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.     

Modulating effects of thyroid state on the induction of biotransformation enzymes by 2,3,7,8-tetrachlorodibenzo-p-dioxin

In this study we investigated to what extent the induction of detoxification enzymes by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is modulated by concomitant TCDD-induced changes in thyroid state. Euthyroid (Eu) male Sprague-Dawley rats, surgically thyroidectomized (Tx) rats and Tx rats receiving substitution doses of 3,3',5-triiodothyronine (Tx+T3) or thyroxine (Tx+T4) by osmotic minipumps were treated with a single ip injection of 10 μg TCDD/kg/bwt or with vehicle (corn oil). Three days after TCDD administration, rats were sacrificed and blood and livers were collected for analysis. Total hepatic cytochrome P450 (CYP) content was increased by ≈50% by TCDD in all groups but was not affected by thyroid state. In Eu rats, TCDD increased CYP1A1/1A2 activity 90-fold, CYP1A1 protein content 52-fold and CYP1A1 mRNA levels ≈5.8-fold. Similar findings were obtained in Tx, Tx+T3 and Tx+T4 rats except that TCDD-induced CYP1A1 activity was significantly decreased in Tx rats. NADPH cytochrome P450 reductase activity was not affected by TCDD but was decreased in Tx rats, which may explain the diminished TCDD-induced CYP1A1 activity in Tx rats. Hepatic p-nitrophenol UDP-glucuronyltransferase (UGT) activity was induced ≈4-fold by TCDD in Eu rats. Similar basal and TCDD-induced activities were observed in Tx+T3 and Tx+T4 rats, but TCDD-induced activities were significantly lower in Tx rats. TCDD did not have a significant effect on overall glutathione-S-transferase (GST) activity or hepatic GST 2-2, 3-3 or 4-4 protein levels but produced a marked increase in GST 1-1 protein levels. Thyroid state did not affect basal or TCDD-induced GST activity or subunit pattern. Iodothyronine sulfotransferase (ST) activity was not affected by TCDD treatment and was slightly but not significantly lower in Tx rats than in Eu, Tx+T3 and Tx+T4 rats. These results suggest that the changes in thyroid hormone levels associated with TCDD treatment have little modulating effects on the induction of hepatic detoxification enzymes in Sprague-Dawley rats exposed to this compound.     

The interaction of L-triiodothyronine and 2,3,7,8-tetrachlorodibenzo-p-dioxin on Ah-receptor-mediated hepatic Phase I and Phase II enzymes and iodothyronine 5'-deiodinase in thyroidectomized rats.

Across all levels of L-triiodothyronine (L-T3) treatment, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in increased hepatic cytochrome P-450-associated activities of 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxyresorufin O-dealkylase (EROD) and aryl hydrocarbon hydroxylase (AHH). The treatment of thyroidectomized rats with L-T3 at physiologic replacement levels in concert with TCDD produced an increase in ECOD, EROD and AHH activity above that seen with only TCDD. TCDD as well as L-T3 enhanced the activity of hepatic 1-naphthol glucuronyl transferase (NGT). In addition, the combined effect of L-T3 and TCDD resulted in similar levels of induction of NGT at both physiologic and supraphysiologic doses of L-T3. TCDD treatment resulted in elevated serum T3 levels at both physiologic and supraphysiologic levels of L-T3. One TCDD dose inhibited hepatic microsomal 3,3',5'-triiodothyronine (reverse T3) 5'-deiodinase activity by 61% in thyroidectomized, T3-untreated rats. The inhibition of 5'-deiodinase activity was partially overcome by increasing the T3 dose.     

Changes in thyroid hormones and thyroxine glucuronidation in hamsters compared with rats following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin

In rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds, serum thyroxine (T4) is depressed. Since hamsters are relatively insensitive to TCDD-induced lethality, the effects of TCDD on several parameters of thyroid status were measured in hamsters as a comparison with the more sensitive rat. At 7 days after ip injection of TCDD, there was a dose-dependent increase in serum 3,5,3'-triiodothyronine (T3) and T4 in hamsters to a maximum level 200% of control; the ED50 was approximately 10 micrograms/kg. Hamsters receiving 100 micrograms/kg lost up to 4% of their body weight but began to recover after about 3 weeks. Serum T4 in these animals was elevated compared to pair-fed and ad libitum controls throughout the 53-day experiment, although it also began to recover after Day 21. This was in direct contrast to the marked reduction of T4 in rats exposed to lower doses of TCDD. T3 was significantly higher in TCDD-treated hamsters than in pair-fed controls on Days 2-7, and TSH was also elevated on Days 2-21. Reverse T3, like T4, was increased by TCDD in hamsters whereas it was decreased in rats. Hepatic microsomal UDP-glucuronosyltransferase (GT) activity was measured using T4 as substrate (T4-GT). On a whole liver basis, T4-GT was induced by TCDD by the same proportion in both rats and hamsters (170-180% of controls) although absolute activities in rats were 3- to 4-fold higher than in hamsters. This similarity in T4-GT inducibility by TCDD suggests that there are likely mechanisms in addition to T4-GT induction which account for the species-specific alterations in T4. Thus, while the response of thyroid hormones to TCDD differed qualitatively, effective doses in hamsters were higher than in rats, suggesting that these changes, although secondary, may correlate more directly with toxicity than does enzyme induction (whose ED50s are similar in both species). An understanding of the mechanism of this species difference may be helpful in unravelling the primary mechanisms of TCDD toxicity.     

Thyroid hormones modulate the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

These experiments examine the role of thyroxine (T4) and triiodothyronine (T3) on the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The first experiment is continuation of a study reported previously (Rozman et al., 1984). In this experiment, 60 male Sprague-Dawley rats were divided into 6 equal groups. Four groups of rats were thyroidectomized by 3 mCi Na131 l/kg rat. Five weeks later 2 of the thyroidectomized and 1 of the nonthyroidectomized groups of rats received ip 100 micrograms TCDD/kg body weight in corn oil/acetone, whereas 3 corresponding groups of rats served as vehicle controls. Two days after dosing and every 7 d thereafter, 1 thyroidectomized control group and 1 thyroidectomized TCDD-dosed group were given ip 105 micrograms T4/kg body weight. Mortality and body weight were monitored. The course of TCDD toxicity was similar in nonthyroidectomized and thyroidectomized T4-treated rats but was different in thyroidectomized animals without T4 replacement therapy. At d 90 after TCDD dosage, mortality was still lower and the mean time to death was increased (p less than 0.01) in this group of rats compared to nonthyroidectomized or thyroidectomized T4-treated rats. However, administration of T4 starting at d 91 after dosing with TCDD resulted within 2 wk in the same final mortality in thyroidectomized rats as in nonthyroidectomized or thyroidectomized T4-treated animals, indicating that thyroid hormones modulate the time course of the wasting syndrome but do not affect the ultimate mortality figure. Body weight loss was much slower in thyroidectomized (approximately 1 g/d) than in nonthyroidectomized or thyroidectomized T4-treated rats (approximately 8 g/d). In the second experiment the three vehicle control groups of the first experiment were used. Nonthyroidectomized vehicle controls and thyroidectomized T4-treated controls were maintained as before, whereas thyroidectomized controls received T3 at 5 micrograms/kg daily. One month later each rat was dosed with TCDD at 100 micrograms/kg in corn oil/acetone. Toxicity of TCDD was similar in nonthyroidectomized, thyroidectomized T4-treated, and thyroidectomized T3-treated rats as judged by mortality, body weight, and food intake, indicating no difference between T3 and T4 in the modulation of TCDD toxicity.     

Phenobarbital, beta-naphthoflavone, clofibrate, and pregnenolone-16alpha-carbonitrile do not affect hepatic thyroid hormone UDP-glucuronosyl transferase activity, and thyroid gland function in mice

The effects of representative liver enzyme inducers such as clofibrate (CLO), phenobarbital (PB), pregnenolone-16alpha-carbonitrile (PCN), and beta-naphthoflavone (NF) on hepatic microsomal thyroxin (T4)- UDP-glucuronosyl transferase (UGT) and triiodothyronine (T3)- UGT activities and thyroid function were evaluated in OF-1 male mice after a 14-day po administration. CLO, PB, and PCN induced histological liver hypertrophy, increases in liver weights, in microsomal protein and cytochrome P450 contents as well as increases in specific UGT activities. Despite this, no significant changes in T4-UGT and T3-UGT activities occurred after treatment by any of these compounds. Furthermore, no significant changes in serum T4 and T3 levels were observed and thyroid histology was not affected. NF treatment induced microvacuolation of hepatocytes but did not affect any of the other tested parameters. The results show that, in contrast to the widely described effects in rats, liver enzyme inducers do not affect hepatic thyroid hormone metabolism and thyroid function in mice, suggesting that this species should be less sensitive to thyroid tumor promotion by hepatic microsomal enzyme inducers than rats.     

UDP-glucuronosyltransferase inducers reduce thyroid hormone levels in rats by an extrathyroidal mechanism.

As many microsomal enzyme inducers have been shown to reduce thyroid hormone levels, this study was conducted to determine if this reduction is produced by directly blocking the synthesis of thyroid hormones, or by indirectly increasing the biotransformation and deactivation of thyroxine (T4) by microsomal enzymes. Surgically thyroidectomized male rats received thyroid hormone replacement therapy by implanted osmotic minipumps, resulting in T4 and T3 serum levels that were similar to those observed in euthyroid controls. Three days after minipump implantation (Day 0), rats were fed diets containing four UDP-glucuronosyltransferase (UDP-GT) inducers: phenobarbital (PB), 3-methylcholanthrene (3MC), pregnenolone-16 alpha-carbonitrile (PCN), or polychlorinated biphenyls (PCB) for 10 days. PB, 3MC, and PCN reduced total (Days 3-10) and free (Days 7-10) T4 serum concentrations 30-50%, whereas PCB produced a 70-75% reduction in total and free serum T4 (Days 3-10). Treatment with PB, PCN, and PCB decreased levels of total T3 (Days 7-10). UDP-GT activity toward T4 was increased by PB, 3MC, PCN, and PCB 270, 400, 570, and 660%, respectively, and was found to correlate with serum T4 levels (total and free). These results demonstrate that reduction of thyroid hormone levels by microsomal enzyme inducers is produced in part by an extrathyroidal mechanism, quite possibly an increase in T4 glucuronidation.     

Effects of short-term in vivo exposure to polybrominated diphenyl ethers on thyroid hormones and hepatic enzyme activities in weanling rats.

Polybrominated diphenyl ethers (PBDEs), used as flame retardants, are ubiquitous environmental contaminants. PBDEs act as endocrine disruptors via alterations in thyroid hormone homeostasis. We examined thyroid hormone concentrations and hepatic enzyme activity in weanling rats exposed to three commercial PBDE mixtures: DE-71, DE-79, and DE-83R. Female Long-Evans rats, 28 days old, were orally administered various doses of DE-71, DE-79, or DE-83R for 4 days. Serum and liver samples were collected 24 h after the last dose and analyzed for serum total thyroxine (T(4)), triiodothyronine (T(3)), thyroid-stimulating hormone (TSH), hepatic microsomal ethoxy- and pentoxy-resorufin-O-deethylase (EROD and PROD), and uridinediphosphate-glucuronosyltransferase (UDPGT) activities. The PBDE-treated groups did not exhibit significant changes in body weight; however, increased liver weights, as well as 10- to 20-fold induction in EROD and 30- to 40-fold induction in PROD were found in the DE-71-- and DE-79--treated animals. DE-71 and DE-79 caused dose-dependent depletion of T(4), accompanied by up to 3- to 4-fold induction in UDPGT activities. Serum total T(4) was decreased a maximum of 80% for DE-71 and 70% for DE-79 in the highest dose, with benchmark doses (BMDs) of approximately 12.74 mg/kg/day for DE-71 and 9.25 mg/kg/day for DE-79. Dose-related effects in serum T(3) levels were less apparent, with maximal reductions of 25-30% at the highest dose for both DE-71 and DE-79. The two mixtures showed no effect on serum TSH levels. Benchmark dose analysis revealed that the two mixtures were comparable in altering thyroid hormone levels and hepatic enzyme activity. DE-83R was not effective in altering any of the measured parameters. The present study suggests that short-term exposure to some commercial PBDE mixtures interferes with the thyroid hormone system via upregulation of UDPGTS:     

Activity of thyroid hormone-inducible enzymes following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) caused a depletion of serum thyroxine, but paradoxically did not change L-3,5,3'-triiodothyronine (T3) levels in serum of rats. The activities of the thyroid-regulated enzymes alpha-glycerol phosphate dehydrogenase (GPD) and malic enzyme (ME) were determined in livers of normal and thyroidectomized (THX) rats treated with 0.1 to 100 nmol TCDD/kg body weight. Mitochondrial GPD activity did not change significantly as a function of TCDD dose in either normal or THX rats. ME activity was induced by TCDD in a dose-dependent fashion, but only in non-THX animals. The absence of ME induction in THX rats treated with TCDD indicates that TCDD is not intrinsically thyromimetic. The dependence of ME induction on thyroid hormones is much like the thyroid-hormone-dependent, multihormonal induction of ME by insulin and glucocorticoids. However, TCDD had no additive or synergistic effects on induction of ME activity in THX rats fed T3. A 30% decrease in steady-state plasma T3 levels of T3-fed animals treated with TCDD relative to T3-fed controls suggested that T3 catabolism was more rapid in TCDD-treated rats than controls. Thus a thyroid-hormone-dependent, multihormonal interaction is suggested as the basis for induction of ME by TCDD, but a strictly T3-dependent process has not been ruled out.     

Changes in steroid hormone metabolism in rat liver microsomes following administration of 2,3,7,8-tetrachlorodibenzo-p-dioxine (TCDD).

Treatment of male and female rats with 4 × 20 μg/kg of 2,3,7,8-tetrachlorodibenzo-p-dioxine (TCDD) led to 2–3-fold increases in liver microsomal cytochrome P450 levels concomitantly with changes in liver microsomal metabolism of 4-androstene-3,17-dione, 5α-androstane-3α,17β-diol and 4-pregnene-3, 20-dione. The changes were most pronounced in female rats where some hydroxylase activities increased 3–5-fold. It is suggested that the observed effects following TCDD administration may be related to the endocrine symptoms sometimes seen in patients exposed to this drug.     

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.     

6-Methyl-1,3,8-trichlorodibenzofuran as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist: inhibition of the induction of rat cytochrome P-450 isozymes and related monooxygenase activities

In addition to being one of the most toxic chemicals known, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent inducer of rat liver microsomal cytochrome P-4501A1 (P-450c). Previous studies have demonstrated that a high affinity, low capacity cytosolic receptor (the Ah receptor) mediates the activity of TCDD to induce cytochrome P-4501A1, which catalyzes benzo[a]pyrene hydroxylation [aryl hydrocarbon hydroxylase (AHH]) and 7-ethoxyresorufin O-dealkylation (EROD). The results of the present study indicate that 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) effectively competes with [3H]TCDD for binding to the Ah receptor in rat liver cytosol. The concentration of MCDF effecting 50% displacement of [3H]TCDD was 4.9 X 10(-8) M, which is approximately 50 times greater than the EC50 for unlabeled TCDD (approximately 1 X 10(-9) M). However, in contrast to TCDD, MCDF was only a weak inducer of AHH and EROD activity in rat hepatoma H-4-II cells in culture. When co-incubated, MCDF diminished in a concentration-dependent manner the ability of TCDD to induce AHH and EROD activity in vitro. Treatment of rats with 20-200 mumol/kg MCDF in vivo had little or no effect on liver microsomal AHH and EROD activity, whereas treatment of rats with 16 nmol/kg TCDD caused a 6- and a 70-fold induction of AHH and EROD activity, respectively. When co-administered, MCDF diminished by approximately 50% the ability of TCDD to induce AHH and EROD activity in vivo. The partial antagonism produced by 50 mumol/kg MCDF could be partially overcome by doubling the dosage of TCDD from 16 to 32 nmol/kg. Immunochemical analysis of rat liver microsomes revealed that treatment of rats with 20-200 mumol/kg MCDF caused little or no induction of cytochromes P-4501A1 and P-4501A2 (P-450d), whereas these isozymes were induced 33- and 5-fold, respectively, in rats treated with 16 nmol/kg TCDD. When co-administered, MCDF diminished by approximately 50% the ability of TCDD to induce cytochrome P-4501A1 in vivo, which established that MCDF was not simply acting as an inhibitor of AHH and EROD activity. MCDF also antagonized the ability of TCDD to induce cytochrome P-4501A2, which suggests that the induction of both cytochromes P-4501A1 and P-4501A2 is regulated by the Ah receptor. These results indicate that MCDF binds with high affinity to the Ah receptor in rat liver cytosol and competitively blocks the binding of TCDD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nature of the enhancement of hepatic uridine diphosphate glucuronyltransferase activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats

After single low-level oral doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to rats, hepatic microsomal p-nitrophenol (PNP) glucuronyltransferase activity was elevated approximately 6-fold, whereas the hepatic glucuronyltransferase conjugating testosterone or estrone was unaffected. Solubilized and purified PNP glucuronyltransferase and steroid glucuronyltransferases from control and TCDD-treated rats exhibited the same relative activities (TCDD:control) as when the enzymes were bound to the endoplasmic reticulum. Elevation of PNP glucuronyltransferase was still evident 73 days after a single oral dose of 25 μg TCDD/kg. Female rats were more susceptible to TCDD actions on liver microsomal PNP glucuronyltransferase than males. The effects of TCDD treatment on PNP glucuronyltransferase appeared to be related to increased amounts of liver enzyme for the following reasons: (1) K_m values for PNP and UDPGA were unchanged by TCDD treatments; (2) the magnitude of the TCDD-induced increase of PNP glucuronyltransferase activity was the same whether enzyme activity was measured in the presence or absence of Mg²⁺ or Triton X-100; (3) TCDD, when added in in vitro, had no detectable effect on enzyme activity; (4) TCDD treatment of rats did not change total hepatic microsomal phospholipid or cholesterol contents: (5) pH optima were unaffected by TCDD treatment; (6) solubilization of enzyme was not accompanied by a change in the TCDD induction effect: and (7) actinomycin D appeared to block the initial phase of induction.     

Hepatic Ah-receptor levels and the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatic microsomal monooxygenase activities in a TCDD-susceptible and -resistant rat strain

Previous studies have shown that in two inbred strains of mice, straightforward correlations exist among the number of hepatic Ah-receptors, enzyme inducibility by TCDD, and lethality of TCDD. Here, studies were conducted in two strains of rats (Han/Wistar and Long-Evans) which differ widely in susceptibility to the lethal effects of TCDD, to determine if these are general phenomenona in TCDD toxicity. The total number of specific binding sites (Ah-receptors) for [3H]TCDD proved to be approximately equal in the livers of both rat strains. Likewise, no notable difference was detected in the effect of TCDD on the activities of 7-ethoxyresorufin O-deethylase, 7-ethoxycoumarin O-deethylase, and ethylmorphine N-demethylase or on the amount of cytochrome P-450 in hepatic microsomal fractions. Immunoblot analysis was carried out with monoclonal antibodies (Mabs). Mab 1-7-1 directed against rat liver 3-methylcholanthrene (MC)-inducible P-450 recognized forms P-450c and P-450d in TCDD-treated rats in a dose-dependent fashion and to a similar extent in both strains. In contrast, Mab 2-66-3 (against phenobarbital-inducible P-450) did not recognize any proteins in either strain, confirming the conclusion that TCDD elicits a MC-type induction of hepatic cytochrome P-450 in both strains of rats. Thus, it seems that the correlations observed in mice do not hold in rats and therefore should not be generalized. The parameters measured in the present study are causally unrelated to the mechanism of lethal action of TCDD in these rat strains.     

Thyroid status and thermogenesis in rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin

Several key aspects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity resemble the effects of hypothyroidism, while in other ways the toxic responses are characteristics of hyperthyroidism. Whether thyroid dysfunction plays a role in TCDD toxicity remained unknown, however. We therefore determined the dose-related effects of TCDD treatment on plasma concentrations of L-thyroxine (T4), 3,5,3'-triiodo-L-thyronine (T3), and thyroid-stimulating hormone (TSH), and compared these changes with signs of TCDD toxicity. We also determined whether indices of functional thyroid status (and thermogenesis) were altered in response to TCDD treatment. Young adult male Sprague-Dawley rats were given single oral doses of TCDD (6.25-100 micrograms/kg) and evaluated 1 week later. Toxicity, measured by decreases in feed intake and body weight, ranged from minimal to severe. Plasma concentrations of T4 were greatly reduced at all doses tested, while T3 was increased in a dose-related fashion (up to 35%). TSH was elevated but was inversely proportional to dose. Thyroid histology was unremarkable, and TCCD treatment had little effect on the ability of rats to raise serum T4, T3, and TSH concentrations in response to acute cold stress. TCDD treatment caused a slight (8%) decrease in basal metabolic rate, yet comparable decreases were seen in pair-fed control animals. Thermogenesis, as measured by O2 consumption and colonic temperatures in rats exposed to various ambient temperatures, was only marginally affected. In summary, although thyroid hormone concentrations were markedly altered, rats given doses of TCDD sufficient to cause overt toxicity appeared to be essentially euthyroid. These results do not support proposals by other researchers that altered thyroid status is a major contributor to TCDD toxicity and/or a key response to TCDD exposure.     

Comparison of the effects of propylthiouracil, amiodarone, diphenylhydantoin, phenobarbital, and 3-methylcholanthrene on hepatic and renal T4 metabolism and thyroid gland function in rats

We studied the effects of propylthiouracil (PTU), amiodarone (AMIO), diphenylhydantoin (DPH), phenobarbital (PB), and 3-methylcholanthrene (MC) on thyroid histomorphology, on the hepatic and renal enzymes involved in endogenous and exogenous metabolism, and on the plasma levels and pharmacokinetics of thyroid hormones after 7 and 14 days of treatment. PTU and PB, by decreasing both serum tetraiodothyronine (T4) and triiodothyronine (T3), induced a massive increase in serum thyrotropin (TSH) and thus induced thyroid hypertrophy. AMIO and MC, by decreasing respectively serum T3 and T4, also induced an increase of TSH, but to a lesser extent, not sufficient to induce thyroid hypertrophy. Hepatic 5'-deiodinase activity was decreased in all treated rats. Inhibition of this enzyme by PTU was demonstrated in vitro; AMIO also decreased the enzyme activity by a still unelucidated mechanism, which probably requires intact cell plasma membranes, whereas in PB- and MC-treated rats the decrease in enzyme activity certainly resulted from decreased serum concentrations of T4. In PTU-treated rats, and probably in MC-treated rats, decreases in circulating thyroid hormones were primarily due to impairment of synthesis and/or of secretion by the thyroid. In contrast, in PB-treated rats, the decrease in serum thyroid hormone levels seems to be due to increased excretion of these hormones, as T4 serum clearance was significantly increased. PB, a microsomal enzyme inducer, increased the cytochrome b5 and P450 content as well as the cytochrome P450-dependent O-depentylation of pentoxyresorufin. The other type of enzyme inducer, MC, did not affect cytochrome b5 and P450 levels, but did increase the cytochrome P450 dependent O-deethylation of ethoxyresorufin. PB increased the glucuronidation of morphine, whereas MC increased the glucuronidation of 1-naphthol. However, serum T4 clearance, mainly determined by its hepatic conjugation rate, was increased only in PB-treated rats. It appears from this study that the close metabolic relationship between the liver/kidney and the thyroid should be taken into consideration when the findings of chronic toxicology and carcinogenicity studies are interpreted.     

Biphasic response for hepatic microsomal enzyme induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin in C57BL/6J and DBA/2J mice

The induction of the murine hepatic microsomal cytochrome P-450 monooxygenase system by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied over a wide range of doses, including those associated with acute toxicity. Studies were conducted in two inbred strains of mice which vary at the Ah receptor and at a number of other genetic loci. C57BL/6J mice possess a high-affinity Ah receptor and are responsive to enzyme inductive effects of TCDD, whereas DBA/2J mice do not possess a high-affinity receptor and are less responsive to TCDD. In a dose-response study, 7-ethoxyresorufin O-deethylase (EROD) activity appeared to be maximally induced in C57BL/6J and DBA/2J mice at 7 days following exposure to 3 and 30 micrograms of TCDD/kg respectively. Very similar results were reported previously for the induction of aryl hydrocarbon hydroxylase activity in these strains of mice. However, at higher doses of TCDD (at least 45 micrograms/kg for C57BL/6J and 300 micrograms/kg for DBA/2J), EROD activity was further increased (2-fold) from the apparent maximal (plateau) level, resulting in an unusual biphasic log dose-response relationship. EROD activity remained at these elevated rates in both strains for doses approaching and exceeding the respective LD50 values for each strain. To further characterize this biphasic induction phenomenon, cytochrome P-450 content, benzo[a]pyrene metabolism, and EROD and NADPH-cytochrome P-450 reductase activities were measured 1, 3 and 7 days after TCDD administration to C57BL/6J (3 and 150 micrograms/kg) and DBA/2J (30 and 600 micrograms/kg) mice. Maximal responses occurred in both strains at 3 days for all doses. In both strains, TCDD produced a dose-dependent increase in cytochrome P-450 content, EROD, and benzo[a]pyrene metabolism. Furthermore, a 2-fold induction of reductase activity was observed in each strain following exposure to the respective high doses. Induction of cytochrome P1-450 and P3-450 was also measured by Western immunoblot, using antisera raised against the homologous rat isozymes. In both strains, TCDD produced a dose-related increase in two protein-staining bands recognized by anti-P-450BNF-B (P1-450) and anti-P-450BNF/ISF-G (P3-450) respectively. The extended induction of hepatic microsomal monooxygenase activities at the respective high doses of TCDD appears to be due, in part, to increases in NADPH-cytochrome P-450 reductase activity and cytochromes P1-450 and P3-450 content. Significant alterations in the expression of the cytochrome P-450 monooxygenase system following exposure to high doses of TCDD may be associated, in part, with the delayed acute toxicity reported at this level of exposure.     

Fipronil-induced disruption of thyroid function in rats is mediated by increased total and free thyroxine clearances concomitantly to increased activity of hepatic enzymes

Fipronil is a widely used phytosanitary product and insecticide for pets. In the rat, fipronil can disrupt thyroid function by decreasing plasma concentrations of total thyroxine (T4) likely through increased T4 clearance. However, the mechanism of fipronil action on thyroid function remains unclear. The goals of the present study were to evaluate the effects of fipronil on thyroid hormone (TH) concentrations and elimination in the rat under well characterized plasma exposure to fipronil and its main metabolite fipronil sulfone. In thyroid-intact female rats, fipronil treatment (3 mg/(kg day) per os for 14 days) decreased both total and free TH plasma concentrations concomitantly to increased thyroid stimulating hormone plasma concentrations. A T4-free euthyroid-like model consisting of thyroidectomized rats treated with tri-iodothyronine (12 microg/(kg day), sc) was developed to evaluate both total and free T4 clearances. In this model, fipronil treatment induced a twofold increase in total and free T4 clearances. The same fipronil treatment increased antipyrine clearance in thyroid-intact rats suggesting an increase in the activity of cytochrome P450 enzymes. Finally, this treatment was also associated with an increase in hepatic microsomal 4-nitrophenol UDP-glucuronosyltransferase activity involved in T4 glucuronidation. Thus, fipronil-induced thyroid disruption results from an increased rate of T4 elimination likely mediated by increased hepatic enzyme activity. Plasma concentrations of fipronil sulfone were at least 20-fold higher than those of fipronil. This highlights the need to further investigate the contribution of fipronil sulfone to the fipronil-induced thyroid disruption.     

Toxicity and effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) on hepatic cytochrome P450 in F344 rats

The subacute toxicity and effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) on hepatic microsomal cytochrome P450 (P450) were investigated in male and female F344 rats. Rats were given 0.25, 0.5, and 1.0% terbutol for 28 days. Liver weights of male and female rats increased at all dose levels. The compound did not affect activity or amount of serum biochemical markers related to hepatic damage. The concentrations of terbutol in rat serum were less than 0.1 microM, and its major metabolites in serum were 2,6-di-tert-butyl-4-carboxyphenyl N-methyl-carbamate and 2,6-di-tert-butyl-4-carboxyphenol. In male rats, P450 and cytochrome b5 (b5) contents, and NADPH cytochrome c reductase (fp2) activity in liver microsomes were increased about 2-fold by 1% terbutol administration for 7 to 28 days. Among the P450-dependent monooxygenase activities in liver microsomes, 7-benzyloxyresorufin-O-debenzylase (BROD) activity was greatly increased by 100-fold, and 7-ethoxyresorufin-O-deethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), and aminopyrine-N-demethylase (APND) activities were elevated 2- to 3-fold. 7-Methoxyresorufin-O-deethylase (MROD), erythromycin-N-demethylase (EMND), estradiol 2-hydroxylase (ED2H), chlorzoxazone 6-hydroxylase (CZ6H), and lauric acid omega-hydroxylase (LAOH) activities were unchanged. For the activities of testosterone hydroxylation, testosterone 16beta-hydroxylase (T16BH) activity was markedly increased by 30-fold, and testosterone 6beta-hydroxylase (T6BH) and testosterone 7alpha-hydroxylase (T7AH) activities were slightly elevated. Testosterone 2alpha-hydroxylase (T2AH) activity was not affected. Terbutol 4-methylhydroxylase (T4MH) activity was increased 9-fold by 1% terbutol. In an immunoinhibition study, T4MH activity in liver microsomes from 1% terbutol-treated rats was decreased about 50% by polyclonal anti-rat CYP2B1, whereas polyclonal anti-rat CYP2A1 and CYP2C11 did not affected the activity. These results indicate that terbutol increased CYP2B subfamily in rat liver microsomes, and that the compound did not cause serious hepatic damage.     

Immunohistochemical localization of thyroid stimulating hormone induced by a low oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin in female Sprague-Dawley rats

We have investigated how a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects thyroid hormone regulation, especially in relation to the localization of thyroid stimulating hormone (TSH) in the pituitary and that of thyroxin (T4) of the thyroid in the rat. Female Sprague-Dawley rats were given a single oral administration of TCDD ranging from 1.0 to 4.0 microg/kg body weight (bw), and then tissue specimens were removed on day 7 post-administration. Thyroid hormone concentrations were measured in serum, and the expression of the TCDD-responsive genes, UDP-glucuronosyltransferase-1 (UGT1) and cytochrome P4501A1 (CYP1A1) were examined in the liver. TCDD administration resulted in an increase in both immunostaining intensity and the number of TSH-positive cells in the anterior pituitary. T4 was found to localize only in the follicular lumen of the thyroid in vehicle-treated control rats, while TCDD administration caused a foamy change in the colloid of some follicles, an indication of accelerating the biosynthesis of T4 in the thyroid. By morphometrical analysis, the ratio of parenchymal/lumenal area of the thyroid was found to increase in response to TCDD. TCDD treatment as low as 2.0 microg TCDD/kg bw induced a significant decrease in both serum total T4 (TT4) and free T4 (FT4) concentrations in the rats, with a significant increase in serum TSH levels in the 4.0 microg TCDD/kg bw rats. Serum total triiodothyronine (TT3) level was unchanged in all groups. The UGT1 gene was significantly induced at a TCDD dose as low as 1.0 microg/kg bw in a dose-dependent manner. TCDD concentrations in the serum, liver and adipose tissues were detected in a dose-related fashion. The present immunohistochemical results clearly support the earlier biochemical findings on the perturbation of the thyroid-pituitary axis by TCDD and suggest that UGT1 is an immediate target of a low TCDD exposure that triggers the perturbation.