Treated With 2,3,7,8-tetrachlorodibenzo-p-dioxin Research Articles

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 correspond to 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). Extrapolations of the results obtained at higher doses to very low exposures is not justified with respect to the effects induced by TCDD on the immune system of marmosets. At lower doses the effect is clearly reversed.

The possible role of phospholipase A2 in hepatic microsomal lipid peroxidation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats

The induction of lipid peroxidation in hepatic microsomes of rodents treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is well documented. The potential mechanisms involved in TCDD-induced microsomal lipid peroxidation were investigated, using selected inhibitors and free radical scavengers in vitro. Rats were treated with 40 micrograms TCDD/kg orally as a single dose. Inhibitors of phospholipase A2, including a variety of phenothiazines, dibucaine, imipramine, and verapamil, inhibited in vitro microsomal lipid peroxidation in response to TCDD administration. In addition, the lipoxygenase inhibitor quercetin, and the hydrogen peroxide scavenger aminopyrine inhibited lipid peroxidation with microsomes from TCDD-treated rats. The singlet oxygen scavenger beta-carotene, the cytochrome P-450 substrate benzphetamine, and the cyclooxygenase inhibitor indomethacin produced moderate enhancement of hepatic microsomal lipid peroxidation. The results suggest that activation of phospholipase A2 may play a critical role in the metabolic events associated with hepatotoxicity and ultimate cell death produced by TCDD. The results also support the involvement of hydrogen peroxide in TCDD-induced microsomal lipid peroxidation.

The effect of TCDD on Acyl CoA: Retinol acyltransferase activity and vitamin a accumulation in the kidney of male sprague‐dawley rats

Previous studies have shown that rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) show signs of toxicity that are similar to the responses of animals to a vitamin A-deficient diet. These include hypophagia, loss of body weight, loss of hepatic vitamin A, and accumulation of renal retinoids. Male Sprague-Dawley rats treated with 10, 30, or 100 nmol/kg of TCDD accumulated renal vitamin A, with retinyl palmitate concentrations reaching 8 times those of control animals, similar to that of male rats fed a vitamin A-free diet for 26 days. Acyl CoA:retinol acyltransferase (ACARAT) activities in both TCDD-treated rats and rats fed a vitamin A-free diet for 26 days were similarly elevated, and were strongly and positively correlated with the renal retinyl palmitate concentrations. Retinol concentrations in the kidneys of rats treated with TCDD or fed a vitamin A-free diet were only slightly elevated when compared to control rats. We suggest that accumulation of retinyl esters in the kidneys of rats treated with TCDD or fed a vitamin A-free diet occurs as a result of increased rates of retinol esterification.

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 ("helper-inducer 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 cell/microliter 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.(ABSTRACT TRUNCATED AT 400 WORDS)

In Vitro Effects of Methylsulfonyl Polychlorinated Biphenyls and 7,8‐Benzoflavone on Aryl Hydrocarbon Hydroxylase Activity in Human Lymphoblastoid Cells

The effects of 11 isomers of methylsulfonyl polychlorinated biphenyls (MSF-PCBs) on the aryl hydrocarbon hydroxylase (AHH) activity were examined at a fixed dose of 1.5 micrograms/ml by using cultured lymphoblastoid cells. One of the isomers 3-MSF-3',4,4',5-tetraCB, as well as 4-MSF-3,3',4',5-tetraCB and 4-MSF-3,3',4',5,5'-pentaCB, effectively reduced the AHH activity when added to the culture medium, especially that previously treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce the AHH activity. However, the inhibitory potencies exerted by the above three MSF-PCB compounds were smaller than that by 7,8-benzoflavone (BNF). When added to the culture medium simultaneously with TCDD or 3-methylcholanthrene (MC), 3-MSF-3',4,4',5-tetraCB blocked the enhancement of the AHH activity by TCDD but did not that by MC. This is in contrast to BNF which blocked the increases due to both TCDD and MC. On the other hand, 3-MSF-3',4,4',5-tetraCB added directly to the reaction mixture for the TCDD-induced AHH activity showed little influence, while BNF decreased the same activity. These results imply that the effect of 3-MSF-3',4,4',5-tetraCB on the AHH activity is different from that of BNF.

Nad(P)H:quinone oxidoreductase (DT-diaphorase) in chick embryo liver: Comparison to activity in rat and guinea pig liver and differences in co-induction with 7-ethoxyresorufin deethylase by 2,3,7,8-tetrachlorodibenzo- p-dioxin

NAD(P)H: quinone oxidoreductase (EC 1.6.99.2; DT-diaphorase) was present in the liver of 18- and 19-day-old chick embryos as assayed both by reduction of resonifin and by the more traditional assay, reduction of 2,6-dichlorophenolindophenol (DCPIP). Both reductions had the classic characteristics of DT-diaphorase: they were equally supported by NADPH and NADH and almost entirely inhibited by dicumarol. Chick embryo liver DT-diaphorase was entirely cytosolic. It was undetectable in the microsomal and mitochondrial fractions. Chick embryo liver cytosol and mitochondrial fractions contained an enzyme oxidizer of resorufin but not of DCPIP. The k m for NADPH for resorufin reductase was an order of magnitude higher in chick embryo than in rat or guinea pig cytosol (1 mM vs 0.1 mM). Resorufin reductase activity was higher for chick embryo than for rat or guinea pig cytosols: V max (nmol resorufin reduced per mg cytosolic protein per min ± SEM) 355 ± 28 for chick embryo, 159 ± 10 for guinea pig and 68 ± 28 for rat. The V max for DCPIP reduction was also twice as high in chick embryo as rat liver cytosol. In the chick embryo, 7 days after treatment with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) at 6.4 μg/kg egg (1 nmol/egg) mortality was increased 2.4-fold, hepatic DT-diaphorase 1.3-fold, and 7-ethoxyresorufin deethylase (7-EROD) 72-fold over control levels. At 32 μg/kg, mortality was increased 4.2-fold, DT-diaphorase 2.3-fold and 7-EROD 100-fold. In the guinea pig, 5 days after treatment with TCDD at 10 μg/kg, TCDD toxicity was also evident (loss of body weight and thymus weight); there was no change in DT-diaphorase as measured by resorufin reduction, confirming by a different assay the observation of Beatty and Neal ( Biochem Pharmacol 27: 505–510, 1978) that TCDD does not induce DT-diaphorase in guinea pig liver, and 7-EROD was increased 8-fold. In contrast, in the rat, 7 days after exposure to TCDD at 10 μg/kg, there was no evidence of toxicity, DT-diaphorase was increased close to 7-fold and 7-EROD, 100-fold. The results demonstrate that avian liver contains DT-diaphorase and show that the extent to which DT-diaphorase is part of the pleiotypic response of the liver to an Ah ( aryl hydrocarbon) receptor ligand is species dependent. They also suggest that DT-diaphorase induction and TCDD toxicity may be inversely related. The possibility that DT-diaphorase protects against TCDD toxicity and participates in species differences in sensitivity to TCDD toxicity warrants further investigation.

Effect of 2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin on the Hepatic Distribution of Iron, Copper, Zinc, and Magnesium in Rats

The distribution of iron, copper, zinc, and magnesium in hepatic subcellular fractions of male and female rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was determined. Animals received 40 micrograms TCDD per kilogram per day for three days by mouth (po) or the vehicle and were killed seven or nine days posttreatment. Iron, copper, zinc, and magnesium were determined by atomic absorption spectroscopy. The iron content of liver from female animals was twofold higher than male animals. The administration of TCDD increased the iron content of mitochondria in female and male rats and decreased iron content of microsomes of both sexes. Significant increases occurred in the copper content of whole liver, mitochondria, and cytosol of male rats and in whole liver and cytosol of female rats. Decreases in the copper content of the microsomes of male rats were observed following TCDD treatment; however, TCDD produced no changes in the zinc content of hepatic subcellular fractions of either sex. The magnesium content of female TCDD-treated rats increased in whole liver, mitochondria, and cytosol, while the magnesium content of microsomes was not altered. With respect to the subcellular distribution of iron, copper, zinc, and magnesium, TCDD produces differential effects. The altered distribution of some cations may contribute to the broad range of effects of TCDD.

Inducers of cytochrome P-450d: Influence on microsomal catalytic activities and differential regulation by enzyme stabilization

The interaction of isosafrole, 3,4,5,3′,4′,5′-hexabromobiphenyl (HBB) and hexachlorobiphenyl (HCB) with cytochrome P-450d was evaluated by characterization of estradiol 2-hydroxylase activity. Displacement of the isosafrole metabolite from microsomal cytochrome P-450d derived from isosafrole-treated rats resulted in a 160% increase in estradiol 2-hydroxylase. The increase was fully reversed by incubation with 1 μ m HBB. Although isosafrole is capable of forming a complex with many different cytochrome P-450 isozymes, it appears to bind largely to cytochrome P-450d in vivo as was demonstrated by measuring the enzymatic activity of microsomal cytochromes P-450b, P-450c, and P-450d from isosafrole-treated rats. When estradiol 2-hydroxylase was measured in rats treated with increasing doses of HCB, there was a gradual decrease in microsomal enzyme activity despite a 20-fold increase in cytochrome P-450d. The ability of cytochrome P-450d ligands to stabilize the enzyme was investigated in two ways. First, cytochromes P-450c and P-450d were quantitated immunochemically in microsomes from rats treated with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), at a dose which maximally induced total cytochrome P-450, followed by a single dose of a second inducer. The specific content of cytochrome P-450d was significantly increased when isosafrole or HCB was the second inducer but not when 3-methylcholanthrene was the second inducer. Second, the relative turnover of cytochrome P-450d was measured by the dual label technique. Following TCDD treatment, microsomal protein was labeled in vivo with [ 3H]leucine, the second inducer was given and protein was again labeled 3 days later with [ 14C]leucine. A higher ratio of 3 H 14 C in the cytochrome P-450d from isosafrole + TCDD- and HCB + TCDD-treated rats relative to TCDD (control)-treated rats suggested that isosafrole and HCB were able to retard the degradation of cytochrome P-450d, presumably by virtue of being tightly bound to the enzyme.

Tissue-specific expression of the mouse dioxin-inducible P(1)450 and P(3)450 genes: differential transcriptional activation and mRNA stability in liver and extrahepatic tissues.

Expression of the P(1)450 and P(3)450 genes was examined in liver and five extrahepatic tissues of mice after they were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3-methylcholanthrene. All six tissues were shown to have increased P(1)450 and P(3)450 mRNA concentrations after treatment with these inducers. P(3)450 mRNA induction was more sensitive than P(1)450 mRNA induction to small doses of TCDD in liver, kidney, and lung. When transcription run-on assays were compared with mRNA prevalence, control P(3)450 mRNA in liver, kidney, and lung was shown to be 20 to 30 times more stable than control P(1)450 mRNA. After TCDD treatment the increases in mRNA concentrations did not necessarily parallel the increases in transcriptional rate. Thus, the inducer appeared to enhance mRNA stability in some instances. This was evident for liver P(1)450 mRNA, in which an 8-fold rise in transcription was associated with a 27-fold increase in mRNA content, and for kidney P(3)450 mRNA, in which a 2-fold rise in transcription was accompanied by a 12-fold increase in mRNA content. In the kidney and lung of control and TCDD-treated mice, transcriptional rates of the P(3)450 gene were at least 10-fold less than those of the P(1)450 gene. These data indicate that even though both genes are controlled by the same receptor, striking tissue-specific differences in transcription and mRNA stabilization affect the final mRNA concentrations.

Tissue-specific expression of the mouse dioxin-inducible P(1)450 and P(3)450 genes: differential transcriptional activation and mRNA stability in liver and extrahepatic tissues.

Expression of the P(1)450 and P(3)450 genes was examined in liver and five extrahepatic tissues of mice after they were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3-methylcholanthrene. All six tissues were shown to have increased P(1)450 and P(3)450 mRNA concentrations after treatment with these inducers. P(3)450 mRNA induction was more sensitive than P(1)450 mRNA induction to small doses of TCDD in liver, kidney, and lung. When transcription run-on assays were compared with mRNA prevalence, control P(3)450 mRNA in liver, kidney, and lung was shown to be 20 to 30 times more stable than control P(1)450 mRNA. After TCDD treatment the increases in mRNA concentrations did not necessarily parallel the increases in transcriptional rate. Thus, the inducer appeared to enhance mRNA stability in some instances. This was evident for liver P(1)450 mRNA, in which an 8-fold rise in transcription was associated with a 27-fold increase in mRNA content, and for kidney P(3)450 mRNA, in which a 2-fold rise in transcription was accompanied by a 12-fold increase in mRNA content. In the kidney and lung of control and TCDD-treated mice, transcriptional rates of the P(3)450 gene were at least 10-fold less than those of the P(1)450 gene. These data indicate that even though both genes are controlled by the same receptor, striking tissue-specific differences in transcription and mRNA stabilization affect the final mRNA concentrations.

Ingestion of soil contaminated with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) alters hepatic enzyme activities in rats

Female rats were treated with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in either corn oil or contaminated soil from the Minker site in Missouri. Eight doses ranging from 0.015 to 5 μg TCDD/kg were used in the corn oil group; the range was 0.015 to 5.5 μg TCDD/kg in the TCDD-contaminated soil group. Rats in a third group were given equal amounts of soil uncontaminated with TCDD. No acute toxicity or effects on body weight gain were observed at these doses. In general, equivalent doses of TCDD in corn oil or TCDD in soil produced similar increases in hepatic aryl hydrocarbon hydroxylase activity (AHH) and UDP glucuronyltransferase activity although effects were slightly greater in the TCDD-corn oil groups. In the corn oil groups, the induction of AHH ranged from about 30-fold at the highest dose to twofold at the lowest dose studied. TCDD also caused an increase in cytochrome P-450 concentration and a shift in spectral peak from 450 to 448 nm. There was no effect of TCDD on ethylmorphine N-demethylase, consistent with previous reports. Liver concentrations of TCDD (mean ± SD) in the 5-μg/kg groups were 40.8 ± 6.3 ppb in the TCDD-corn oil group and 20.3 ± 12.9 ppb in the TCDD-contaminated soil group. Our results suggest that the bioavailability of TCDD in soil in rats is approximately 50%. Therefore, ingestional exposure to TCDD-contaminated soil may constitute a significant health hazard in view of its extremely high toxicity and relatively high bioavailability.

Regulation of aromatic oxidation of estradiol-17β in maternal hepatic, fetal hepatic and placental tissues: Comparative effects of a series of inducing agents

The effects of nine separate inducers of cytochrome P-450-dependent monooxygenases on the hydroxylation of estradiol-17β (E 2) were investigated in near-term pregnant rats. Isosafrole exhibited highly effective inducing properties in the maternal liver (20-fold and 5-fold increases in 4- and 2-hydroxylase activities respectively). Pregnenolone 16α-carbonitrile produced approx 20- and 30-fold increases in measured respective rates of 4- and 2-hydroxylase activities in fetal hepatic tissues; isosafrole produced only 2-fold increases in the same reaction. Only minor changes or slight increases in estrogen hydroxylation rates were observed in maternal hepatic, fetal hepatic or placental tissues following treatment with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) or other potent 3-methylcholanthrene (MC)-like inducing agents (β-naphthoflavone, MC, caffeine). Phenobarbital exhibited relatively weak inducing properties and exposure of pregnant rats to ethanol from days 3–19 of gestation was without statistically significant effects on the parameters investigated. Rat placentas exhibited extremely low estrogen hydroxylase activities irrespective of pre-exposure of pregnant rats to the inducers studied. The results suggested separate regulatory controls for estrogen 2- and 4-monooxygenase activities even though relatively high correlation between the two reaction were generally observed in all three tissues.

Reversible inhibition of in vitro epithelial cell proliferation by 2,3,7,8-tetrachlorodibenzo- p-dioxin

Subconfluent cultures of a mouse epithelial cell line, which after prolonged subculturing exhibited an elevated saturation density as compared to the original cell line, were treated with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Cultures of cells with or without TCDD grew at equal rates until confluency was reached. At confluency, cultures treated with as little as 10 −11 m TCDD showed a decline in cell proliferation relative to controls as demonstrated by cell enumeration and supported by reduced [ 3H]thymidine incorporation (both by liquid scintillation spectrometry of whole culture and autoradiography of individual cells). After 14 days of exposure, the saturation density of the treated culture was about 50% of the control culture. This TCDD-induced, increased sensitivity to density-dependent inhibition of replication (DDIR) was accompanied by a change from a fusiform morphology in the high-saturation-density control cells to a flat cobblestone appearance in the treated low-saturation-density cells. The nondividing cultures treated for 14 days with 10 −11 m TCDD had the same viability as control cultures. Upon trypsin suspension and reseeding, these formerly quiescent cultures were again capable of growing to high cell density and of again showing susceptibility to TCDD-induced changes in cell growth and morphology. Evidence is presented to suggest that this reversible increase in sensitivity to DDIR and the morphological change are not a consequence of cell growth inhibition. This system may provide the basis for an in vitro model to study the effect of TCDD on the control of replication of these cells.

Opposing effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone on growth and differentiation of cultured malignant human keratinocytes.

The human keratinocyte line SCC-13, derived from a squamous cell carcinoma of epidermis, was examined for effects on growth and differentiation upon treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Inhibition of growth was observable at 0.1 nM and maximal in the concentration range of 1-100 nM TCDD, but was completely antagonized by addition of hydrocortisone to the growth medium. TCDD was found to inhibit several aspects of keratinocyte differentiation that are stimulated by hydrocortisone. In confluent cultures, accumulation of keratin protein and transglutaminase activity were suppressed as well as spontaneous envelope formation and envelope competence. This phenomenon occurred without significant effect of TCDD on depletion of hydrocortisone from the medium. We conclude that the response of SCC-13 cells to TCDD depends upon hormonal conditions in culture and that this agent can interfere with cellular responses to normal physiological conditions, thereby altering the differentiation program ordinarily observed.

Intestinal absorption of nutrients in rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Impairment of active intestinal absorption of glucose and leucine was observed in rats 2-3 wk after oral treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (5 and 100 mg/kg). At the higher dose level used this response was complicated by the effects of severely reduced food consumption. Malabsorption of specific nutrients may help occasion the body wasting seen in many animals after acute exposure to TCDD.

Toxicity of 2,3,7,8-tetrachlorodibenzo- p-dioxin in the Golden Syrian hamster

Lethality, pathology, and various clinical chemical parameters were assessed in the hamster following a single ip or po treatment with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). A single dose, 50-day LD 50 of greater than 3000 μg TCDD/kg, ip, was obtained for male and female hamsters while the LD 50 of orally administered TCDD was found to be 1157 μg/kg. Thus, the hamster appears to be the least sensitive mammalian species to the lethal effect of TCDD that has yet been investigated. TCDD treatment generally reduced the rate of body weight gain, with orally treated hamsters exhibiting the greatest reduction in rate. Thymic atrophy was the most consistent pathologic finding in TCDD treated hamsters. No histopathological changes were seen in the liver, spleen, kidneys, adrenals, or heart. Moderate to severe ileitis and peritonitis were found in many of the hamsters which died following oral treatment with TCDD. This lesion usually affects the distal ileum and consists of a marked hyperplasia of the mucosal epithelium with mild to severe hemorrhaging and necrosis. The intestinal lesion probably contributed in part to the greater lethality of TCDD in orally treated hamsters. A significant increase in serum alkaline phosphatase, bilirubin, protein, iron, cholesterol, and a decrease in serum albumin, chloride, urea nitrogen, and triglycerides were found in hamsters following both ip and po treatment with TCDD.

Blood clearance tests for detecting 2,3,7,8-tetrachlorodibenzo-p-dioxin hepatotoxicity in rats and rabbits.

The objective was to determine if a blood clearance test would detect liver dysfunction in rats and rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Guinea pigs were included as a negative control, because TCDD does not produce detectable hepatotoxicity in this species. TCDD was given as a single dose to male rats (25 microgram/kg, po), rabbits (25 microgram/kg, ip) and guinea pigs (2 microgram/kg, ip) and liver function was assessed 10 days later by determining blood clearance of indocyanine green (ICG) and ouabain. Activity in serum of sorbitol dehydrogenase (SDH), glutamic pyruvic transaminase (SGPT) and gamma glutamyl transpeptidase (gamma GTP) were also measured and light microscopy performed on the liver. The results showed that hepatotoxicity in the rabbit could be detected by a reduction in ICG blood clearance and an elevation in SDH activity. In the rat, ouabain blood clearance was reduced and SDH activity elevated. None of the liver function tests were altered in the guinea pig. These results underline the usefulness of blood clearance tests using ICG and ouabain in detecting TCDD hepatotoxicity in animals.

Purification of the major cytochrome P-450 of liver microsomes from rabbits treated with 2,3,7,8-tetrachlorodibenzo- [formula omitted]-dioxin (TCDD)

Cytochrome P-450 was isolated in highly purified form from liver microsomes of adult male rabbits treated with 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). Preparations average 17.8 ± 0.8 nmoles cytochrome P-450 per mg protein and have an estimated molecular weight of 54,500. The visible absorption spectrum of the purified cytochrome displays absorption spectral maxima characteristic of high spin forms of cytochrome P-450. When reconstituted with highly purified NADPH-cytochrome P-450 reductase, this cytochrome catalyzes the hydroxylation of acetanilide and the O-deethylation of 7-ethoxyresorufin, two activities induced by TCDD.

Resolution of two forms of cytochrome P-450 from liver microsomes of rabbit treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Two forms of cytochrome P-450 with different substrate specificities were isolated from liver microsomes of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A specific antibody was produced toward the major form of the cytochrome. The antibody inhibits microsomal acetanilide hydroxylation (80%). It does not cross-react with the minor fraction of the cytochrome or inhibit the hydroxylation of 3,4-benzpyrene or coumarin, the N-demethylation of aminopyrine or the O-deethylation of 7-ethoxycoumarin catalyzed by rabbit liver microsomes. The major form has an estimated Mr = 54,000 and displays an n-octylamine difference spectrum with an absorption maximum at 426 nm and a minimum at 391 nm. When reconstituted, this cytochrome catalyzes acetanilide hydroxylation at a higher rate than microsomes or the minor fraction. The n-octylamine difference spectrum of the minor fraction displays an absorption maximum at 431 nm and a minimum at 410 nm. When reconstituted, this fraction catalyzes the hydroxylation of 3,4-benzpyrene and the O-deethylation of 7-ethoxycoumarin. The two cytochromes appear to be distinct entities and function in different catalytic pathways.