The role of iron in the toxicity of 2,3,7,8-tetrachlorodibenzo-(p)-dioxin (TCDD)

The effects of TCDD on normal and iron-deficient mice fed a casein-based diet were compared. Liver damage, porphyria, and thymic atrophy were studied in C57B1/6J mice, chloracne and liver damage in the HRS/J strain. Sequential pathologic changes also were studied in C57B1/6J mice fed laboratory chow and treated with TCDD (25 μg/kg/week). Steatosis was present by Day 4; cell swelling, necrosis, and disorganization of the acinar architecture followed with maximal effects from weeks 4 to 8. Widespread necrosis of liver and cholangiofibrosis were late changes. The disturbance of porphyrin metabolism was localized to zone III.Mice fed a low iron diet supplemented with 0.05% $\text{w}\text{w}$ Fe and treated with TCDD (25 μg/kg/week × 12 weeks) developed porphyria after 15 weeks; venesected mice not supplemented with iron had not developed porphyria by 20 weeks. In general, an interaction was noted between TCDD and iron as judged by the rapidity of onset and severity of the porphyria. Iron-deficient and iron-supplemented groups of C57B1/6J mice received 37.5 μg/kg of TCDD. After 5 days there was an equal decrease in thymus weight in both groups of animals but histologic evidence of liver damage was less in the low iron mice. TCDD (45 μg/kg) was applied to the skin of iron-deficient and iron-supplemented hairless (HRS/J) mice. After 3 weeks chloracne developed irrespective of iron status; liver porphyrin was normal in the low iron animals but increased 100-fold in iron-supplemented mice in which histologic damage was also more severe.In general, iron deficiency did not decrease the activity of the hepatic mixed-function oxygenase systems. We conclude that iron deficiency protects against porphyria due to TCDD but that protection by iron deficiency against histologic damage to the liver is incomplete. In contrast, iron deficiency did not affect thymic atrophy or chloracne caused by TCDD.     

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): results of a 13-week oral toxicity study in rats.

Rats were given 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at dosages of 0, 0.001, 0.01, 0.1, or 1.0 μg/kg, 5 days/wk for 13 wk. Doses of 1.0 μg TCDD/kg/day caused some mortality, inactivity, decreased body weights and food consumption, icterus, increased serum bilirubin and alkaline phosphatase, pathomorphologic changes in the liver, lymphoid depletion of the thymus and other lymphoid organs, increased urinary excretion of porphyrins and delta-aminolevulinic acid, and minimal alterations of some hematopoietic components. Morphological evidence of a functional suppression of the reproductive organs was consistent with either a direct toxic effect of this dose of TCDD, or an indirect toxic effect associated with the poor physical condition of these rats. Doses of 0.1 μg TCDD/kg/day caused decreased body weights and food consumption, and slight degrees of liver degeneration and lymphoid depletion. Other effects seen only in females given this dose level included increases in urinary excretion of coproporphyrin and delta-aminolevulinic acid and increases in serum alkaline phosphatase and bilirubin. Effects seen only in males given this dose level included a depression of some hematologic parameters (packed cell volume, red blood cells, and hemoglobin). In rats given 0.01 or 0.001 μg TCDD/kg/day, all parameters were essentially unaffected, except for a slight increase in the mean liver-to-body-weight ratio in rats given 0.01 μg TCDD/kg/day. This slight increase in relative liver weight was not considered of any toxicological significance. These data indicate that no discernible ill effects occurred in rats given 0.01 or 0.001 μg TCDD/kg 5 days/wk for 13 wk.     

Immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in strains of mice with different susceptibility to induction of aryl hydrocarbon hydroxylase

Mouse strains with different susceptibility to aryl hydrocarbon hydroxylase (AHH) induction and with different levels and/or affinity for a specific cytosolic binding protein (“receptor”) were used to investigate the immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Humoral antibody production was strongly inhibited in $\text{C57B1}\text{6}$ and $\text{C3H}\text{HeN}$ mice (more susceptible strains) with very low, single doses of TCDD (1.2 μg/kg), while other strains ($\text{DBA}\text{2}$ and AKR) required higher doses (at least 6 μg/kg) to be partially suppressed. Longer exposure (8 weeks) did not increase the sensitivity of $\text{DBA}\text{2}$ mice. A good correlation between the degree of enzyme inducibility and immunosuppression was observed in studies with B6D2F1 mice and backcrosses. Similar results were obtained with 2,3,7,8-tetrachlorodibenzo-furan (TCDF), the most powerful competitor for TCDD “receptor” in vitro and in vivo. TCDD immnotoxic effects appeared to be associated with the presence of a specific cytosolic binding protein which mediates AHH induction.     

Tissue distribution,excretion, and metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the Golden Syrian hamster

The hamster has been reported to be the least sensitive mammalian species to the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The fate of a single dose of [³H]- or [¹⁴C]TCDD (650 μg/kg, ip or po) was assessed in male hamsters for up to 35 days following treatment. The greatest content (percentage dose/g tissue) of radioactivity was found in the liver, adipose tissue, and adrenals. The radioactivity in liver and adipose tissue was identified as unmetabolized TCDD. The rate of ³H or ¹⁴C elimination in urine and feces suggested a first-order process. Similar half-life of elimination ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) values of 12.0 ± 2.0 and 10.8 ± 2.4 days (mean ± SD) were obtianed with ip administered [³H]- and [¹⁴C]TCDD, respectively. With both [³H]- and [¹⁴C]TCDD, approximately 35 and 50% of the radioactivity was eliminated in urine and feces, respectively. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for po administered [³H]TCDD was 15.0 ± 2.5 days. High-pressure liquid chromatography of the urine and bile of animals receiving [¹⁴C]TCDD revealed one major and several minor radioactive peaks, none of which corresponded to [¹⁴C]TCDD. The apparent absence of TCDD metabolites in extracts of liver or adipose tissue indicates that the biotransformed products of TCDD are readily excreted in urine and bile. The enhanced rate of metabolism and excretion of TCDD in hamsters relative to other species may in part contribute to, but not totally explain its unusual resistance to TCDD toxicity.     

Enhanced liver DNA synthesis in partially hepatectomized rats pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on liver DNA synthesis was studied in rats after a $\text{1}\text{3}$ hepatectomy. The rats were maintained on a controlled feeding schedule and were treated with 5 μg/kg of TCDD or acetone/corn oil (control). Five days after treatment a $\text{1}\text{3}$ hepatectomy was performed and at designated times thereafter liver DNA synthesis was measured by [³H]thymidine incorporation into DNA. The main finding was that liver DNA synthesis was increased 8- to 10-fold by TCDD over that which was observed in control rats. This increase occurred after a latency period that was appropriate for the regenerative liver DNA synthesis response. Other experiments showed that increased incorporation of thymidine in TCDD-treated rats could be blocked by hydroxyurea, an inhibitor of semiconservative DNA synthesis, and that the increased incorporation was secondary to increased DNA synthesis and not increased thymidine kinase activity. Thus, hepatocytes in TCDD-treated rats respond in a quantitatively different manner than control rats to the same proliferative signal, $\text{1}\text{3}$ hepatectomy. Liver DNA synthesis in nonhepatectomized TCDD-treated rats tended to be greater than control, but the difference was not statistically significant.     

Comparison of the T Cell-Independent Antibody Response of Mice and Rats Exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmentalcontaminant that produces adverse effects on the immune systemof experimental animals. In this study, the effect that TCDDhas on the antibody plaque-forming cell (PFC) response to theT cell-independent (TI) antigen trinitrophenyl-lipopolysaccharide(TNP-LPS) was compared in adult female B6C3F1 mice and F344rats. Mice or rats were given a single intraperitoneal injectionof TCDD at doses ranging from 1 to 30 µg/kg, 7 days priorto immunization with TNP-LPS by intravenous injection. Threedays later body, spleen, thymus, and liver weights were measuredand the PFC response to TNP-LPS was determined. Thymus weightswere decreased at 10 and 30 µg TCDD/kg, whereas spleenweights were decreased and liver weights increased in mice dosedat 3,10, and 30 µg/kg. Mice dosed at 10 and 30 µgTCDD/kg had suppressed PFC responses and serum hemagglutinationliters. In rats, thymus weights were decreased and liver weightsincreased at 3, 10, and 30 µg TCDD/kg; however, the PFCresponse and serum hemagglutination titers to TNP-LPS were suppressedonly at 30 µg/kg TCDD. TCDD did not affect splenic lymphocytesubsets evaluated by flow cytometry. These results indicatethat TCDD suppresses the TI antibody response to TNP-LPS inboth B6C3F1 mice and F344 rats, with mice more sensitive tosuppression by TCDD than rats.     

The effect of altered hepatic function on the toxicity, plasma disappearance and biliary excretion of diethylstilbestrol

The present investigation was prompted by the observation that the toxicity of diethylstilbestrol (DES) was about 140 times greater in bile duct-ligated (BDL) rats than in control rats when dimethylsulfoxide was the vehicle (24-hr LD50: 0.75 vs 100 mg/kg, respectively). The DES was also more toxic (about 70 times) in the BDL rats when ethyl alcohol was the solvent (0.47 vs 34 mg/kg), and was 5 times more toxic in the BDL rats when administered in propylene glycol (100 vs 530 mg/kg). Since it appeared that altered hepatic function markedly alters the acute toxicity of DES, the plasma disappearance and biliary excretion of DES (0.118 mg/kg, iv) were measured in control rats and in rats with altered hepatic function (produced by surgical removal of two-thirds of the liver, ip injection of 1 ml CCl₄/kg, or bile duct ligation). All three procedures of altering hepatic function decreased the plasma disappearance of DES. In the BDL rats, the total plasma concentration of DES did not decrease over the 2-hr period, suggesting that even when the biliary route of excretion is blocked, other routes are not efficent for the excretion of DES. During the furst 15 min after DES administration, the biliary excretory rate of DES was 2.1 μg/min/kg in control rats and 1.2 μg/min/kg in the rats that had a $\text{2}\text{3}$ hepatectomy or CCl₄. Therefore, impaired hepatic function increased the plasma concentration and toxicity of DES as it decreased its conjugation and biliary excretion.     

The influence of malnutrition on the toxicity of 2,3,7,8-tetrachlorodiobenzo-p-dioxin (TCDD) in rats

A single oral dose of 2,3,7,7-tetrachlorodibenzo-p-dioxin (TCDD) (20 or 40 μg/kg body wet) administered to rats resulted in reduced body weight gain, water intake, degenerative changes in the liver and involution of the thymus. Although a restricted diet alone caused thymic involutions it was shown that TCDD at 10 or 20 μg/kg body wt accentuated this atrophy. Microscopically, a marked atrophy of the thymus and swelling and necrosis of the liver were found in the malnourished animals. The effects of TCDD were also tested in rats fed either 3.5%, 26% or 55% protein. There was no clear relation between the protein level and the influence of TCDD on the thymus. However, the effect of TCDD on body weight and the liver was more severe in rats given low protein diet. These effects were not reversed by high protein diet. Since it is unlikely that the influence of TCDD on the thymus is related to malnutrition, other mechanisms, such as depletion of energy reserves, a direct effect of TCDD on the thymus ior a mediation by the hepatotoxic action may be involved.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) distribution and cytochrome P4501A induction in young adult and senescent male mice

While the developmental toxicology of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its congeners has received considerable attention, the impact of advanced age on the biochemical effects and the pharmacokinetics of dioxins remains largely undetermined. In the present investigation, TCDD tissue distribution and cytochrome P4501A (CYP1A) induction were characterized in male C57BL/6N mice aged 10 weeks and 28 months at 7 days after administration of single oral [³H]TCDD doses ranging from 0.015 to 15 μg/kg body wt. Determinations of hepatic marker enzyme activities for CYP1A1 (ethoxyresorufin O-deethylation, EROD) and 1A2 (acetanilide-4-hydroxylation, ACOH) indicated that the dose response curves for EROD induction by TCDD were nearly identical for the 2 age groups, but the ACOH induction response was greater in old mice. After receiving the 15 μg/kg dose, an increase ( ~ 35%) in relative liver weight was observed 7 days after dosing in the 10-week mice, but not in the aged mice, and the hepatic concentration of TCDD was ~ 25% greater in young than old mice. No age difference was found in hepatic nuclear concentrations of TCDD. A dose-dependent increase in liver:fat tissue concentration ratios was noted at both ages, and adipose tissue and blood concentrations of TCDD did not vary significantly with age. In old mice however, TCDD concentrations in skin, kidney and muscle were all approximately twice those of young mice at the 15 gmg/kg dose. These results suggest that advanced age may have differential effects on Ah receptor-mediated enzyme induction, while increased TCDD concentrations in certain tissues may have toxicological implications for older animals.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin: Acute oral toxicity in hamsters

The acute oral toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was evaluated in hamsters. Male golden Syrian hamsters received 0 (corn oil only), 300, 600, 1000, 3000, or 6000 μg TCDD/kg body wt by single-dose gavage. To facilitate dosage, TCDD was fed as a suspension in acetone/corn oil (1:9). The 55-day single-dose oral LD50 was 5051 μg/kg (3876–18,487 μg/kg, 95% confidence interval). Deaths occurred from Day 9 through Day 43 following single-dose administration. Initially, no hamsters exhibited adverse effects related to dosage with TCDD, but 4–5 weeks following dosage, hamsters of the 1000, 3000, and 6000 μg/kg dose groups began to develop unkempt hair coats. A dose-related depression of mean body weight when compared to control weights was apparent in hamsters of the 1000, 3000, and 6000 μg/kg dose groups by 3 weeks posttreatment. Gross necropsy revealed the primary target organs affected in the hamster to be the same as in other laboratory species, namely, liver and thymus; this was accompanied by a slight decrease in adipose tissue reserves. No other significant effects were noted upon necropsy. Based on these results, the hamster appears to be much less sensitive than other laboratory species to the acute oral toxicity of TCDD.     

Sensitivity to suppression of cytotoxic T cell generation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on the Ah genotype of the murine host

Susceptibility of mice to a variety of toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is genetically determined by the Ah locus. To determine if immunotoxicity following TCDD exposure was also regulated by the Ah locus, we tested the ability of low dose TCDD (4 ng/kg) to suppress the generation of allospecific cytotoxic T cells (CTL) by lymphocytes from “susceptible” $\text{C57Bl}\text{6}$, and “resistant” $\text{DBA}\text{2}$, and from C57B1/6XDBA/2J F₁ hybrid mice in vitro. To determine if TCDD acted directly on the “susceptible” lymphoid cells, the immune response of $\text{C57B1}\text{6}\text{→}\text{DBA}\text{2}$ and $\text{DBA →}\text{C57B1}\text{6}$ bone marrow chimeras was also measured. $\text{C57B1}\text{6}$ and F₁ mice proved susceptible to suppression consistent with the dominant effect of Ah. Susceptibility to suppression in chimeric mice, however, was determined by the Ah genotype of the host and not by the genotype of the grafted lymphomyeloid cells. Mixing experiments demonstrated that suppression of CTL generation by TCDD was due to suppressor T cells. The frequency of CTL precursors was not affected by TCDD. These results are consistent with the idea that TCDD acts by an Ah locus-dependent mechanism to indirectly promote development of suppressor T cells that block the generation of CTL from their precursors.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on plasma disappearance and biliary excretion of foreign compounds in rats

Plasma disappearance and biliary excretion of ouabain, phenol-3,6-dibromphthalein (DBSP), and sulfobromophthalein (BSP) were determined in rats, 2, 10, 20, 25, or 40 days after oral administration of a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 10 or 25 μg/kg. Treatment with either dose of TCDD increased the plasma concentration of ouabain and decreased its excretion into bile. Both effects could be detected 2 days after TCDD and reached a peak between 10 and 20 days, and a slight recovery trend was seen at 40 days. The magnitude of the depression in ouabain excretion was directly related to the dose of TCDD. Plasma disappearance and biliary excretion of DBSP and BSP were affected differently by TCDD treatment than was ouabain. The low dose of TCDD enhanced initial biliary excretion of DBSP and BSP 25 days after TCDD. On the other hand, the high dose depressed biliary excretion and increased the plasma concentration of the dyes at 10 days. The depression in DBSP and BSP biliary excretion after the high dose of TCDD was of lesser magnitude and shorter duration than that of ouabain, being fully recovered after 25 days. Both doses of TCDD decreased body weight and increased liver wet weight. Bile flow was decreased by TCDD and the magnitude of depression was directly related to the dose of TCDD. The time course of the depression in bile flow was similar to that for the depression in ouabain excretion. Light microscopic examination of the liver 10 and 20 days after TCDD revealed swelling and occasional necrosis of individual hepatocytes and aggregations of mononuclear inflammatory cells in periportal areas. Our findings reveal that TCDD depresses the biliary excretion of ouabain in a dose-related manner and emphasize the long duration of the effect.     

Three-generation reproduction study of rats given 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the diet

A three-generation reproduction study was conducted to evaluate the effects of chronic, low-level ingestion of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Sprague-Dawley rats were maintained continuously on diets providing dose levels of 0, 0.001, 0.01, or 0.1 μg TCDD/kg/day. No significant toxicity was noted in the f₀ rats of either sex during the 90 days of TCDD ingestion prior to mating. Significant decreases in fertility and neonatal survival were observed in the f₀ generation rats receiving 0.1 μg TCDD/kg/day; these effects precluded continuation of this high dose level in subsequent generations. At 0.01 μg TCDD/kg/day, fertility was significantly decreased in the f₁ and f₂ but not f₀ generations. Other indications of toxicity seen at 0.01 μg TCDD/kg/day included decreases in litter size at birth, gestation survival (proportion of pups born alive), and neonatal survival and growth. Among the rats receiving 0.001 μg TCDD/kg/day, no effect on fertility, litter size at birth, or postnatal body weight was observed in any generation. No consistent effect on neonatal survival was observed at 0.001 μg TCDD/kg/day. In summary, the reproductive capacity of rats ingesting TCDD was clearly affected at dose levels of 0.01 and 0.1 μg TCDD/kg/day, but not at 0.001 μg TCDD/kg/day, through three successive generations.     

Metabolic rate of phenacetin and of paracetamol in dogs before and after treatment with phenobarbital or skf 525 A

In six male mongrel dogs the apparent biological half life ($\text{t}\text{2}$) in plasma of intravenously injected phenacetin decreased from 34·6 to 27·2 min after treatment with phenobarbital (25 mg/kg/day for 8 days) (P < 0·025). In the same dogs, the apparent $\text{t}\text{2}$ of intravenously injected phenazone decreased from 78·6 to 32·4 min (P < 0·05). Treatment with SKF 525 A (25 mg/kg) increased the $\text{t}\text{2}$ of phenazone in three other dogs from 70·6 to 246·7 min, whereas the $\text{t}\text{2}$ of phenacetin remained unaffected.The concentration of phenacetin at zero time of intravenous injection increased from 29·5 ± 5·9 S.E. to 43·5 ± 12·6 μg/ml plasma (P < 0·2) after phenobarbital treatment; pretreatment with phenobarbital, however, had no influence on the mean concentration of phenazone at 0 time. SKF 525 A did not influence the zero time concentration of either phenacetin or phenazone.Beagle liver microsomal protein and cytochrome P-450 concentrations increased from 3·2 to 4·2 mg/ml and from 1·0 to 4·1 n-mole/mg protein, respectively, after phenobarbital treatment. Treatment with phenobarbital had no influence on the rates of formation of paracetamol and p-phenetidin nor was the apparent K_m of phenacetin affected.The binding of phenacetin to blood constituents increased from 50 per cent at 5 μg/ml to 59 per cent at 1 μg phenacetin/ml. In the presence of 30 μg phenobarbital/ml blood, the binding of phenacetin at 1 μg/ml blood decreased by 22 per cent.It is concluded that the decrease of the apparent $\text{t}\text{2}$ of phenacetin in dog plasma after phenobarbital treatment may be due to a change in the apparent volume of distribution and/or to some stimulation of an NADPH-dependent enzyme system of the liver less affected by treatment with SKF 525 A than in other species.     

Toxicological studies on 4-(hexadecylamino)benzoate (PHB), an agent with anti-atherosclerotic properties,in the rat

The subacute oral toxicity of sodium 4-(hexadecylamino)benzoate (PHB) was studied in male and female Sprague-Dawley rats. The animals were given PHB 0, 10, 30, 100 or 300 $\text{mg}\text{kg}$ body weight as a 3% gum arabic suspension for 13 weeks. During PHB administration all animals on the highest dose level died or were killed because of loss of weight: there were deaths also in the 100 $\text{mg}\text{kg}$ group. PHB caused a leukocytopenia which was significant only at the highest dose level. The hemoglobin decreased in the two lowest dose groups. In the highest dose group the histological liver pictures were pathological. Acidophilic bodies and karyorrhexis indicating severe liver cell damage occurred at this level. In the groups receiving 30, 100 and 300 $\text{mg}\text{kg}$ there were foci with poorly distinguishable liver cells, mono-nuclear cells and some neutrophiles. PHB had no noticeable toxic effects on the other organs or parameters measured.     

Tissue Distribution,Metabolism, and Excretion of 14C-TCDD in a TCDD-Susceptible and a TCDD-Resistant Rat Straina

A comparative study was carried out in the most TCDD-resistant [Han/Wistar (H/W), LD50 > 3000 μg/kg] and the most TCDD-susceptible [Long-Evans (L-E), LD50 about 10 μg/kg] rat strain to assess the significance of kinetic factors in TCDD toxicity. Young adult males of both strains were administered 5 μg/kg (1.9 μCi/kg) ¹⁴C-TCDD intraperitoneally. Four rats per strain were killed at 4 hr, 1, 4, 8, 16, and 32 days after exposure. A total of 22 tissues along with blood and serum were sampled for liquid scintillation counting. From half of the animals, daily urine and faeces were also analyzed. In addition, 3 rats per strain were given 50 μg/kg (19 μCi/kg) ¹⁴C-TCDD and prepared for whole-body autoradiography after 1, 4 or 8 days. The livers of two rats per strain killed at 4 hr, 4 or 16 days, and the excreta from two rats of both strains collected on days 1-4, 5-8, 13-16, and 29-32 after exposure were analyzed for metabolites of TCDD by high pressure liquid chromatography. The label was mainly excreted in faeces as metabolites of TCDD, and the half-life of elimination was 20.8 (L-E) or 21.9 (H/W) days. A very similar overall distribution pattern was observed in both strains irrespective of dose, and the liver was the major site of accumulation. Practically all liver ¹⁴C-activity was found as the parent compound. Moderate strain-related differences were observed in the thyroid, thymus, prostate, adrenals, and brown and white fat, where lower values were recorded in H/W rats. Site-dependent variation was detected in the brain. The results do not support the view that TCDD metabolism or disposition would have a major impact on the strain difference in TCDD lethality.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) effects on hepatic microsomal cytochrome P-448-mediated enzyme activities.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatic microsomal mixed function oxidase (MFO) enzyme systems were examined in female rats. Although TCDD had little effect on NADPH-cytochrome c reductase activity and cytochrome P-450 content, the activities of the cytochrome P-448-mediated enzymes benzo[α]pyrene hydroxylase, ethoxyresorufin O-deethylase, and biphenyl 2-hydroxylase were greatly increased. Three months after a single oral dose of 2 μg/kg TCDD, the cytochrome P-450 content and benzo[α]pyrene hydroxylase and ethoxyresorufin O-deethylase activities were still significantly increased. In addition, the microsomal metabolism of the novel substrate 4,4′-dimethylbiphenyl was greatly increased by TCDD pretreatment. Low dose studies revealed that the ED50 of TCDD induction of benzo[α]pyrene hydroxylase was 0.63 μg/kg and the lowest dose of TCDD which caused a significant increase in enzyme activity was 0.002 μg/kg. Studies in which [1,6-³H]TCDD was used to determine the extent of hepatic uptake of orally administered TCDD at the lowest effective dose of 0.002 μg/kg lead to the estimate that only 65 molecules of TCDD per hepatocyte were required to produce a measurable increase in benzo[α]pyrene hydroxylation. These results attest to the specificity and persistence of TCDD in the induction of cytochrome P-448-mediated enzyme activities in rat liver. The small number of molecules required to induce benzo[α]pyrene hydroxylase suggests that TCDD is among the most potent MFO-inducing agents yet demonstrated in mammalian liver.     

The fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin following single and repeated oral doses to the rat.

Rats were given a single oral dose of 1.0 μg of [¹⁴C]-2,3,7,8-Tetrachlorodibenzo-p-dioxin ([¹⁴C]TCDD)/kg/day, or repeated oral doses of 0.01, 0.1, or 1.0 μg of [¹⁴C]TCDD/kg/day Monday through Friday for 7 weeks. Following a single oral dose of 1.0 μg of [¹⁴C]TCDD/kg, ¹⁴C activity could be detected only in feces, but not in urine. The half-life of ¹⁴C activity in the body was 31 ± 6 days. Twenty-two days after the single oral dose, concentrations of ¹⁴C activity were located principally in liver and fat. Following repeated oral doses the major route of excretion was via the feces; urine contained 3–18% of the cumulative dose of ¹⁴C activity by 7 weeks. The half-life of ¹⁴C activity in the body of these rats was 23.7 days. Assuming a one compartment open model, 76.2% of steady-state concentrations were achieved in the whole body after 7 weeks. ¹⁴C activity in liver and fat approached steady-state values at a rate similar to the whole body. Radioactivity in the liver was identified as TCDD by gas chromatography-mass spectrometry and could be extracted from liver tissue with organic solvents. The results of this study in rats indicate that TCDD approaches steady-state concentrations in the body within 13 weeks, and the rate constant defining the approach to steady-state concentrations is independent of the dosage of TCDD over the dose range of 0.01?1.0 μg of TCDD/kg/day.     

Effects of ethinyl estradiol on bile secretion and liver microsomal mixed function oxidase system in the mouse

In the search for an animal model suitable for the study of ethinyl estradiol (EE) induced alterations of bile secretion, we have investigated the effects of three different doses (50–500–5000 μg/kg body wt., orally for 10 days) of EE on bile flow and composition and on liver microsomal mixed function oxidase system in female Albino-Swiss mice. No difference of bile flow was found between control and EE-treated mice. The decrease of bile acid secretion was dose-related and significant in animals treated with 500 and 5000 μg/kg of EE. Cholesterol output was similar in control and EE-treated animals. The molar ratio of bile acid to biliary cholesterol was significantly lower in all groups of EE-treated mice as compared with controls. The specific activities of 3,4-benzpyrene hydroxylase, aniline hydroxylase and NADPH cytochrome-c-reductase, as well as the content of cytochromes P-450 and b₅ decreased proportionally, in a dose dependent manner and significantly after 500 and 5000 μg/kg of EE. Our data indicate that mice, following EE, develop a lithogenic bile without obvious cholestasis. Moreover, they demonstrate a decrease of liver microsomal enzyme activities and cytochromes and suggest a relationship between the impairment of liver microsomal mixed function oxidase system and the changes of bile lipid composition.