Martha W. Harris

The comparative toxicity of chlorinated dibenzo-p-dioxins in mice and guinea pigs

Abstract Mice and guinea pigs were given a single oral dose of various chlorinated dibenzo-p-dioxins (CDD) to establish and compare the LD50 and clinical and pathologic manifestations of toxicity. It was apparent that the 2,3,7, and 8 positions must be chlorinated to achieve the greatest degree of toxicity. Additional chlorine atoms at an ortho position reduced toxicity but not nearly to the degree caused by deletion of a chlorine atom at one of the lateral positions. A decrease in body weight gain was the most sensitive clinical parameter and animals severely intoxicated showed a marked weight loss, especially guinea pigs. The median time to death at a LD50 was 17 to 20 days in guinea pigs and 22 to 25 days in mice. Doses 10 times greater did not markedly shorten this period. At the toxic dose the spectrum and severity of lesions and organ weight effects were similar for all homologs and isomers within the same animal species; however, there were interspecies differences. The thymus was greatly reduced in size in both animal species due to a reduction in the number of cortical lymphocytes. Significant macroscopic and histopathologic hepatic effects including porphyria were observed only in the mouse and were found at does levels well below the LD50. Hyperplasia of the transitional epithelium in the urinary tract was found in guinea pigs. There was a reduction of total serum protein in the mouse due to lower levels of α-globulin. Other lesions were generally interpreted to be a secondary response to debilitation.

Testicular toxicity and reduced Sertoli cell numbers in neonatal rats by di(2-ethylhexyl)phthalate and the recovery of fertility as adults.

Neonatal and adult rats (1, 2, 3, 6, and 12 weeks of age) were given five daily oral doses of di(2-ethylhexyl) phthalate (DEHP) (0, 10, 100, 1000, 2000 mg/kg) and histological changes in the testes were examined 24 hr after the last dose. Relative testis weights were reduced at doses of 1000 mg/kg in 1, 2, 3, and 6-week-old but not in 12-week-old rats, while doses of 2000 mg/kg were fatal to suckling rats and caused decreased relative testis weight but not death in 6- and 12-week-old rats. In neonatal rats (1 week old), DEHP (1000 mg/kg) caused a 35% decrease in Sertoli cell numbers while 2- and 3-week-old rats showed losses of spermatocytes but not of Sertoli cells. The 6- and 12-week-old rats showed loss of both spermatids and spermatocytes at 1000 and/or 2000 mg/kg. Total testicular zinc concentrations were decreased in 12-week-old but not in suckling (3-week) or weaned (6-week) rats. The results support the hypothesis that the Sertoli cell is the primary testicular target of phthalate ester toxicity since effects were observed at an age when only Sertoli cells were present. Fertility was assessed in mating trials in adult male rats after neonatal exposure to DEHP on Days 6-10. Although Sertoli cell number was reduced 24 hr after the last dose, the numbers were normal at 6 and 13 weeks of age. However, at 6 weeks there was a dose-related decrease in maturation of the spermatids in the tubules. There were no consistent changes in fertility, implantation rate, or numbers of live fetuses in untreated females mated with the DEHP-treated males. However, there were decreases in testis weight and testicular spermatid numbers at 13 and 19 weeks but not at 11, 12, 16, or 23 weeks of age. Therefore, a loss of Sertoli cells due to DEHP exposure neonatally did not affect the fertility of the rats as adults, but may have caused subtle effects on sperm production.

COMPARATIVE TOXICITY OF THREE HALOGENATED DIBENZOFURANS IN GUINEA PIGS, MICE, AND RHESUS MONKEYS

The chlorinated dibenzofurans have been reported to be present in a variety of polychlorinated biphenyls (PCBs)’” and in pentachlorophenol: They were also detected in the PCB-contaminated rice oil that was responsible for the human intoxication referred to as ‘Y~sho’.’.~ Polychlorinated dibenzofurans were also detected in tissues of patients with ‘Yusho’.’ A polychlorinated dibenzofuran fraction extracted from PCB’s was found to be highly toxic in chickens.* A mixture of polychlorinated dibenzofurans also caused toxic effects when fed to rats? 2,3,7,8Tetrachlorodibenzofuran (TCDF) has been evaluated for toxicity in chicks,” guinea pigs,” and mice.” The results reported in this manuscript extend earlier studies with TCDF in guinea pigs and mice, report the toxic effects in rhesus monkeys, and describe the toxic effects of 2,3,4,7,8-pentachlorodibenzofuran (PCDF) and 2,3,7&tetrabromodibenzofuran (TBDF) in guinea pigs.

Examination of bone marrow, immunologic parameters and host susceptibility following pre- and postnatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The effects of pre/postnatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on various immunological, bone marrow and host susceptibility assays were examined in B6C3F1 hybrid mice. Exposure was accomplished by maternal dosing on Day 14 of gestation and again on Days 1, 7, and 14 following birth, employing dosages of 0, 1.0, 5.0 or 15.0 micrograms/kg body weight. The 15.0 micrograms/kg dosage was lethal to 70% of the offspring with the remainder of that dosage group revealing overt toxicity. Bone marrow toxicity occurred in both the 15.0 and 5.0 micrograms/kg dosage groups as evidenced by bone marrow hypocellularity and depressed colony formation of macrophage-granulocyte progenitor cells and pleuripotent stem cells. Evidence was presented that depression of lymphoproliferative responses following mitogen stimulation in TCDD-immunosuppressed mice was due to a functional defect of lymphocyte activation rather than suppressor cell activity. Administration of either Listeria monocytogenes or syngeneic PYB6 tumor cells in mice exposed to relatively low levels of TCDD during pre- and postnatal development increased their susceptibility to either bacterial or tumor challenge.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on lipid profiles in tissue of the Fischer rat.

Female Fischer 344 rats were given single oral doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 10, 50 or 100 microgram/kg, and sacrificed 1, 3, 10, 14 or 21 days later. The fatty livers caused by a sub-lethal dose of TCDD involved a temporary increase in triglyceride and free fatty acid levels, with a persistent decrease in levels of sterol esters. In contrast, the fatty livers resulting from a lethal dose of TCDD involved a large increase in cholesterol esters and free fatty acids, with little change in triglyceride levels. These changes appeared to result in part from damage sustained by lysosomes. TCDD also altered the lipoprotein composition of the serum, the fatty acid composition of various lipid classes in liver and serum, and the ultrastructure of the liver (formation of myeloid bodies). A rapid, dose-dependent effect of TCDD, was the elevation of levels of organic-soluble fluorescent pigment in the heart. This pigment was found to match a previously characterized fraction of lipofuscins in fluorescence spectrum and chromatographic properties. The relatioship of these observations to a possible mechanism of toxicity for TCDD involving radical-induced lipid peroxidation is discussed.

Retinoic acid and 2,3,7,8-tetrachlorodibenzo-p-dioxin selectively enhance teratogenesis in C57BL/6N mice☆

TCDD is one of the most toxic man-made compounds and an extremely potent teratogen in mice. Many of its toxic symptoms resemble those seen during vitamin A deficiency. Vitamin A and its derivatives, such as alltrans-retinoic acid (RA), are also teratogenic in mice, as well as many other species. Both TCDD and RA produce cleft palate in susceptible strains of mice. However, while TCDD produces hydronephrosis, RA does not, and TCDD does not produce limb bud defects while RA does. To determine whether TCDD and RA would enhance or antagonize the teratogenic effects of the other compound, C57BL/6N dams were treated po on Gestation Day (gd) 10 or 12 with 10 ml corn oil/kg containing TCDD (0-18 micrograms/kg), RA (0-200 mg/kg), or combinations of the two chemicals. Dams were killed on gd 18 and toxicity and teratogenicity assessed. Coadministration of TCDD and RA had no effect on maternal or fetal toxicity beyond what would be expected by either compound alone. Cleft palate was induced by RA at lower doses on gd 10 than on gd 12, but by TCDD at lower doses on gd 12 than on gd 10. Sensitivity to TCDD-induced hydronephrosis was similar on both gd 10 and 12. The limb bud defects were only observed when RA was administered on gd 10, not when given on gd 12. No other soft tissue or skeletal malformations were related to administration of TCDD or RA. No effect of TCDD was observed on the incidence or severity of limb bud defects induced by RA, nor did RA influence the incidence or severity of hydronephrosis induced by TCDD. However, the incidence of cleft palate was dramatically enhanced by coadministration of the xenobiotic and vitamin. On both gd 10 and 12, the dose-response curves for cleft palate induction were parallel, suggesting some similarities in mechanism between the two compounds. However, combination treatment resulted in a synergistic response that varied with the stage of development and was tissue specific.

Teratogenic potency of TCDD, TCDF and TCDD-TCDF combinations in C57BL/6N mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibenzofuran (TCDF) cause the same spectrum of fetal anomalies in C57BL/6N mice. Pregnant dams were treated with TCDD, TCDF and combinations of the 2 compounds on gestation day 10, and examined for maternal and fetal effects on day 18. The fetal kidneys were the most sensitive target for teratogenicity. The dose response for cleft palate induction fit the probit model for both compounds, suggesting that TCDD was approximately 30 times more potent than TCDF. The interaction between these 2 compounds was consistent with a model for additive toxicity.

Teratogenic effects of polychlorinated dibenzofurans in combination in C57BL6N mice

Abstract Polychlorinated dibenzofurans (PCDFs) are highly toxic environmental contaminants which have been involved in several incidents of human poisoning. Two congeners, 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF) and 1,2,3,4,7,8-hexachlorodibenzofuran (HCDF), have been shown to persist in the tissues of victims of accidental ingestion from Japan and Taiwan. The teratogenicity of these compounds, both alone and in combination, was assessed in C57BL 6N mice. Pregnant mice were treated with 10 ml/kg corn oil containing no PCDFs, 4-PeCDF (0–30 μg/kg), HCDF (0–300 μ/kg), or a combination of the two on gestation Days 10–13, followed by necropsy on gestation Day 18. Maternal and fetal toxicity were assessed and selected soft tissues were examined for abnormalities. Both chemicals caused hydronephrosis and cleft palate in the absence of any overt toxicity. Hydronephrosis occurred at doses approximately fivefold lower than those causing cleft palate. The combination of 4-PeCDF and HCDF was additive for terata based on responses predicted by probit analysis. In addition, the combination of 2,3,4,5,3′,4′-hexachlorobiphenyl (0–60 mg/kg), a structurally related compound also present in PCDF poisoning victims, and 4-PeCDF appears additive. Thus, these chemicals, which cause toxic effects similar to those of 2,3,7,8-tetrachlorodibenzo- p -dioxin, are additive in the induction of fetal anomalies in the mouse.

Characterization of the Peak Period of Sensitivity for the Induction of Hydronephrosis in C57BL/6N Mice following Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an extremely potent teratogen in mice. Hydronephrosis and cleft palate are the most sensitive measures of teratogenicity in mice following exposure to TCDD and other structurally related polyhalogenated aromatic hydrocarbons. Despite a relatively long half-life, investigators have identified a critical window for the induction of cleft palate in C57BL/6N mice. To characterize the critical period for renal teratogenesis, pregnant C57BL/6N mice were treated once by gavage with 0-24 micrograms TCDD/kg body wt on Gestation Day (GD) 6, 8, 10, 12, or 14. All dams were killed on GD 18, and the fetuses were examined for the presence of hydronephrosis and cleft palate. Maternal liver-to-body weight ratios were significantly elevated above controls on all days, while maternal weight gain was unaffected. Fetal mortality was increased relative to controls only at 24 micrograms TCDD/kg on GD 6. There was no significant difference in fetal body weights between control and TCDD-treated fetuses. The incidence of cleft palate increased in a dose-related fashion from GD 6 to GD 12, and identification of GD 12 as the critical window for induction of clefting of the hard palate was confirmed. Hydronephrosis was observed at all dose levels, regardless of exposure day, and the incidence was close to 100% at 3 micrograms TCDD/kg and higher doses on GD 12 and earlier. At all doses on GD 14, both the incidence and severity of hydronephrosis were decreased relative to all other days. There was a dose-related increase in the severity of the renal lesion on each day, but between GD 6 and 12 severity was constant. Thus, while palatal sensitivity to TCDD increased with gestational age between GD 6 and 12, there was no difference among these days in development of hydronephrosis. The data suggest, however, that on GD 14 the urinary tract may be less sensitive to TCDD.

Teratogenicity of three polychlorinated dibenzofurans in C57BL6N mice

Polychlorinated dibenzofurans (PCDFs) are widespread environmental contaminants which have been detected in human tissues and implicated in several poisoning incidents. Their toxic effects are similar to those observed with other related halogenated aromatic hydrocarbons such as TCDD. The teratogenic effects of three PCDFs, 1,2,3,7,8-pentachlorodibenzofuran (1-PeCDF), 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), and 1,2,3,4,7,8-hexachlorodibenzofuran (HCDF), were assessed in C57BL/6N mice. Pregnant mice were exposed on Gestation Days 10-13 to 10 ml corn oil/kg containing PCDFs. The dams were killed on Gestation Day 18 and maternal and fetal toxicity were assessed. All three compounds were highly teratogenic, with very steep and parallel dose-response curves for the two diagnostic indicators of dioxin-like teratogenicity, hydronephrosis, and cleft palate. 4-PeCDF was the most teratogenic with an ED50 of 36 micrograms/kg for cleft palate and 7 micrograms/kg for hydronephrosis. 4-PeCDF was approximately 4 times as potent as 1-PeCDF and 10 times as potent as HCDF. The teratogenic responses occurred at a dose below that where any obvious maternal or fetal toxicity was detected. Thus, these three compounds cause teratogenic responses similar to those seen with TCDD but are only 1/10 to 1/100 as potent.