GO)-catalyzed conversion of glyoxylate to oxalate was strongly inhibited by physiological concentrations of glycolate and lactate. In contrast, the LDH-catalyzed conversion of glyoxylate to oxalate was only marginally affected by physiological concentrations of lactate and unaffected by physiological glycolate concentrations. This inhibition pattern suggests that LDH, not GO, catalyzes this conversion in vivo. Alanine inhibited oxalate synthesis by converting the bulk of the glyoxylate to glycine. On exposure to high alanine concentrations, however, inhibition was not complete and peroxisomes were able to convert sufficient glycolate to oxalate to account for daily endogenous oxalate production. NADH was a potent inhibitor of oxalate production by LDH by increasing glycolate formation from glyoxylate. Glycine was an ineffective source of glyoxylate, and an alkaline pH, a high-glycine concentration, and a prolonged incubation time were required to obtain a detectable synthesis. These results suggest that oxalate synthesis will be modulated by the metabolic state of the liver and resultant changes in NADH, lactate, and alanine levels.

Glucagon increases urinary oxalate excretion in the guinea pig.

Factors that influence hepatic oxalate synthesis are poorly defined. Hormones are important regulators of hepatic metabolism and could potentially be involved. The effects of hyperglucagonemia were examined in guinea pigs injected with either saline or pharmacological doses of glucagon for 4 days. Glucagon treatment increased mean urinary oxalate excretion by 77% in male and 34% in female animals. The levels of hepatic peroxisomal enzymes involved in oxalate synthesis declined with glucagon treatment, but experiments with isolated peroxisomes indicated that oxalate synthesis in vitro was unaffected. Glucagon decreased hepatic alanine levels by 66%, lactate by 69%, and pyruvate by 73%, but glycolate and glyoxylate levels were unaffected. This decrease in alanine would substantially lower the activity of alanine-to-glyoxylate aminotransferase activity in vivo and make more glyoxylate available for oxalate synthesis. The decrease in lactate and pyruvate concentrations would stimulate the enzymatic conversion of glyoxylate to oxalate and may account for the increase in oxalate synthesis without an increase in glyoxylate concentration. These results demonstrate that hepatic oxalate synthesis is influenced by metabolic changes and that alterations in hepatic alanine, lactate, and pyruvate concentrations may be important elements.