Metabolism and disposition of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rainbow trout

Accumulation, tissue distribution, and depuration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-derived 3H were studied in fingerling rainbow trout fed a diet containing 494 ppt [3H]TCDD for 13 weeks followed by the same diet without TCDD for 13 weeks. This exposure did not cause fin rot, cutaneous hemorrhage, reduced growth rate, or an increase in relative lethality in TCDD-exposed fish. Visceral fat, carcass, skin, and pyloric caeca and all fatty tissues, accounted for greater than 90% of the TCDD-derived 3H in the fish after the 13-week exposure period. The remaining TCDD-derived radioactivity was distributed to skeletal muscle, gill, gastrointestinal tract, liver, kidney, heart, and spleen. High-pressure liquid chromatographic analysis of 3H in skeletal muscle, liver, kidney, carcass, and visceral fat showed that it was primarily due to TCDD (greater than or equal to 98%) and not metabolites (less than or equal to 2%). The t1/2 for whole-body depuration of TCDD-derived 3H was 15 weeks, and individual organ t1/2 values ranged from 8 to 19 weeks. To determine if rainbow trout metabolize TCDD, adult fish were injected with [14C]TCDD (60 micrograms/kg, ip), and gallbladder bile, liver, skeletal muscle, and kidney were analyzed 1 week later. While only the parent compound was found in the tissues, bile contained at least three TCDD metabolites and the parent compound. beta-Glucuronidase treatment of the bile suggested that at least one TCDD metabolite was a glucuronide conjugate.     

Metabolism and disposition of 2,3,7,8-tetrachlorodibenzo-p-dioxin in guinea pigs

Marked interspecies variability exists in the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), with the guinea pig being the mammalian species most sensitive to the acute toxicity of TCDD. The metabolism and disposition of TCDD was investigated in guinea pigs for 45 days following a single exposure to purified [3H]TCDD (0.56 microgram/kg, ip). Guinea pigs included in the toxicokinetic study gained body weight, maintained a normal relative body composition, and exhibited no gross signs of toxicity during the 45-day study. Approximately 36% of the dose of TCDD-derived 3H remained in the adipose tissue at 45 days following exposure to [3H]TCDD, while the liver, pelt, and skeletal muscle and carcass each contained about 7% of the administered dose. Although most of the TCDD-derived radioactivity in liver, kidney, perirenal adipose tissue, and skeletal muscle represented unchanged TCDD, from 4 to 28% of the 3H was associated with metabolites of TCDD. This unexpected finding suggests that TCDD metabolites are not efficiently excreted from guinea pigs. The urinary and fecal excretion of TCDD-derived radioactivity followed apparent first-order kinetics, with an elimination half-life of 93.7 +/- 15.5 days (mean +/- SD). HPLC analysis of urine and bile from [3H]TCDD-treated guinea pigs showed that all of the radioactivity represented metabolites of TCDD, indicating that these routes of elimination are dependent on prior metabolism of TCDD. However, 70 to 90% of the radioactivity in fecal samples was found to represent unmetabolized TCDD throughout the 45-day excretion study. The presence of TCDD in feces and its absence in bile suggest that the fecal excretion of unchanged TCDD resulted from the direct intestinal elimination of the lipophilic toxin. Furthermore, the cumulative excretion of TCDD-derived radioactivity over 45 days indicated that 74.3% of the 3H was excreted in feces as unchanged TCDD, while 25.7% of the 3H was excreted in urine and feces as TCDD metabolites. Thus, TCDD is primarily eliminated unchanged in the feces of guinea pigs, indicating that the metabolism of TCDD does not play a major role in the ultimate elimination of the toxin from the guinea pig. This may in part explain the relatively long excretion half-life for TCDD in the guinea pig and may contribute to the remarkable sensitivity of the guinea pig to the acute toxicity of TCDD.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin toxicity in yellow perch (Perca flavescens)

Growth, mortality and morphologic lesions in juvenile, hatchery-reared yellow perch, Perca flavescens, were studied after treatment with graded single doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 1-125 micrograms/kg, intraperitoneally). TCDD doses of 25 and 125 micrograms/kg caused 95% mortality by 28 d after treatment, without decreasing body weight. A TCDD dose of 5 micrograms/kg resulted in progressive loss of body weight with cumulative mortality of 80% by 80 d posttreatment. Periodic handling stress did not affect the time course of mortality or cumulative percent lethality in TCDD-treated perch. Fin necrosis, petechial cutaneous hemorrhage, and ascites occurred in perch treated with 5 micrograms/kg or more of TCDD. Thymic atrophy, decreased hematopoiesis in the head kidney, fibrinous pericarditis, focal myocardial necrosis, submucosal gastric edema, and hyperplasia of the epithelium of gill filaments and lamellae occurred in perch dosed with 25 or 125 micrograms/kg. Dose-related splenic lymphoid depletion occurred in perch receiving 5 micrograms/kg or more TCDD, and hepatocyte lipidosis occurred in groups treated with doses of 1 microgram/kg or more TCDD. Thus yellow perch are as responsive to the acute toxic effects of TCDD as some of the more sensitive mammalian species, and neither loss of body weight nor histologic lesions in TCDD-treated perch are sufficient to explain mortality.     

Species Differences in 2,3,7,8-Tetrachlorodibenzo-p-dioxin Toxicity and Biotransformation in Fish

Species Differences in 2,3,7,8-Tetrachlorodibenzo-p-dioxin Toxicityand Biotransformation in Fish. KLEEMAN, J. M., OLSON, J. R.,AND PETERSON, R. E. (1988). Fundam. Appl. Toxicol 10, 206-213.Rainbow trout, yellow perch, carp, bluegill, largemouth bass,and bullhead were treated with graded doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD; 1, 5, 25, or 125 µg/ kg) or vehicle, ip. The lethalpotency of TCDD tended to be greater in yellow perch, carp,and bullhead than in the other three species (LD50 80 days post-treatment,3-5 versus 10-16 µg/kg, respectively). All species treatedwith the highest dose of TCDD (125 µg/kg) displayed alatency period of 1-4 weeks prior to death; longer latency periodswere produced by lower lethal doses. Effects of TCDD treatmenton body weight were both species-dependent and dose-dependent.Fin necrosis was observed in all fish species; however, cutaneoushemorrhage was observed only in TCDD-treated perch, carp, andbluegill, and cutaneous hyperpigmentation only in TCDD-treatedcarp and largemouth bass. Gallbladder bile was analyzed forTCDD and its metabolites 7 days after fish were injected with[¹⁴C]TCDD (60 µg/kg, ip). At least three TCDD metabolitesin addition to the parent compound were found in the gallbladderbile of all six species. In addition, the retention time ofthe major biliary TCDD metabolite (determined by HPLC) was similarin all species except yellow perch. ß-Glucuronidasetreatment of the bile from largemouth bass and bluegill suggestedthat at least two of the TCDD metabolites were glucuronide conjugates.Thus, species differences exist in the lethal potency, signsof overt toxicity, and biotransformation of TCDD among freshwaterfish.     

Subchronic Exposure of [3H]- 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in female B6C3F1 mice: relationship of steady-state levels to disposition and metabolism.

The present study of subchronic low exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at or near steady-state levels tries to emulate the most probable mode for human exposure, dietary consumption. This study is the first and most intensive pharmacokinetic study to be reported with repeated dosing, multiple times, and multiple doses examining disposition of TCDD-derived radioactivity and CYP1A activities in mice. For time-course relationships, animals were dosed (daily, Monday-Friday) with 0, 1.5, or 150 ng [3H]TCDD/kg for 4, 8, 13, or 17 weeks and also for 13 weeks followed by 4 weeks with no dosing. For dose-response relationships, animals were dosed for 13 weeks (daily, Monday-Friday) with 0, 0.15, 0.45, 1.5, 4.5, 15, 45, 150, or 450 ng [3H]TCDD/kg. Additional animals dosed for 13 weeks (daily, Monday-Friday) with 1.5 or 150 ng [(3)H]TCDD/kg were housed in metabolism cages. Time- and dose-dependencies of TCDD were confirmed in all measured tissues. Liver/fat (L/F) concentration ratios ranged from 0.2-3.4 (low to high dose). Hepatic CYP1A1 enzymatic activity increased (p < 0.05) starting at 0.15 ng/kg/day with L/F of 0.2 and body burden of 2.8 ng TCDD/kg body weight. By examining TCDD exposures at or near steady state, this study reports for the first time and provides direct evidence of low-dose effects on a measured reversible response at body burdens that are within background levels of the general human population. In addition, this study emphasizes cumulative effects of daily dosing and suggests the importance of tissue dosimetry or body burden for a persistent chemical such as TCDD.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the distribution and biliary excretion of polychlorinated biphenyls in rats.

Plasma disappearance, biliary excretion, and tissue distribution of two polychlorinated biphenyls (PCBs), 2,5,2′,5′-[³H]tetrachlorobiphenyl (4-CB) and 2,4,5,2′,4′,5′-[³H]hexachlorobiphenyl (6-CB), was determined in rats 10 days after oral administration of a single 10 or 25 μg/kg dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Plasma disappearance of both PCBs was not altered by TCDD treatment, but biliary excretion was depressed. Associated with the depressed excretion was a reduction in bile flow and concentration of 4-CB- and 6-CB-derived ³H in bile. Treatment with TCDD resulted in less PCB being distributed to the skin and a greater percentage of the dose being accumulated in the liver. The content of PCB-derived ³H in skeletal muscle, adipose tissue, and urine was similar in control and TCDD-treated rats. Extraction of bile with hexane showed that the majority of biliary radioactivity was in the form of polar metabolites and that the proportion of parent PCB (hexane-extractable radioactivity) to polar metabolites was not altered by TCDD treatment. In rats given 4-CB and sacrificed 1 hr later, the majority of radioactivity in the liver was hexane extractable, and the smaller amount of hepatic radioactivity due to polar 4-CB metabolites was greater in the TCDD treatment group than in the control group. When biliary metabolites of 4-CB were administered, biliary excretion of the metabolites was depressed in TCDD-treated animals. Thus, TCDD treatment impairs the initial and main excretory pathway for PCB elimination in the rat—biliary excretion—and alters the distribution of PCBs to the skin and liver.     

Impairment of metabolic capacities in copper and calcium contaminated wild yellow perch (Perca flavescens)

This study examined variations in resting oxygen consumption rate (ROCR), post-exercise oxygen consumption rate, relative scope for activity (RSA), liver and muscle aerobic and anaerobic capacities (using citrate synthase (CS) and lactate dehydrogenase, respectively, as indicators), and tissue biosynthetic capacities (using nucleoside diphosphate kinase (NDPK) as an indicator), in wild yellow perch from four lakes varying in copper (Cu) and cadmium (Cd) contamination. Liver Cu and Cd concentrations largely reflected environmental contamination and were positively correlated with liver protein concentrations and NDPK activities. Our results suggest that metal contamination leads to an upregulation of liver protein metabolism, presumably at least in part for the purpose of metal detoxification. In contrast, muscle NDPK activities decreased with increasing liver Cd concentrations and NDPK activities. There was a 25% decrease in ROCR for a doubling of liver Cu concentrations and a 42% decrease in RSA for a doubling of liver Cd concentrations in the range studied. Cu contamination was also associated with lower muscle CS activities. Our results support previous findings of impaired aerobic capacities in the muscle of metal-contaminated fish, and demonstrate that this impairment is also reflected in aerobic capacities of whole fish. The evidence presented suggests that mitochondria may be primary targets for inhibition by Cu, and that Cd may reduce gill respiratory capacity. Muscle aerobic and anaerobic capacities were inversely related. This work indicates that metal exposure of wild yellow perch leads to a wide range of disturbances in metabolic capacities.     

Metabolism and disposition of 2,3,7,8-tetrachlorodibenzo-p-dioxin in ring-necked pheasant hens, chicks, and eggs.

The T 1/2 for whole-body elimination of [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) derived radioactivity in ring-necked pheasant hatchlings was 13 d, whereas in adult hen pheasants that were not producing eggs it was 378 d. All TCDD-derived radioactivity in hen tissues was from the parent compound. The oral bioavailability of TCDD in the adult hen pheasant varied with the environmental matrix, with 30% of the dose absorbed from a suspension of earthworms, 33% absorbed from a soil suspension, 41% absorbed from a suspension of paper mill sludge, and 58% absorbed from a suspension of crickets. A cumulative dose of 1.0 micrograms TCDD/kg body weight, administered as weekly doses of 0.1 micrograms/kg for 10 wk, did not adversely affect hen condition or egg production. Under these exposure conditions, hens translocated about 1% of their cumulative TCDD dose to each of the first 15 eggs laid. All of the TCDD-derived radioactivity in the eggs was the parent compound and was confined entirely to the yolk; no TCDD was detected in egg albumin. We conclude that TCDD was more persistent in pheasant hens than in chicks and that egg laying was an important route of elimination in the hen.     

Interactive Effects between 2,3,7,8-Tetrachlorodibenzo-p-dioxin and 2,2',4,4',5,5'-Hexachlorobiphenyl in Female B6C3F1 Mice: Tissue Distribution and Tissue-Specific Enzyme Induction

The distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) was studiedin female B6C3F1 mice. Single doses of TCDD alone (0, 0.1, 1,or 10 µg [³H]TCDD/kg), PCB 153 alone (0, 3.58, 35.8, or358 mg [¹⁴C]PCB 153/kg), and all possible combinations of thesedoses were administered in corn oil, po. At 7 days after dosing,11 different tissues were analyzed for radioactivity. When TCDDwas administered alone, TCDD-derived radioactivity distributedto all tissues in a dose-dependent manner, increasing with dosein the liver, while decreasing (as a percentage of the administereddose) in all other tissues. When PCB 153 was administered alone,the PCB 153 concentration was dose-dependently (percentage ofdose) decreased in liver, skin, lung, adrenals, kidney, andblood; no dosimetric effects were observed in the other organs.Coadminis-tration of low doses of both TCDD and PCB 153 resultedin little or no effect on the distribution of either compound.Interactive effects occurred in the pharmacokinetic behaviorof both compounds only at higher doses. For example, the amountof TCDD in the liver was increased by 358 mg PCB 153/kg. Inmost other organs administration of PCB 153 resulted in a dose-dependentdecrease in the TCDD content Coadministration of PCB 153 with10 µg TCDD/kg increased PCB 153 in the liver, but notin other tissues. These results clearly demonstrate that interactiveeffects on pharmacokinetic behavior occur only at high doses.     

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

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

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.     

Measurements of genotoxic potential of cadmium in different tissues of fresh water climbing perch Anabas testudineus (Bloch), using the comet assay

The present investigation was undertaken to study the induction of DNA damage by CdCl(2) in freshwater climbing perch Anabas testudineus (Bloch) using alkaline single cell gel electrophoresis (comet assay). The DNA damage was measured in the tissue of gill, kidney and liver as the percentage of DNA in comet tails and comet heads in the tissue of the fish specimens exposed to 0.1, 1.0, 2.0mgL(-1) concentrations of CdCl(2). It was found that at all the concentrations of CdCl(2), the liver tissue exhibited significantly (p<0.01) higher DNA damage, followed by kidney and gill tissue. The DNA damage was found to be concentration dependent, with the highest DNA damage at 2mgL(-1) concentration, followed by 1.0 and 0.1mgL(-1). At the concentration of 2mgL(-1) of CdCl(2), the tail and head DNA of liver tissue were 38.81% and 59.49%, in kidney tissue the values were 32.37% and 64.66% whereas in gill tissue the values were 31.30% and 66.40% respectively. This study conclude that comet assay can be used for in vivo laboratory experiment using fish as model for screening the genotoxic potential of cadmium.     

Tissue Distribution and Toxicokinetics of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in Rats after Intravenous Injection

Tissue Distribution and Toxicokinetics of 2,3,7,8-Tetrachlorodibenzo-p-dioxinin Rats after Intravenous Injection. WEBER, L. W. D., ERNST,S. W., STAHL, B. U., AND ROZMAN, K. (1993). Fundam. Appl. Toxicol.21, 523–534. Male Sprague-Dawley rats (240–290 g) received intravenouslya nonlethal (9.25 µg/kg) or a lethal (72.7 µg/kg)dose of ¹⁴C-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)administered as an emulsion. Animals were euthanized between5 min and 16 days (lethal dose) or 32 days (nonlethal dose)after treatment. Tissue distribution was considered completeafter 24 hr, as by this time radioactivity levels in white adiposetissue had reached a maximum. The highest levels of radioactivitywere found in liver (5% of dose/g tissue), followed by whitefat (1% of dose/g tissue); serum was lowest at 0.01% of dose/mlserum. Relatively high levels of radioactivity were also detectedin most known target organs of TCDD toxicity, e.g., brown fat,adrenals, and thyroid. The pattern of organ distribution ofTCDD was essentially the same after the lethal and the nonlethaldose, but did not follow a simple lipophilicity relationship,as levels in liver were higher than those in white fat, andthose in brain were extremely low. A pool of TCDD in liposomesinitially trapped in lung and spleen was redistributed within24 hr mainly to liver and adipose tissue. Affinity of TCDD tostorage fat seemed to play a more important role as a drivingforce for redistribution than did induction of cytochrome P4501A2. The terminal slope of elimination of TCDD from tissuesindicated a half-life of 16 days after the nonlethal dose. Afterthe lethal dose radioactivity declined in all tissues for 2to 8 days and then increased again, reflecting shrinking tissuevolumes as well as remobilization of TCDD caused by the processof body mass wasting. Distribution data for 17 tissues and serumwere subjected to regression analysis and resulted in up totwo uptake phases and up to three elimination phases for a giventissue. After the nonlethal dose TCDD was mainly excreted viafeces; combined urinary and fecal excretions occurred with abiological half-life of 16.3 ± 3.0 days. Much longerhalf-lives were detected in white fat and skin. After the lethaldose, the fecal excretion of TCDD-derived radioactivity decreasedafter 8 days, and urinary excretion increased starting 12 daysafter dosing. Radioactivity in liver and white fat and the extractableportion in feces was mainly unchanged TCDD, as determined bythin-layer chromatography. Radioactivity in urine indicatedthe presence of a metabolite(s) of TCDD only.     

Tissue distribution of diazepam and its metabolite desmethyldiazepam: a human autopsy study

Concentrations of diazepam (DZ) and desmethyldiazepam (DMDZ) were determined quantitatively in the brain, skeletal muscle, heart, liver, lung, fat, adrenal gland, and kidney in 14 autopsied patients who had been treated with DZ or clorazepate (a DMDZ prodrug) during their hospital course. To facilitate interpatient comparisons, all tissue concentrations from the same patient were normalized as ratios to the concentration of DZ or DMDZ found in that patient's skeletal muscle. Tissue uptake ratios were not influenced by gender or chronicity of dosage. Distribution equilibrium was reached in at least two hours. Tissue uptake ratios differed considerably among tissues for DZ and DMDZ. Mean (+/- SE) DZ uptake ratio was highest for adrenal gland (12.1 +/- 5.9), liver (5.9 +/- 1.9), heart (4.3 +/- 1.0), and kidney (4.0 +/- 1.0), with lower values for lung (2.1 +/- 0.5), fat (2.2 +/- 0.4), and brain (1.9 +/- 0.4). Similar patterns were observed for DMDZ, except for significantly lower fat uptake. Extrapolating to an average body composition for a 70 kg man with 16% body fat, the largest fractions of total body stores of DZ would be found in muscle (42%), fat (35%), and liver (12%), with smaller stores in brain (4.3%), lung (3.3%), heart (1.7%), kidney (2.0%), and adrenal gland (0.24%).     

Distribution and excretion of 2,3,7,8-tetrachlorodibenzo-p-dioxin in congenic strains of mice which differ at the Ah locus

The effect of the genetic background on the distribution and excretion of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was examined in two sets of congenic mouse strains in which the congenic pairs differed only at the Ah locus. Male C57BL/6J mice which were either Ahb/Ahd or Ahd/Ahd and female DBA/2J mice which were also Ahb/Ahd or Ahd/Ahd were treated with 500 ng/kg 3H-TCDD and held from 1 to 42 days in individual metabolism cages. Daily excretion and tissue distribution at eight time points were determined. The Ah locus had no effect on the distribution of TCDD-derived radioactivity to the major tissue depots of adipose tissue, skin, kidney, carcass, and blood but seemed to cause elevated levels in the liver. The Ah locus also played no role in either the rate or the extent of urinary and/or fecal excretion. However, the route and rates of excretion did vary between the congenic sets with the male C57BL/6J mice excreting greater amounts of radioactivity in the urine and less in the feces than the female DBA/2J mice. The Ah locus also had no effect on the liver weight or adipose tissue volume in either congenic set. Thus, at the dose level studied, the distribution and excretion of TCDD were primarily governed by the total genetic background rather than the allele present at the Ah locus.     

Cadmium-handling strategies in two chronically exposed indigenous freshwater organisms--the yellow perch (Perca flavescens) and the floater mollusc (Pyganodon grandis)

Laboratory experiments on a variety of aquatic organisms suggest that metallothionein-like proteins (MT) play an important role in the regulation of essential metals, and in the sequestration and detoxification of non-essential metals (e.g., Cd). However, the importance of metallothionein production relative to alternative strategies of metal detoxification, and its effectiveness in metal detoxification, remain largely unexplored in field situations. In the present study we explored metal-handling strategies in an adult benthic bivalve (Pyganodon grandis) and in juvenile yellow perch (Perca flavescens), exposed to Cd in their natural habitat. The two biomonitor species were collected from lakes located along a Cd concentration gradient. Ambient dissolved Cd concentrations were determined by in situ dialysis as a measure of metal exposure. Sub-cellular Cd partitioning was determined in target tissues (bivalve gills and digestive gland; perch liver) by differential centrifugation, and metallothionein was measured independently by a mercury-saturation assay in the bivalve tissues. Malondialdehyde concentrations were measured as a potential indicator of oxidative stress. Ambient dissolved Cd concentrations ranged from 0.06 to 0.57 nM in the nine lakes from which bivalves were collected, and from < 0.3 to 6.7 nM in the eight lakes from which yellow perch were sampled. Bioaccumulated Cd also varied from lake to lake, more so for the bivalve than for the yellow perch; the [Cd]max/[Cd]min ratios for the various tissues decreased in the order: bivalve gill Cd 28 > bivalve digestive gland Cd 18 > perch hepatic Cd 14. In the two lakes that were common to both the bivalve and perch studies, i.e. lakes Opasatica and Vaudray, accumulated Cd concentrations were consistently higher in the bivalve than in the perch. Cadmium-handling strategies were similar in the bivalve digestive gland and perch liver, in that Cd was mainly associated with the heat-stable protein (HSP) fraction. Furthermore, in these organs the contributions from the "mitochondria" and "lysosomes + microsomes" fractions were consistently higher than in the gill tissue. In the bivalve gill, the HSP fraction could only account for a small proportion (10+/-3%) of the total Cd burden, and the metal was instead largely sequestered in calcium concretions (58+/-13%). Along the Cd-exposure gradient, Cd detoxification appeared to be reasonably effective in the bivalve gill and digestive gland, as judged from the protection of the heat-denaturable protein (HDP) fraction. However, in both organs Cd concentrations did increase in potentially metal-sensitive organelles (mitochondria), and malondialdehyde concentrations increased along the exposure gradient in the gills (but not in the digestive gland). Cadmium detoxification seemed less effective in juvenile yellow perch. As total hepatic Cd increased, Cd concentrations increased in all sub-cellular fractions, including the HDP fraction that was well protected in the bivalve. The relative proportions of Cd in the various fractions did not vary appreciably along the exposure gradient and there was no evidence of a threshold exposure concentration below which sensitive metal pools were protected.     

Histological analysis of acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in zebrafish

Previous studies have demonstrated that acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by injection leads to inhibition of caudal fin regeneration in zebrafish. Since the TCDD exposure in these studies is systemic, it is possible that pathology in organs other than the fin could result in inhibition of fin regeneration. Therefore, histopathology of adult zebrafish (Danio rerio) organs was characterized following abdominal cavity injection of a TCDD dose (70ng/g). The most pronounced histopathologic changes 5 days post-injection included lipidosis and hypertrophy of liver hepatocytes and hypertrophy of gill lamellae. Effects of TCDD exposure on immunolocalization of the zebrafish aryl hydrocarbon receptor nuclear translocator (ARNT2), the heterodimer partner of the aryl hydrocarbon receptor (AHR2), and an AHR regulated gene cytochrome P450 1A (CYP1A) was also determined. ARNT2 was immunolocalized to the gastrointestinal tract, gill lamellae, kidney, ventricle of the heart, caudal fin, brain and liver of zebrafish. TCDD exposure had no measurable effect on ARNT2 abundance or localization. CYP1A was immunolocalized in TCDD exposed fish as a biomarker for cells with an activated AHR pathway. CYP1A was not detected in any tissue from vehicle exposed fish. Significant TCDD-dependent induction of CYP1A was detected in the proximal tubules of the kidney, in liver hepatocytes and in the gastrointestinal tract of TCDD exposed fish. Significant but lower TCDD-dependent CYP1A expression was evident in the gill, caudal fin and ventricle of the heart. Overall, TCDD exposure in adult zebrafish leads to histopathology similar to that reported in other fish species, and it appears unlikely that the histopathology in these organs completely explains the inhibition of fin regeneration.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptotic cell death and cytochrome P4501A expression in developing Fundulus heteroclitus embryos

Fundulus heteroclitus embryos were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early development using nanoinjection or water bath exposure. TCDD caused developmental abnormalities that included hemorrhaging, loss of vascular integrity, edema, stunted development and death. The LC(50) and LD(50) of TCDD for Fundulus embryos were approximately 19.7+/-9.5 pg TCDD/microl (water bath) and 0.25+/-0.09 ng TCDD/g embryo (nanoinjection). To identify a possible cause for these developmental abnormalities we analyzed the effects of TCDD on apoptotic cell death and cytochrome P4501A (CYP1A) expression in the embryos. TCDD exposure increased apoptotic cell death in several tissues including brain, eye, gill, kidney, tail, intestine, heart, and vascular tissue. CYP1A expression was also increased in the TCDD-exposed embryos predominantly in liver, kidney, gill, heart, intestine, and in vascular tissues throughout the embryo. There was co-occurrence of TCDD-induced apoptosis and CYP1A expression in some, but not all, cell types. In addition the dose response relationships for apoptosis and mortality were similar, while CYP1A expression appeared more sensitive to TCDD induction.     

Effect of Dose, Time, and Pretreatement on the Biliary Excretion and Tissue Distribution of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the Rat

Effect of Dose, Time, and Pretreatment on the Biliary Excretionand Tissue Distribution of 2,3,7,8-Tetrachlorodibenzo-p-dioxinin the Rat. KEDDERIS, L. B., ANDERSEN, M. E., AND BIRNBAUM,L. S. (1993). Fundam. Appl. Toxicol. 21, 405–411. Previous studies of the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) pretreatment on the biliary excretion and hepatic dispositionindicated that TCDD did not induce its own metabolic elimination.Pretreatment with TCDD did enhance its hepatic uptake. The presentwork was designed to further examine the effects of dose, time,and pretreatment on the tissue distribution and biliary eliminationof [³H]TCDD. Adult male F-344 rats were administered 0 or 100nmol [¹⁴C]TCDD or [³H]-TCDD/kg body weight po 3 days prior tobile duct cannulation and iv injection of 0 or 1 nmol [³H]TCDDor 1, 10, or 100 nmol [¹⁴C]TCDD/kg. Bile was collected for upto 8 hr while rats were maintained under pentobarbital anesthesia.Biliary TCDD and TCDD metabolites were quantified by liquidscintillation spectrometry. In naive animals which receivedno pretreatment, similar rates of excretion (% dose) were observedfollowing iv administration of 1 nmol [³H]TCDD/kg or 10 or 100nmol [¹⁴C-TCDD/kg. Metabolic elimination of highly purified[³H]TCDD (<99%) appeared to be linear with respect to timewith 0.8% of the dose being excreted in the bile over a 5- to8-hr collection period 0 or 24 hr after iv dosing (1, 10, or100 nmol/kg) and 72 hr after oral dosing (100 nmol/kg). In allgroups, higher concentrations of TCDD were found in liver versusfat, and perirenal fat concentrations were elevated relativeto epididymal fat concentrations, probably reflective of theenhanced blood perfusion of the former tissue. Pretreatmentenhanced hepatic concentrations and decreased fat concentrationsof the challenge dose. The time dependence of factors involvedin the dose-related hepatic accumulation of TCDD dispositionwas illustrated by the elevated liver:fat concentration ratiosobserved at the 100 versus 10 or 1 nmol/kg dose at 30 hr, butnot by 5–6 hr after dosing. In studies designed to evaluatethe role of CYP1A2 in the hepatic disposition of TCDD, pretreatmentwith isosafrole, a selective inhibitor of CYP1A2, diminishedhepatic concentrations of [³H]TCDD. In conclusion, TCDD wasmetabolically cleared at a fairly constant rate over an 80-hrperiod, and the rate of elimination was proportional with regardto dose. Studies with isosafrole suggest that TCDD is boundto CYP1A2 in the liver.