Formation of core-shell and hollow nanospheres through the nanoscale melt-solidification effect in the Sm-Fe(Ta)-N system.

Sm-Fe-Ta-N-O nanospheres were synthesized by pulsed-laser deposition from a Sm(13.8)Fe(82.2)Ta(4.0) target in a nitrogen atmosphere. Three structurally and compositionally distinct types were identified: amorphous, core-shell and hollow nanospheres. Amorphous spheres were compositionally homogeneous and completely oxidized. The core-shell spheres were composed of an iron-rich crystalline core with up to 10 at.% interstitially incorporated nitrogen, surrounded by an amorphous and oxidized shell. The hollow spheres were characterized by voids filled with N(2) gas. It was found that the formation of either amorphous or complex nanospheres is defined by an initial Fe/Sm ratio within the molten droplet. The formation of hollow spheres is believed to be related to the general affinity of liquid metals for gas intake. During rapid solidification the dissolved gas in the melt is trapped within the surrounding solid rim, preventing the outwards diffusion of gas. As long as the amount of gas atoms in the melt is kept below its solubility limits it can be completely interstitially incorporated into the solid, thus forming crystalline Fe(N)-rich cores. If the melt contains more than an equilibrium amount of nitrogen it is possible that the gas recombines to form N(2) molecules, which are condensed inside the spheres.

Hybrid FePt/SiO2/Au nanoparticles as a theranostic tool: in vitro photo-thermal treatment and MRI imaging.

We have produced an innovative, theranostic material based on FePt/SiO2/Au hybrid nanoparticles (NPs) for both, photo-thermal therapy and magnetic resonance imaging (MRI). Furthermore, a new synthesis approach, i.e., Au double seeding, for the preparation of Au nanoshells around the FePt/SiO2 cores, is proposed. The photo-thermal and the MRI response were first demonstrated on an aqueous suspension of hybrid FePt/SiO2/Au NPs. The cytotoxicity together with the internalization mechanism and the intracellular fate of the hybrid NPs were evaluated in vitro on a normal (NPU) and a half-differentiated cancerous cell line (RT4). The control samples as well as the normal cell line incubated with the NPs showed no significant temperature increase during the in vitro photo-thermal treatment (ΔT < 0.8 °C) and thus the cell viability remained high (∼90%). In contrast, due to the high NP uptake by the cancerous RT4 cell line, significant heating of the sample was observed (ΔT = 4 °C) and, consequently, after laser irradiation the cell viability dropped significantly to ∼60%. These results further confirm that the hybrid FePt/SiO2/Au NPs developed in the scope of this work were not only efficient but also highly selective photo-thermal agents. Furthermore, the improvement in the contrast and the easier distinction between the healthy and the cancerous tissues were clearly demonstrated with in vitro MRI experiments, proving that hybrid NPs have an excellent potential to be used as contrast agents.

Magnetic interactions and in vitro study of biocompatible hydrocaffeic acid-stabilized Fe-Pt clusters as MRI contrast agents.

A detailed magnetic study of separated Fe-Pt NPs and Fe-Pt clusters was performed to predict their optimal size and morphology for the maximum saturation magnetization, a factor that is known to influence the performance of a magnetic-resonance-imaging (MRI) contrast agent. Excellent stability and biocompatibility of the nanoparticle suspension was achieved using a novel coating based on hydrocaffeic acid (HCA), which was confirmed with a detailed Fourier-transform infrared spectroscopy (FTIR) study. An in vitro study on a human-bladder papillary urothelial neoplasm RT4 cell line confirmed that HCA-Fe-Pt nanoparticles showed no cytotoxicity, even at a very high concentration (550 µg Fe-Pt per mL), with no delayed cytotoxic effect being detected. This indicates that the HCA coating provides excellent biocompatibility of the nanoparticles, which is a prerequisite for the material to be used as a safe contrast agent for MRI. The cellular uptake and internalization mechanism were studied using ICP-MS and TEM analyses. Furthermore, it was shown that even a very low concentration of Fe-Pt nanoparticles (<10 µg mL-1) in the cells is enough to decrease the T 2 relaxation times by 70%. In terms of the MRI imaging, this means a large improvement in the contrast, even at a low nanoparticle concentration and an easier visualization of the tissues containing nanoparticles, proving that HCA-coated Fe-Pt nanoparticles have the potential to be used as an efficient and safe MRI contrast agent.

Delayed acute toxicity of 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD), after oral administration, Obeys Haber's rule of inhalation toxicology.

Eight different doses (2.5 to 10.0 mg/kg) of 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD) were administered acutely to a total of 272 female Sprague-Dawley rats. The doses ranged from a NOAEL for wasting/hemorrhage to supralethal doses. Dose- and time-responses of wasting/hemorrhage, anemia, and cancer were and are being studied as end points of toxicity. The experiments will be continued until the last rat dies. There was a very steep dose- and time-response between the LOAEL for wasting/hemorrhage (2.8 mg/kg) and the third highest dose (4.1 mg/kg) of HpCDD. The dose-and time-responses were nearly symmetrical, obeying Haber's Rule of inhalation toxicology (c x t = constant) even beyond 100% mortality. Introduction of a minimum of 25% body weight loss as a discriminatory criterion to separate wasting from hemorrhage as the primary cause of death reduced variability from 5.8 to 3.2%. An arithmetic plot of the dose and time data resulted in a nearly perfect hyperbola. A logarithmic plot of these data yielded a straight line of similar perfection. Dose-response data at constant times illustrate the shifting of the dose-response curve towards a liminal value, which represents the necessary observation period for this effect. Time-response data at constant doses demonstrate the shifting of the time-response curve towards a liminal value, which represents the LOAEL for the dose-response of this effect. A three-dimensional plot of dose- and time-response data depicts the surface area on which c x t is constant along hyperbolas, in terms of wasting as the end point of toxicity. Surviving rats in all groups started developing anemia 126 days after dosing, but no rat died of wasting/hemorrhage after day 74. Rats surviving anemia began to die of lung cancer as of day 397 after dosing. Thus, although the experiment has been completed as far as dose- and time-responses of wasting/hemorrhage are concerned, it will be about another 2 years before complete dose and time responses will become available for anemia and lung cancer.

Enhanced total body clearance of heptachlor from rats by trans-stilbeneoxide.

The effect of the phase II enzyme inducer, trans-stilbeneoxide (TSO; 400 mg/kg i.p. on 4 consecutive days), on the clearance of intravenously given [14C]heptachlor (2 mg/kg) was investigated in male Sprague-Dawley rats. TSO or vehicle treatment was started 4 days before (group I), immediately after (group II), or 4 days following (group III) heptachlor injection. Rats were sacrificed 6 days after the last TSO or vehicle dosage. In all 3 groups TSO increased cumulative excretion of heptachlor derived material in feces significantly (P less than 0.01) but had no effect on total urinary excretion. Pretreatment (group I) of rats with TSO resulted in elevated fecal excretion of 14C already on the first day after dosing with heptachlor, whereas the effect of the treatment was not apparent in group II and III rats until after 2 days and reached its maximum 3 days after the start of TSO administration. At termination, tissue radioactivity was significantly (P less than 0.05) lower in fat, kidney, brain, skin, heart and muscle, but not in serum, erythrocytes (group II and III), liver (group II and III), and spleen of TSO treated rats as compared to controls. The body burden calculated from the sum of 14C remaining in tissues was reduced to about 27% of control values. The toxicity of TSO may preclude it from considerations as a useful therapeutic agent in the treatment of chlorinated cyclodiene insecticide-exposed individuals. However, this experiment further emphasizes the importance of pharmacokinetic considerations when examining interactions in the disposition of xenobiotics.

The role of time in toxicology or Haber's c x t product.

It happened exactly 100 years ago that Warren established for the first time a quantitative link between dose and time while studying the toxicity of sodium chloride in Daphnia magna (Straus). During this century many toxicologists in different contexts returned to this idea, which has become known as Haber's rule of inhalation toxicology. Most attempts to explore this relationship ended in frustration because of the supposed occurrence of exceptions. Thus, toxicologists concentrated on the quantitative relationship between dose and effect under mostly isotemporal conditions while time took a back seat and was assigned such arbitrary, semiquantitative designations as acute, subacute, subchronic and chronic. Time itself as a quantifiable variable of toxicity was seldom studied and when it was studied, it was often not under isodosic (steady state) conditions as required by theory. A recent analysis of toxicological time indicated the impact of three independent time scales (toxicokinetic, toxicodynamic, exposure frequency/duration) in toxicological studies, which interact with dose and effect to yield the enormous complexity known to every toxicologist. Based on prototypical examples when toxicokinetic (dioxins), toxicodynamic (nitrosamines, benzene) or exposure frequency (methylene chloride, chloroacetic acid, HgCl(2), CdCl(2), etc.) represent the critical time scale, the general validity of the c x t=k concept will be discussed as a starting point for a theory of toxicology. As endpoints of toxicity, (delayed) acute toxicity, blood dyscrasias and cancer will be used to illustrate the critical conditions needed to demonstrate the validity of this theory.

Dose-response and time course of hypothyroxinemia and hypoinsulinemia and characterization of insulin hypersensitivity in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats.

Male Sprague-Dawley rats were administered i.p. various doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in corn oil. At several time points thyroid stimulating hormone (TSH), total thyroxine (TT4), free thyroxine (FT4), total triiodothyronine (TT3), reverse triiodothyronine (rT3) and insulin were determined in serum using radioimmunoassays, and glucose was measured by the glucose oxidase method. TSH and TT3 were not affected by any dose at any time point of measurement. TT4 and FT4 were decreased in a somewhat dose-dependent manner by days 2 to 4 after dosing. Return of TT4 and FT4 to normal values by day 32 after TCDD dosage also occurred in a dose-dependent manner, except in rats that died later. rT3 was also decreased at each dose level early and returned to normal levels in a somewhat dose-dependent fashion. Rats in the 2 highest dose groups became hypoinsulinemic and in the highest dose group also hypoglycemic by day 8 after dosing. Serum insulin and glucose remained suppressed in non-survivors of TCDD until death ensued. In survivors, serum insulin returned to normal values by day 32 after dosing. The hypoinsulinemic state was further characterized by hypersensitivity towards insulin, i.e. injection of an otherwise non-toxic dose of insulin 3 days after administration of 125 micrograms/kg TCDD was lethal to 80% of the rats within 24 h. Insulin hypersensitivity preceded both hypoglycemia and hypoinsulinemia. These findings suggest that hypothyroxinemia and hypoinsulinemia may be part of an adaptive process whereby rats attempt to diminish the toxic insult of TCDD.

Enhanced fecal elimination of stored hexachlorobenzene from rats and rhesus monkeys by hexadecane or mineral oil.

The effect of various dietary treatments on the fecal excretion of [14C]-hexachlorobenzene (HCB) was studied in rats and rhesus monkeys. Cholestyramine and sesame oil failed to influence fecal excretion of HCB and/or metabolites. However, dietary administration of n-hexadecane (5%) increased fecal excretion of radioactivity 4-13-fold in rats and rhesus monkeys. Similarly, mineral oil in the diet (5%) of rhesus monkeys elicited a 6-9-fold increase in fecal excretion of HCB and/or metabolites. As a result of the mineral oil treatment, and enhanced depletion of HCB from blood and also of the stored HCB from adipose tissue was observed. The concentration of HCB in the blood declined in accordance with decreasing storage levels of HCB in adipose tissue. The major site of elimination of HCB and/or metabolites seemed to be the intestine; in particular, the cecum and the colon ascendens. Both hexadecane and mineral oil appeared to stimulate specifically this elimination path way.

Dose and time as variables of toxicity.

Recognizing that if there is no exposure, there is no toxicity leads us to the conclusion that if there is exposure, toxicity can ensue when exposure exceeds a certain dose and/or time and that it will be dependent on toxicokinetics and toxicodynamics. Analysis of the fundamental description of toxicity (dT/dE=dT/dDxdD/dKxdK/dE, where T stands for toxicity, D for toxicodynamics, K for toxicokinetics and E for exposure) yields the recognition of three independent time scales, the first being an intrinsic property of a given compound (what does the chemical do to the organism), which is the dynamic time scale. The second time scale is an intrinsic property of a the organism (what does the organism do to the chemical), which represents the kinetic time scale. The frequency of exposure denotes the third time scale, which is independent of the dynamic and kinetic time scales. Frequency of exposure depends on the experimental design or on nature, but not on the organism or substance. A liminal condition occurs when the frequency becomes so high that it is indistinguishable from continuous exposure. Continuous exposure forces the two other time scales to become synchronized thereby reducing complexity to three variables: dose, effect and one time scale. Keeping one of those variables constant allows for the study of the other two variables reproducibly under isoeffective or isodosic or isotemporal conditions. However, any departure from continuous exposure will introduce the full complexity of four independent variables (dose and kinetic, dynamic and frequency time scales) impacting on the effect (dependent variable) at the same time. The examples discussed in this paper demonstrate how nature in the form of long half-lives provides liminal conditions when either kinetic or dynamic half lives force synchronization of all three time scales. However, for compounds having very short dynamic or kinetic half-lives, only continuous exposure will provide a synchronized time scale. A decision tree-type approach is being used to illustrate how to reduce the enormous complexity generated by five variables (dose, effect and up to three time scales) in toxicology to manageable proportions by identifying and modeling the rate-determining (-limiting) step(s) in the manifestation of toxicity.

Paracelsus, Haber and Arndt.

After a brief overview of the contributions of Paracelsus, Haber and Arndt to the theory of toxicology, examples are provided for quantitative risk/safety assessments using dose (c), time (t) and effect (E) as macroscopic variables of toxicity. The discussion offers explanations for application of the decision tree approach in identifying rate-determining steps in the toxicity of chemicals. Having done so allows for reasonably accurate predictions of cancer incidence (bladder, liver, heart, histiocyte) using Haber's Product under isoeffective conditions and the equation cxt=kxE for isodosic and isotemporal responses.

Subchronic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and their reversibility in male Sprague-Dawley rats.

The hypothesis tested in this experiment is that effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) show identical dose-responses after subchronic as after acute exposure when the dose is corrected for toxicokinetics. Groups of male Sprague-Dawley (S-D) rats were administered orally a total dose of 0, 0.2, 2.3, 11.5, 35, 70 or 115 micrograms/kg of TCDD over a period of 10 weeks at 4 ml/kg of vehicle. Body weight was recorded weekly. One week after the last dose of TCDD one half of the rats was killed and tryptophan 2,3-dioxygenase (TdO), 7-ethoxyresorufin-O-deethylase (EROD) and phosphoenolpyruvate carboxykinase (PEPCK) activities were measured in livers, whereas tryptophan and total T4 (TT4) were determined in serum. The results show that the dose-response for decreased TdO and PEPCK activity and elevated serum tryptophan levels are similar if not the same as the dose-response for subchronic retardation of body weight increase. They also demonstrate that the dose-responses for the induction of EROD activity and the reduction of serum TT4 occurred at much lower doses than those for decreased TdO and PEPCK activities or elevated tryptophan levels and mortality. After a 6-week recovery period, PEPCK and TdO activities in liver as well as tryptophan in serum returned to near control values, whereas EROD activity and serum TT4 still displayed a dose-dependent induction and reduction, respectively, albeit both shifted to the right in accordance with toxicokinetics. These data support the notion that subchronic dose-responses of TCDD are similar to acute dose-responses when corrected for toxicokinetics and that at least some TCDD-induced effects are reversible also in accordance with toxicokinetics.

Decreased liver type I 5'-deiodinase and increased brown adipose tissue type II 5'-deiodinase activity in 2,3,7,8-tetrachlorobibenzo-p-dioxin (TCDD)-treated Long-Evans rats.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied on the activity of type I 5'-deiodinase (5' DI) in liver and of type II 5'-deiodinase (5' DII) in brown adipose tissue (BAT) microsomes of Long-Evans rats. TCDD decreased the activity of liver 5' DI and increased that of BAT 5' DII. It also reduced the concentration of serum total thyroxine (T4) without affecting triiodothyronine (T3). These effects of TCDD on 5'-deiodinases and serum thyroid hormones are compatible with partial hypothyroidism. Based on our results, it is not possible to decide unequivocally whether TCDD affects thyroid hormone status and 5'-deiodination by a direct action or a feedback mechanism. However, these data are important because they demonstrate that TCDD affects thyroid status in a tissue-specific manner and that its effects on thyroid hormone metabolism are much more complex than currently assumed.

Hexadecane enhances non-biliary, intestinal excretion of stored hexachlorobenzene by rats.

[14C]Hexachlorobenzene (100 mg/kg) was orally administered to 4 groups of rats. Ten days later the effects of hexadecane (3 x 5 ml/kg by gavage) and/or bile duct ligation on urinary and fecal excretion and tissue levels of hexachlorobenzene were examined. Hexadecane did not affect urinary excretion of hexachlorobenzene, whereas bile duct ligation tripled it. Each of the 3 treatments (n-hexadecane, bile duct ligation and the combination of the two) resulted in a significant increase in fecal excretion of hexachlorobenzene. Moreover, the combination of hexadecane and bile duct ligation produced a greater increase in fecal excretion of hexachlorobenzene than either treatment alone. Concentrations of hexachlorobenzene in blood, fat and kidney were not affected by any of the treatments, but liver concentrations were reduced significantly by bile duct ligation. Concentrations of hexachlorobenzene in intestinal contents indicate that intestinal-wall passage is the primary route of elimination from the body and that enhancement of elimination occurs mostly distal to the jejunum.

Occupational exposure limits for 30 organophosphate pesticides based on inhibition of red blood cell acetylcholinesterase.

Toxicity and other relevant data for 30 organophosphate pesticides were evaluated to suggest inhalation occupational exposure limits (OELs), and to support development of a risk assessment strategy for organophosphates in general. Specifically, the value of relative potency analysis and the predictability of inhalation OELs by acute toxicity measures and by repeated oral exposure NOELs was assessed. Suggested OELs are based on the prevention of red blood cell (RBC) acetylcholinesterase (AChE) inhibition and are derived using a weight-of-evidence risk assessment approach. Suggested OEL values range from 0.002 to 2 mg/m(3), and in most cases, are less than current permissible exposure levels (PELs) or threshold limit values(R) (TLVs(R)). The available data indicate that experimental data for most organophosphates evaluated are limited; most organophosphates are equally potent RBC AChE inhibitors in different mammalian species; NOELs from repeated exposure studies of variable duration are usually equivalent; and, no particular grouping based on organophosphate structure is consistently more potent than another. Further, relative potency analyses have limited usefulness in the risk assessment of organophosphates. The data also indicated that equivalent relative potency relationships do not exist across either exposure duration (acute vs. repeated) or exposure route (oral vs. inhalation). Consideration of all variable duration and exposure route studies are therefore usually desirable in the development of an OEL, especially when data are limited. Also, neither acute measures of toxicity nor repeated oral exposure NOELs are predictive of weight-of-evidence based inhalation OELs. These deviations from what is expected based on the common mechanism of action for organophosphates across exposure duration and route - AChE inhibition - is likely due to the lack of synchrony between the timing of target tissue effective dose and the experimental observation of equivalent response. Thus, comprehensive interpretation of all toxicity data in the context of available toxicokinetic, toxicodynamic and exposure information for each individual organophosphate in a weight-of-evidence based risk assessment is desirable when deriving inhalation OELs.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on humoral and cell-mediated immunity in Sprague-Dawley rats.

There is much discussion about the occurrence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced effects on the human immune system. Extensive studies have been conducted in mice, but those results cannot explain some of the epidemiological data obtained in exposed humans. Therefore, studies in other laboratory animal species are needed. The aim of these experiments was to examine effects of TCDD on cell- and humoral-mediated immunity in male Sprague-Dawley (SD) rats. A delayed-type hypersensitivity (DTH) assay was used to examine cell-mediated immunity. A time-course study demonstrated that TCDD treatment on day -5 relative to immunization (day 0) produced the greatest effect on cell-mediated immunity. In a dose-response experiment, rats were treated with 1, 3, 10, 20, 30, 40 and 90 micrograms TCDD/kg The effect of TCDD on cell-mediated immunity displayed an inverted U-shaped dose-response curve, in that low doses enhanced and high doses suppressed this immune function. This is the first study to demonstrate an U-shaped dose-response curve of TCDD on the immune system. Primary antibody response to sheep red blood cells (SRBC) was used as endpoint to study the effect of TCDD on humoral immunity. Serum anti-SRBC IgM and IgG levels were measured using an enzyme-linked immunosorbent assay (ELISA). In the dose range examined (10, 20 and 40 micrograms TCDD/kg), serum IgM levels were not affected by TCDD compared to controls at 7 and 14 days after immunization. In contrast, serum IgG levels were dose-dependently elevated both 7 and 14 days after immunization, with a maximum increase of 59% over controls.

Reduced activity of tryptophan 2,3-dioxygenase in the liver of rats treated with chlorinated dibenzo-p-dioxins (CDDs): dose-responses and structure-activity relationship.

The activity of tryptophan 2,3-dioxygenase (TdO) was measured in the livers of male Sprague-Dawley rats after acutely toxic doses (LD20-LD80) of chlorinated dibenzo-p-dioxins (CDDs) with 4 of the up to 7 chlorine substituents occupying the 2,3,7,8-positions. Treatment with toxic doses of CDDs results in voluntary feed refusal of rats. A corresponding involuntary reduction of feed intake in naive animals (pair-feeding) causes elevated levels of TdO activity. In the CDD treated rats, however, TdO activities were dose-dependently reduced. An LD80 reduced TdO activity to about 50% of the level found in the corresponding pair-fed animals. This decrease of TdO activity explains the dose-dependent increase of serum tryptophan, which in turn is the likely cause of voluntary feed refusal observed in CDD-treated rats. The activity of another enzyme which is regulated in a fashion very similar to that of TdO, viz., tyrosine aminotransferase (TAT), was consistently, but not dose-dependently, affected by treatment with CDDs.

Cytokines (IL-1beta and TNFalpha) in relation to biochemical and immunological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats.

Previous studies in different strains of rats and mice have shown that the inhibition of gluconeogenesis as a result of reduced liver phosphoenolpyruvate carboxykinase (PEPCK) activity together with appetite suppression play critical roles in the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Recent immunological studies in rats demonstrated that exposure to low doses of TCDD resulted in an early and enhanced IgG response to immunization with sheep red blood cells (SRBC) and an enhanced delayed-type hypersensitivity (DTH) reaction as well as a positive popliteal lymph node (PLN) response. However, high doses of TCDD suppressed the DTH reaction. This study aimed at examining the involvement of cytokines (IL-1 and TNF) in mediating the above effects. Liver samples from a previous dose-response study on DTH reaction were investigated, in which rats were treated with TCDD (1, 3, 10, 30 and 90 microg/kg) and immunized with an antigen. mRNA levels of IL-1beta were elevated begining at the 1 microg/kg (non-lethal) dosage group with a maximum increase of about 5-fold above controls in the 90 microg/kg (lethal) dosage group. mRNA levels of TNFalpha were also significantly elevated begining at the 30 microg/kg dosage group. These results suggest that at low doses of TCDD, increased IL-1beta could be responsible for immune function stimulation, whereas at high doses of TCDD, greatly elevated TNFalpha and IL-1beta levles may exacerbate or mediate acute toxicity including immune suppression and related biochemical effects. A time course study (60 microg TCDD/kg without immunization) revealed that liver mRNA levels of TNFalpha were significantly elevated starting 24 h, and reaching a maximum 48 h after dosing with TCDD. This change was accompanied by a transient increase of mRNA levels of IL-1beta at day 4 after TCDD dosage. Thus, these data demonstrated that TCDD alone (without immunization) can cause transient increases of mRNA levels of TNFalpha and IL-1beta in liver. Results from these experiments suggest that TCDD-induced cytokine changes may play important roles in various effects of TCDD.

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.

Gonadotropin-releasing hormone (GnRH) partially reverses the inhibitory effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on ovulation in the immature gonadotropin-treated rat.

Several studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has inhibitory effects on ovulation. This action may be the result of either direct effect(s) of TCDD on ovarian function or via altered secretion of pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) which regulate ovarian follicular development and ovulation. To further evaluate the effects of TCDD on pituitary gonadotropins and their regulation, the potential role of gonadotropin-releasing hormone (GnRH) was investigated in the current study. Immature (23-day-old) female Sprague-Dawley rats were dosed with TCDD (32 microg/kg) in corn oil or vehicle alone. Equine chorionic gonadotropin (eCG) was injected subcutaneously (5 IU, sc) 24 h later to induce follicular development. Immediately prior to the expected time of the LH/FSH surges, 54 h after eCG injection, half of TCDD- or corn oil-treated rats were injected with GnRH (2 microg/rat, sc). Blood and ovaries were collected at 54, 56, 58, 60 and 72 h after eCG. Serum concentrations of 17beta-estradiol (E(2)), progesterone (P(4)), LH, and FSH were determined by radioimmunoassay. An indication of ovulation rate was assessed at 72 h after injection of eCG by irrigating the ova from oviducts. TCDD reduced the number of ova in the oviducts by 70-80% (2-3 ova/rat) and this was confirmed by the number of corpora lutea. GnRH partially restored ovulation (6-7 ova/rat) in TCDD-treated rats without reversing its effect on ovarian weight reduction. In controls, the LH and FSH surges at 58 h after eCG were significantly reduced at that time in TCDD-treated rats. However, in rats treated with TCDD and GnRH, a huge LH/FSH surges occurred at 56 h after eCG injection. GnRH alone enhanced E(2) and P(4) serum levels at 56-58 h after eCG injection. In rats treated with both TCDD and GnRH, E(2) secretion was significantly lower at 58, 60, and 72 h when compared with GnRH alone, whereas serum P(4) was only decreased at 72 h after eCG injection. The results indicate that exogenous GnRH induces LH and FSH surges in TCDD-treated rats, but only partially restores the inhibitory effects of TCDD on ovulation.

Reduction of hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is due to decreased mRNA levels.

We have previously shown that the rate of hepatic gluconeogenesis is reduced in TCDD-treated rats and that this decrease in carbohydrate production is associated with a dose-dependent reduction of the activity of PEPCK, the rate limiting enzyme of gluconeogenesis. This derailment of glucose metabolism has been suggested to be the critical lesion in acute TCDD toxicity. To further elucidate the mechanism of decreased PEPCK activity we performed Northern blot analyses using a cDNA probe complementary to a portion of the mRNA coding for PEPCK. We have demonstrated that 4 and 8 days after TCDD treatment (125 micrograms/kg, p.o.) liver PEPCK mRNA in Sprague-Dawley rats was decreased to very low levels as compared to vehicle-treated and pair-fed control animals. This decline of PEPCK mRNA was paralleled by decreased levels of PEPCK protein, as revealed by Western blot analyses and was accompanied by a reduction in the enzymatic activity of PEPCK. These results indicate that the decrease of PEPCK activity by TCDD is most likely the result of decreased expression of the PEPCK gene. These together with previous results also suggest that many of the physiological responses occurring in TCDD-treated animals (reduced feed intake, decreased insulin, increased corticosterone, increased glucagon and cAMP levels) which would normally stimulate PEPCK gene expression, are ineffective. Furthermore tryptophan 2,3-dioxygenase (TdO) activity, which is regulated in a very similar fashion to PEPCK activity, is also reduced after TCDD treatment, suggesting a common mechanism by which TCDD alters the regulation of these enzymes. P-450 1A1 mRNA and related EROD activity were maximally induced under the conditions of these experiments and represent a positive control for TCDD-related alterations of gene expression. However, because of differences in the dose-response characteristics of TCDD-induced reduction of PEPCK activity and induction of EROD activity an involvement of the Ah receptor in the reduction of PEPCK activity cannot be postulated.