Pharmacokinetics and biological activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Wistar rats were treated initially with very high single doses of ¹⁴C-2,3,7,8-TCDD (either 75 or 25 g/kg body wt) followed by weekly maintenance doses of 15 and 5 g/kg body wt, respectively. ¹⁴C-radioactivity was measured in various organs over a period of 22 weeks. 1) 75 g TCDD/kg body wt (followed by the maintenance doses) was lethal for all the rats within a period of 9 weeks. While the concentration of ¹⁴C-TCDD equivalents in liver and thymus stayed reasonably constant during this period in the surviving rats, the concentration in adipose tissue and kidneys clearly increased in the same animals. 2) The dose of 25 g TCDD/kg body wt (followed by weekly doses of 5 g/kg body wt) proved to be a just tolerable dose over a period of 22 weeks for our strain of rats. 3) Within the individual variabilities the TCDD concentrations in the investigated organs showed no clear-cut decline, indicating that the animals were exposed to fairly constant levels of TCDD throughout the study. Thus, this dosing regime is suitable for maintaining constant TCDD exposure during long-term studies.Abbreviations TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin     

Polyhalogenated dibenzo-p-dioxins and dibenzofurans and the immune system. 1. Effects on peripheral lymphocyte subpopulations of a non-human primate (Callithrix jacchus) after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Monoclonal antibodies were used to analyse the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on peripheral lymphocytes from marmosets (Callithrix jacchus) following injections of single low doses of TCDD. A reduction of the percentage and the total number of lymphocytes among the white blood cells was seen 3 weeks after a single administration of 300 ng TCDD/kg body wt, but not following 167 ng TCDD/kg or less. After treatment of marmosets with the single subcutaneous dose of 10 ng TCDD/kg body wt, we observed an average decrease of about 20% in the percentage of CD4⁺ cells in the venous blood of treated animals. This change was not seen at the time of maximum absorption (2 weeks after the injection), but was demonstrable after 4 weeks and reached its maximum 15 weeks after the injection. The time course of the changes suggests an indirect effect (possibly via the thymus). Due to the considerable inter- and intra-individual variability in the number of peripheral lymphocytes the effect was less convincing on the basis of the total CD4⁺ cells per microliter blood. There was a significant concomitant increase in the percentage of cells with the CD8⁺ marker. A closer analysis of the lymphocyte subpopulation involved revealed a predominant effect on the cells with the CD4CDw29 (leu 3a⁺4B4⁺) surface marker (helperinducer cells). Subsequent to a dose of 10 ng TCDD/kg body wt the percentage of these cells was reduced by 50% or more when compared with the (24) controls. On an absolute basis (number of cells/l blood) these CD4⁺CDw29⁺ cells were clearly reduced in only two out of four marmosets. There was no significant effect on the percentage of the CD4⁺CD45R⁺ (leu3a⁺2H4⁺) subpopulation (suppressor-inducer cells). The ratios: CD4⁺CDw29⁺/CD4⁺CD45R⁺, or CD4⁺CDw29⁺/CD8⁺ appear to be convenient measures for monitoring the effect described. Since the difference between the percentage of CD2⁺ cells minus the sum of CD4⁺ plus CD8⁺ cells was found to be increased following rather high doses (167 ng TCDD/kg body wt or more), this suggests that immature cells (CD2⁺ CD4-CD8-) are released into the periphery as a result of the exposure to TCDD. Furthermore, a decrease in the percentage of CD20⁺ (B₁ ⁺) cells (about 50% when compared with controls) was observed in the treated animals following a single dose of 10 ng TCDD/kg body wt or higher. Subsequent to a dose of 10 ng TCDD/kg body wt the percentage of CD56⁺ (NKH1⁺) cells seemed to be slightly increased. The dose-response for the effects observed was rather poor. At present it cannot be decided whether the changes have to be considered as adverse health effects. All of the animals involved were apparently healthy and did not exhibit, e.g. any infections. However, the deviations described certainly represent biologicaleffects induced at very low dose levels. From the data available up till now, a single dose of 10 ng TCDD/kg body wt seems to be the lowest-observed-effect-level (LOEL) in the marmoset for all the effects studied. More extensive experiments within the dose range between 1 and 10 ng TCDD/kg body wt are under way in our laboratory. When peripheral lymphocytes of TCDD-treated animals were incubated in vitro with a mitogen (PWM = poke weed mitogen) an exaggeration of thedecrease in the percent of cells with the CD4 surface marker and a concomitant increase in the percentage of CD8⁺ cells was observed. This seems to be an especially sensitive experimental approach for demonstrating biological effects (but not necessarily adverse health effects) of this class of substances. Such an effect was demonstrated and found to be statistically significant already 2 weeks after a single injection of 10 ng TCDD/kg body wt in comparison to vehicle-treated controls.     

Polyhalogenated dibenzo-p-dioxins and dibenzofurans and the immune system. 4. Effects of multiple-dose treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on peripheral lymphocyte subpopulations of a non-human primate (Callithrix jacchus)

Non-human primates (Callithrix jacchus) were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) over a period of 30 weeks, and lymphocyte subpopulations of venous blood were monitored using monoclonal antibodies and flow cytometry (FACScan). There was no clear-cut change in the total lymphocyte population during this study. In the first part of the study the new-world monkeys (marmosets) were treated for 24 weeks with a weekly dose of 0.3 ng TCDD/kg body wt. At the end of this treatment period a level corresponding to an actual cumulative dose of about 2.5–2.7 ng TCDD/kg body wt was expected. The percentage and the absolute number of the CD4⁺CDw29⁺ cells (helper inducer or memory cells) surmounted the physiologically occurring increase. Concomitantly the percentage of the CD4⁺CD45RA⁺ cells (suppressor-inducer or naive cells) decreased. There was, at the same period, no change in the total T cell population (CD2⁺ cells) or in the cells carrying the CD8 or the CD4 epitope. When increasing the weekly dose to 1.5 ng TCDD/kg body wt, a transient increase in the percentage and the absolute number of the CD8⁺CD56⁺ cell population (cytotoxic T cells) was observed 3 weeks after the increase in dosing. At this time the expected decrease in the percentage or the absolute number of CD4⁺CDw29⁺ cells was just detectable and this decline was at its maximum 6 weeks after switching to the higher weekly doses. The reduction in the percentage and the absolute number of CD4⁺CDw29⁺ cells persisted 5 weeks after discontinuation of the dosing, but this cell population was again within normal limits 7 weeks later. Because the two subpopulations are changed in opposite directions, the ratio CD4⁺CDw29⁺/CD4⁺CD45RA⁺ is a very sensitive measure of the effect induced by TCDD. There was a pronounced decrease in the percentage of the CD20⁺ cells (B₁ cells), but their percentage and number rapidly normalized, in contrast to the CD4⁺CDw29⁺ cells, when the dosing was discontinued. At the end of the treatment period the apparent body burden was calculated to correspondt an actual dose of about 9–10 ng TCDD/kg body wt. Such an actual dose level might be assumed to be reached under steady-state conditions in chronic experiments with daily doses of about 135 pg TCDD/kg body wt (assuming a half-life for TCDD in the marmoset of 6–8 weeks). Er trapolations of the results obtained at higher doses to very low exposures is not justified with respect to the effect induced by TCDD on the immune system of marmosets. A lower doses the effect is clearly reversed.     

Reproductive toxicity and toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Possible effects on the next generation after long-term exposure (subcutaneous administration) of male rats to very high doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied. Two dose regimes were applied: TCDD-25 (initial dose: 25 g/kg body wt; maintenance dose: 5 g/kg body wt, once weekly) and TCDD-75 (initial dose: 75 g/kg body wt; maintenance dose: 15 g/kg body wt). Male rats were treated for 10 weeks before mating and then throughout the entire 12 week mating period. They were mated to unexposed virgin females. One group of pregnant females was used for teratological evaluations, and another group was allowed to deliver. No significant differences were observed in the number of implantations or fetuses per litter, and resorption rate, and fetal weight between the controls and TCDD-treated groups. No gross-structural anomalies occurred in any of the fetuses sired by TCDD-treated males. In the TCDD-25 group an increased frequency of two types of variations was observed which also occur in controls: incompletely ossified fingers (TCDD-25=5.1%, controls=2.6%), and incompletely ossified ossa zygomatica (TCDD-25=1.8%, controls=0.5%). In the TCDD-25 group a slight but statistically significant increase was observed in the rate of stillbirths (TCDD-25=1.3%, controls=0.1%), apparently due to an unusually low frequency occurring in the controls (overall historical controls=0.6%). There was no difference in postnatal mortality (TCDD-25=1.3%, controls=1.3%). Taken together, despite the very high doses of TCDD used, the data do not provide evidence for biologically significant paternally-mediated developmental toxicity in the fetuses and newborn.     

Pharmacokinetics and biological activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat liver and adipose tissue, and hepatic ethoxyresorufin O-deethylase (EROD) activity were studied subsequent to a single subcutaneous injection of TCDD. Two types of experiments were performed to study: (a) time-dependent changes following a single injection of 300 ng TCDD/kg body wt (points 1-4), and (b) dose-dependent changes measurable after 7 days following a single injection (points 5-7). 1. Absorption of TCDD following a single subcutaneous injection was about 90% after 3 days and 98% after 5 days. 2. Following a single dose of 300 ng TCDD/kg body wt peak concentrations were: liver (after 3 days): 4.7 +/- 0.9 ng/g wet wt, and adipose tissue (after 7 days): 0.82 +/- 0.07 ng/g wet wt. 3. T1/2 of TCDD in liver was 13.6 days over the total experimental period (from day 10 to 91 of the study), apparently with an initial faster phase: 11.5 days (from day 10 to 49), and a slower period at the end of the experiment: 16.9 days (from day 49 to 91); in adipose tissue the t1/2 was 24.5 days (from day 14 to 91 of the study). 4. Maximum induction of EROD in the liver was observed (14-fold at 300 ng TCDD/kg body wt) 3-7 days following the injection; the activity was decreased to about one third of the maximum 3 weeks after the injection; increase in total cytochrome P-450 at this dose was only about 1.4-fold at the induction maximum. 5. The ratio of the TCDD concentrations in liver and adipose tissue increased considerably between doses of 3 ng TCDD/kg body wt (ratio: about 0.74) and 3000 ng TCDD/kg body wt (ratio: about 7.7). 6. The extent of EROD induction in the liver increased dose dependently. A significant effect was first observed with a dose of 3 ng TCDD/kg body wt (activity about +32% above control activity). The corresponding tissue concentration was about 10 pg TCDD/g liver wet wt. 7. An almost perfect linear relationship exists (when using a double-log plot) between the hepatic TCDD concentration and the EROD activity for tissue concentrations ranging from 40 to 30,000 pg TCDD/g wet wt.     

Reproductive toxicity and toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin

The effects of a single dose of TCDD on the testis were studied in rats. The animals were treated (subcutaneously) once with TCDD doses of 0, 0.5, 1.0, 3.0, 5.0 g/kg body weight. Doses of 3.0 or 5.0 g TCDD/kg reduced the number of spermatids/testis significantly (60% of the controls). Electron microscopic inspection revealed that both doses led to a dissolution on the germinal epithelium. Altered germ cells at all developmental stages occurred in all testes evaluated. Doses of 0.5 or 1.0 g TCDD/kg did not induce any effects in the testis; therefore, under these experimental conditions of single exposure to rats the dose of 1.0 g TCDD/kg can be considered as NOAEL.     

Reproductive Toxicity and Tissue Concentrations of Low Doses of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin in Male Offspring Rats Exposed Throughout Pregnancy and Lactation

The effects of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the reproductive system of male offspring rats were examined. The dams were treated subcutaneously 2 weeks prior to mating and throughout mating, pregnancy, and lactation. They received an initial loading dose of 25, 60, or 300 ng TCDD/kg body wt, followed by a weekly maintenance dose of 5, 12, or 60 ng TCCD/kg body wt (TCDD 25/5, TCDD 60/12, and TCDD 300/60). Three dams per group were killed on Gestation Day 21 and the fetuses were removed. The concentration of TCDD in the maternal liver and fat was measured. After birth, developmental landmarks in male rats were monitored. At weaning, the concentration of TCDD in the offspring liver and testis was determined. Effects on male reproduction were studied on Postnatal Days (PND) 70 and 170. At weaning, the concentration of TCDD in the offspring liver was 0.24, 0.39, and 1.78 ng/g in the TCDD 25/5, TCDD 60/12, and TCDD 300/60 groups, respectively. In the testes, the concentration of TCDD was 0.25 ng/g in the TCDD 25/5 and TCDD 60/12 groups and 0.28 ng/g in the TCDD 300/60 group. The number of sperm per cauda epididymis was reduced in TCDD groups at puberty and at adulthood. Daily sperm production was permanently decreased as was the sperm transit rate in the TCDD-exposed male rats, thus increasing the time required by the sperm to pass through the cauda epididymis. Moreover, the male rats of the TCDD groups showed an increased number of abnormal sperm when investigated at adulthood. Similarly, mounting and intromission latencies were significantly increased in the TCDD 25/5 and TCDD 300/60 groups. In the highest dose group, serum testosterone concentration was decreased at adulthood. Likewise, in this dose group permanent changes including pyknotic nuclei and the occurrence of cell debris in the lumen were revealed. The lowest adverse effect level and the no observed effect level can be estimated to be substantially lower than the estimated daily dose of the lowest dose which is 0.8 ng/kg body wt/day. Sperm parameters were more susceptible than the other end points investigated. However, the question as to whether such doses exposed throughout gestation and lactation induce subtle changes in humans remains to be determined.     

Reproductive toxicity and pharmacokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin

A study on the reproductive toxicity of ¹⁴C-TCDD in male rats was performed. Two dose regimes were applied subcutaneously: TCDD-25 (initial dose: 25 g/kg body wt; maintenance dose: 5 g/kg body wt) and TCDD-75 (initial dose: 75 g/kg body wt; maintenance dose: 15 g/kg body wt); the maintenance dose was administered once weekly. The rats were treated for 10 weeks before they were mated and throughout the entire mating period. The dose regime TCDD-75 led to a mortality rate of 93% within a period of 16 weeks. The first animals died during 4 weeks, and an LD₅₀ was reached after 8 weeks. The dose regime TCDD-25 did not cause any mortality over a period of 12 weeks; but an LD₁₀ was reached within 13–20 weeks. The body weight was significantly decreased in both groups treated with TCDD after 1 week of treatment. It stabilized in the TCDD-25-group 4 weeks after treatment and stayed at this level until the end of the treatment period. The most significant finding is the delayed fertilization by the treated males; 15% of the males were found to be sterile. The mating index (84%) and fertility index (14 ± 11 days) of the TCDD-25-group were lower when compared with controls (95%, 8 ± 5), but the pregnancy index was not reduced. Application of the chosen TCDD doses led to clear-cut morphological changes of the testes. The Sertoli cells were changed (increased occurrence of vacuoles, swelling of endoplasmatic cavities), and the contact between the Sertoli cells and spermatogonia was disturbed, which might indicate an inhibited maturation of spermatozoa percursors.Abbreviations TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin     

Peri- and postnatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin: effects on physiological development,reflexes, locomotor activity and learning behaviour in Wistar rats

 Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the development of rat offspring were studied after administration of a loading dose of 300 or 1000 ng TCDD/kg body wt on day 19 of pregnancy, followed by weekly maintenance doses of 120 or 400 ng TCDD/kg body wt. The dose regimens led to a fluctuation of average TCDD concentrations in the liver of the offspring of 4.9–14.9 ng/g (TCDD1000/400 group) or 1.4–6.3 ng/g (TCDD300/120 group) during the course of the experiment. In both TCDD-exposed groups the body weight of the offspring was significantly lower on postnatal day 7 (PND 7); in the high dose group from PND 7 to PND 31. Some landmarks of postnatal development were retarded in the exposed groups; in particular, the vaginal opening was delayed for several days in both TCDD-exposed groups. The TCDD-exposed animals revealed a reduced ability to remain on a rotating rod. During reflex testing, the rate of successfully responding animals was higher in the exposed groups. No statistically significant differences in the locomotor activity between controls and TCDD-exposed offspring were detectable under our experimental conditions. In a discrimination learning test no effects on the learning ability were found. However, TCDD-exposed offspring showed an increase in unanswered trials during critical phases of the task. They also exhibited increased locomotor activity in a novel environment; prior to an amphetamine challenge dose of 1 mg/kg body weight. Amphetamine-induced activity was decreased in a dose-dependent manner. Received: 2 May 1994 / Accepted: 13 July 1994     

The effect of L-cysteine on the portion-selective uptake of cadmium in the renal proximal tubule

Cadmium (Cd), co-administered with an excess of L-cysteine, accumulates rapidly in the kidneys of the rat. After subcutaneous (s. c.) injection of 3 mol CdCl₂/kg body wt the concentrations of Cd in the blood and kidneys increase with the dose of cysteine over the range 0.06–5.0 mmol/kg body wt. At cysteine doses of less than 1.5 mmol/kg body wt the ratio of the concentrations of Cd in the outer medulla and cortex of the kidney remains the same as that after the injection of Cd alone. This ratio, however, is more than doubled at dose levels of 5–10 mmol cysteine/kg body wt. Hepatic uptake of Cd is unaffected by doses of cysteine below 1.5 mmol/kg body wt but decreases markedly at higher doses. In animals that are dosed simultaneously with 5 mmol cysteine/kg body wt, renal uptake of ¹⁰⁹Cd is known to occur in the straight segments of the proximal tubules. At a dose level of less than 1.5 mmol cysteine/kg body wt the present autoradiographical studies show that ¹⁰⁹Cd is taken up predominantly by the proximal convoluted tubules of the kidney cortex. At the critical dose level (1.5 mmol/kg body wt), cysteine decreases the retention of Cd at the s. c. injection site, but probably has little effect on the distribution of Cd between protein and other carrier molecules in the blood. This distribution, however, is altered at higher cysteine dose levels. It is suggested that, under the latter conditions, stable Cd-cysteine complexes are formed in the blood and are filtered readily through the glomeruli. These complexes are taken up in the kidney at the sites of cysteine reabsorption which, by studies with L-[³⁵S]-cysteine, are identified as the straight segments of the proximal tubules.     

Genetically mediated induction of aryl hydrocarbon hydroxylase activity in human lymphoblastoid cells by polychlorinated dibenzofuran isomers and 2,3,7,8-tetrachlorodibenzo-p-dioxin

Aryl hydrocarbon hydroxylase(AHH)-inducing potency of toxic polychlorinated aromatic hydrocarbons such as polychlorinated dibenzofuran (PCDF) isomers, 3,4,5,3,4,5-hexachlorobiphenyl (HCB) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in human lymphoblastoid cell lines with different AHH inducibility for 3-methylcholanthrene (3-MC) obtained from healthy subjects. Each of the cell lines was treated with eitht individual PCDF isomers, TCDD, and HCB at doses of 1.9–15 ng/ml of culture medium, 1.9–7.5 ng/ml and 95 ng/ml, respectively. Lymphoblastoid cell lines were arbitrarily classified into three groups based on their AHH inducibilities with 3-MC (2.5 M); low (3-MC/ control=I<3), middle (3<=I<6) and high (I>=6). Degrees of the enzyme inducibilities of the organochlorine compounds proportionally increased with those for 3-MC. AHH inducibilities with 2,3,4,7,8-pentachlorodibenzofuran(2,3,4,7,8-PCDF), 1,2,3,4,6,7-hexachlorodibenzofuran(1,2,3.4,6,7-HCDF) and 1,2,3,4,7,8-hexachlorodibenzofuran(1,2,3,4,7,8-HCDF) were comparable to those of TCDD at doses of 7.5 ng/ ml, and about twice as high as those of 2,3,7,8-tetrachlorodibenzofuran (TCDF), at the same dose, HCB, at a dose of 95 ng/ ml, did not induce enzyme activity. The experimental evidence indicated that AHH inducibility by the organochlorine compounds reflected the genetic susceptibility of the cells to the phenomenon of induction, and PCDF isomers found at relatively high concentrations in tissues of mammals exerted the highest values of AHH induction.Part of this work was presented at the 53rd annual meeting of the Japanese Society for Hygiene, April 5–7, 1983, Osaka, Japan     

Effects of vitamin E on reactive oxygen species-mediated 2,3,7,8-tetrachlorodi-benzo-p-dioxin toxicity in rat testis

This study was undertaken to investigate whether treatment with vitamin E protects rat testis from oxidative stress induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Male rats of Wistar strain were administered TCDD at doses of 1, 10 and 100 ng kg(-1) body wt. day(-1) for 45 days. Other groups of animals were co-administered TCDD (1, 10 and 100 ng kg(-1) body wt. day(-1)) and vitamin E (20 mg kg(-1) body wt. day(-1)) for 45 days. Animals administered TCDD and those co-administered TCDD and vitamin E did not show any significant change in body weight. Administration of TCDD decreased the weights of the testis, epididymis, seminal vesicles and ventral prostate. The daily sperm production decreased in the animals administered TCDD from the control values of 22.19 +/- 2.67 to 13.10 +/- 3.16 x 10(6). There was a significant decline in the activities of superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase with concomitant increased levels of hydrogen peroxide and lipid peroxidation. Co-administration of TCDD and vitamin E did not show any significant changes in the weights of the testis, epididymis, seminal vesicles and ventral prostate. The daily sperm production remained unchanged in the animals co-administered TCDD and vitamin E. The activities of antioxidant enzymes and the levels of hydrogen peroxide and lipid peroxidation did not change in the animals co-administered TCDD and vitamin E. The results suggested that administration of TCDD induces oxidative stress in testis, and vitamin E could impart a protective effect against TCDD-induced oxidative stress.     

Dose-related effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in C57BL/6J and DBA/2J mice

The dose-related effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied in B6D2F1/J (B6D), C57BL/6J (C57), and DBA/2J (DBA) mice. A 14-fold difference in lethality was observed in C57 and DBA mice, based upon 30-day LD50 values of 182 and 2570 micrograms TCDD/kg body wt, respectively. The 30-day LD50 for B6D mice was 296 micrograms TCDD/kg body wt. A progressive loss of body weight in all strains of mice was observed during the 30-day LD50 studies, with maximal weight losses of 24.7, 34.0, and 33.4% prior to death of C57, B6D, and DBA mice, respectively. In separate experiments, it was found that decreased feed consumption did not contribute to weight loss in C57 mice exposed to lethal or sublethal doses of TCDD until the animals were moribund. Time-course studies in C57 mice treated with 200 micrograms TCDD/kg body wt indicated that decreases in serum glucose and triglyceride concentrations and increases in hepatic triglyceride content occurred within 4 to 8 days of exposure, and were maximally altered within 17 to 21 days postexposure, concomitant with a 25% body weight loss. C57 mice fasted for 24 to 96 hr lost 18% of body weight and also exhibited alterations in glucose and lipid parameters; however, these changes were substantially different than the effects of TCDD exposure. In concert, these observations demonstrate that decreased feed consumption (hypophagia) does not account for weight loss and changes in carbohydrate and lipid metabolism in TCDD-treated C57 mice. Dose-response experiments resulted in comparable changes in glucose and lipid parameters when DBA mice were exposed to 10-fold higher doses of TCDD than C57 mice. Parallel LD50 responses and parallel changes in carbohydrate and lipid metabolism, at 10- to 15-fold differences in dose range, are indicative of a common mechanism of toxicity in TCDD-treated C57 and DBA mice.     

Relative Sensitivities of 2,3,7,8-Tetrachlorodibenzo-p-dioxin-lnduced Cypla-1 and Cypla-2 Gene Expression and Immunotoxicity in Female B6C3F1 Mice

Improvements in risk assessment require better linkage of exposureto response by the determination of target tissue dose. Therelative sensitivity of several responses in female B6C3F1 micewas compared on the basis of administered and target tissuedose spanning 3 orders of magnitude. Twenty-four hours afteradministration, [³H]TCDD was detected in the heart, spleen,kidney, uterus, thymus, lung, and liver, and the highest concentrationswere noted in the liver, uterus, and lung. At doses from 5 to25 ng/kg, hepatic [³H]TCDD levels associated with the cytosolicand nuclear subcellular fractions increased from 12 to 62% ofthe total liver levels and then decreased at higher doses. Atthe two lowest doses used in the enzyme induction study, 5 and10 ng/kg, the levels of specifically bound nuclear Ah receptorcomplex liganded with [³H]TCDD were 2.3 and 2.5 fmol/mg protein.Slightly higher levels of nuclear Ah receptor complex were observedat doses between 25 and 100 ng/kg (i.e., 3.6 to 4.2 fmol/mgprotein) and a steep dose-dependent increase in nuclear Ah receptorlevels was noted at doses of 500, 1000, and 5000 ng/kg (8.0,39.3, and 92.8 fmol/mg protein, respectively). The dose-dependenteffects of [³H]TCDD on hepatic Cypla-1 and Cypla-2 mRNA levels,ethoxyresorufin O-deethylase (EROD) activity, and the splenicantibody plaqueforming cell (PFC) response to sheep red bloodcells were also determined; the latter response was determined9 days after administration of TCDD. Statistically significantinduction of hepatic Cypla-1 was observed at lower doses (25ng/kg) than any other marker, followed by induction of ERODand PFCs expressed per spleen or per 10⁶ cells which was observedat 100 ng TCDD/kg and at higher doses. Cypla-2 was elevatedsignificantly relative to control at doses <1000 ng/kg. TheED50 value for PFCs/10 cells was the lowest of the variablesanalyzed and was not statistically significantly different fromcontrol (91 ± 92 ng/kg). A 50% increase in Cypla-2 andCypla-1 mRNA levels was observed at doses of 736 ± 132and 1630 ± 431 ng/kg, respectively. Due to variabilityin response in PFCs/spleen and the submaximal induction of ERODactivity, ED50 values could not be calculated for these responses.The analyses indicate that the immunosuppressive response (whennormalized for the number of spleen cells) may be depressedby administered doses as low as 90 ng TCDD/kg body weight. A50% increase in Cypla-1 or Cypla-2 was observed at higher administereddoses (1630 or 736 ng/kg, respectively). This suggests thatthe immunosuppressive response is depressed at lower doses ofTCDD than the other variables studied.     

Drug level of d- and l-amphetamine during intravenous self-administration

Rats were allowed to self-administer dextro and levo isomers of amphetamine in doses of 0.25, 0.50, 0.75 and 1.0 mg/kg/injection for 6 h/day. Total body level of drug was calculated at the time of responding for each drug injection. Body level of amphetamine initially increased and then decreased (0–2 h), and thereafter remained relatively constant for the remainder of the experimental session (2–6 h). During 2–6 h of self-administration, calculated whole body levels of both d- and l-amphetamine remained relatively constant across injection doses. In another study, blood was removed several times during 2–6 h at the time of responding for drug injection. Again, no difference in blood level of ¹⁴C-amphetamine was found across a range of injection doses. Mean blood levels were 0.48 g/ml for l-amphetamine and 0.18 g/ml for d-amphetamine. Drug intake averaged 2.0 mg/kg/h for l-amphetamine and 0.79 mg/kg/h for d-amphetamine.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibenzofuran (TCDF) in pregnant C57BL/6N mice: distribution to the embryo and excretion

The distribution and excretion of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlordibenzofuran (TCDF) were studied in pregnant C57BL/6N mice following an oral dose of 30 g/kg ¹⁴C-TCDD and 800 g/kg ¹⁴C-TCDF on gestation day 11. The distribution in maternal blood and liver and excretion in urine and feces was similar to that previously reported in males of the same strain. However, the rates of elimination were more rapid in pregnant females for both chemicals. This was more pronounced for TCDD than for TCDF. At all time points examined, the levels of radioactivity in the individual embryos were below 0.5% of the total TCDD dose and below 0.05% of the total TCDF dose. Assuming that all radioactive material found in embryos was unmetabolized compound, no more than 2.6 ng (8 pmoles) TCDD and 6.4 ng (21 pmoles) TCDF per g tissue were detected. In light of recent findings which strongly suggest a direct effect of TCDD and related compounds on embryonic palatal tissue, our data clearly support the potent teratogenic effect of TCDD and TCDF on the development of the secondary palate.     

Low doses of 2,3,7,8-tetrachlorodibenzo- p-dioxin increase transforming growth factor beta and cause myocardial fibrosis in marmosets ( Callithrix jacchus)

Epidemiological studies have suggested an association between exposure to dioxins and cardiovascular morbidity and mortality. However, cardiotoxic effects of low doses of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in animals have not been reported so far. We studied the hearts of male marmosets ( Callithrix jacchus)after treatment with single subcutaneous doses of 1, 10 or 100 ng TCDD/kg body weight or vehicle (toluene/DMSO 1+2 v/v, 100 microl/kg body weight). The animals were killed 2 or 4 weeks after treatment. Tissue samples of left ventricular myocardium were stained with picrosirius red and examined histologically along with quantitative image analysis. Extracellular matrix proteins were additionally analysed by western blotting. Monkeys showed no overt signs of toxicity nor did their relative heart weights differ significantly depending on treatment. Histology revealed an increase of picrosirius red-positive area above control values in 2 of 4 (1 ng TCDD/kg body weight), 6 of 12 (10 ng/kg) and 6 of 10 (100 ng/kg) marmosets. Western blotting confirmed these histological findings showing an increase of collagen, fibronectin and laminin in the hearts of TCDD-treated animals. Western blotting additionally showed an increased concentration of transforming growth factor beta1 (TGF-beta1) as well as TGF-beta receptor type I which could be a functional link to the effects on extracellular matrix. Our findings might explain the association of TCDD exposure with increased cardiovascular mortality observed in epidemiological studies and should stimulate further research on the role of changes in the extracellular matrix in the toxic effects of dioxins and related substances on other organs.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on serum concentrations and the uterotrophic action of exogenous estrone in rats

Experiments were conducted to determine whether the short-term administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in low doses alters the disposition and action of exogenous estrogen. Ovariectomized rats were pretreated with TCDD, 1 μg/kg body wt/day for 5 days, or with vehicle. Following the administration of estrone, 10 μg/100 g body wt/day for 4 days, serum estrone concentrations were 57% higher in TCDD-pretreated animals. Aryl hydrocarbon hydroxylase (AHH) activity in liver microsomes was increased 20-fold 5 days following the last dose of TCDD, data which confirm that monooxygenase activity was altered during the course of estrone administration. Since estrone disposition and/or metabolism appears to be decreased by TCDD exposure, experiments were performed to determine if TCDD altered the uterotrophic response to estrone. When various doses of estrone were administered to control animals, serum estrone values increased with the administered dose without any apparent conversion to 17β-estradiol. Estrone administered in doses of 0.4, 2.0, and 10.0 μg/100 g for 4 days elicited a dose-dependent increase in uterine wet weight. Control and TCDD-pretreated animals showed no differences in the uterine weight increase observed at each dose of estrone. Thus, the uterine growth response to exogenous estrone was not altered by any change in estrogen disposition elicited by short-term administration of TCDD.     

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

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