The Effect to Pretreatment on the Biliary Excretion of 2,3,7,8-Tetrachlorodibenzo-p-dioxin, 2,3,7,8-Tetrachlorodibenzofuran, and 3,3',4,4'-Tetrachlorobiphenyl in the Rat

The Effect of Pretreatment on the Biliary Excretion of 2,3,7,8-Tetrachlorodibenzo-p-dioxin,2,3,7,8-Tetrachlorodibenzofuran, and 3,3',4,4'-Tetrachlorobiphenylin the rat. MCKINLEY, M. K., KEDDERIS, L. B., and BIRNBAUM,L. S. (1993). Fundam. Appl. Toxicol. 21, 425–432. The laterally halogenated chemicals 2,3,7,8-tetrachlorodibenzofuran(TCDF) and 3,3',4,4'-tetrachlorobiphenyl (TCB) exhibit the samespectrum of toxic effects as 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD), the prototype and most toxic member of the halogenatedaromatic hydrocarbon family. Metabolism of all three compoundsappears to be the rate-limiting step for excretion, which isprimarily via the bile into the feces. Therefore, the biliaryelimination of TCDF, TCDD, and TCB was examined as an indirectmeasure of metabolism. Male F344 rats were anesthetized withpentobarbital, the bile duct was cannulated, and 0.1 µmol[³H]TCDD, [¹⁴C]TCDF, or [¹⁴C]TCB/kg body wt was administerediv. Bile was collected for 0–8 hr while the animals werekept under anesthesia. To determine if TCDF was able to induceits own metabolism in vivo, a single dose of 1.0 µmolTCDF/kg was administered to rats by oral gavage 3 days priorto iv injection of 0.1 or 0.3 µmol [¹⁴C]TCDF/kg. Biliaryexcretion and hepatic concentrations of [¹⁴C]TCDF were significantlyincreased in the pretreated animals. These results suggest anautoinduction of TCDF metabolism. Essentially all biliary [¹⁴C]TCDFradioactivity was attributable to metabolites. High-pressureliquid chromatography profiles of biliary radioactivity from0 to 4 hr were qualitatively different between naive and pretreatedrats. To determine if pretreatment with TCDD altered the metabolismof TCDF and vice versa, a single dose of 1.0 µmol TCDF/kgor 0.1 µmol TCDD/kg was administered by oral gavage 3days prior to iv injection of 0.1 µmol [³H]TCDD or [¹⁴C]TCDF/kg,respectively. TCDD pretreatment increased the metabolism andhepatic concentrations of [¹⁴C]TCDF in pretreated rats, whilebiliary elimination and hepatic concentrations of [³H]TCDD wereunaffected by pretreatment with TCDF. In a fourth experiment,the ability of TCDD to alter the metabolism of TCB, a laterallysubstituted polychlorinated biphenyl (PCB), was examined. Pretreatmentwith TCDD increased the metabolism of [¹⁴C]TCB by approximatelytwofold, but no differences in hepatic concentrations were seendue to the rapid elimination of TCB. Rats pretreated with Aroclor1254, a commercial mixture of PCBs, demonstrated significantlyincreased metabolism of [¹⁴C]TCB compared to the naive group.Therefore, under these experimental conditions, induction ofTCDF metabolism occurred in the rat upon pretreatment with eitherTCDD or TCDF at doses which also elicited enhanced hepatic uptake.TCDD and Aroclor 1254 induced the metabolism of TCB. These findingssuggest that repeated exposure to a chemical or to a mixtureof these halogenated aromatic hydrocarbons can result in morerapid metabolism and elimination from the body than followinga single, acute exposure.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin Pretreatment of Female Mice Altered Tissue Distribution but Not Hepatic Metabolism of a Subsequent Dose

2,3,7,8-Tetrachlorodibenzo-p-dioxin Pretreatment of Female MiceAltered Tissue Distribution but Not Hepatic Metabolism of aSubsequent Dose. CURTIS, L. R., JCERKVLIET, N. I., BAECHER-STEPPAN,L., AND CARPENTER, H. M. (1990). Fundam. Appl. Toxicol 14, 523–531.Lipid partitioning of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)inadequately explains its tissue distribution since higher concentrationsoccur in liver than fat except at high doses. This study providesIn vivo evidence that an inducible, saturable system plays apredominant role in disposition of [¹⁴C]TCDD in female miceat doses between 5 and 20 Mg/kg. Female C57BL/6J mice were gavagedwith 0, 5, or 15 µg, TCDD/kg, received a subsequent gavageof 5 or 20 11% [¹⁴C]TCDD after 6 days, and were killed 1 daylater. In mice pretreated with 5 and 15MgTCDD/ kg and subsequentlydosed with 20 µg [¹⁴C]TCDD/kg, liver weight and [¹⁴C]TCDDconcentration increased. Total liver [¹⁴C]TCDD burden increasedabout 50% in both pretreatment groups. Concentrations of [¹⁴C]TCDDin kidney, fat, heart, lung, gastrointestinal tract, but notplasma or splenic lymphocytes, decreased in a reciprocal manner.Alterations in absorption, concentrations of polar metabolitesof [¹⁴C]TCDD in liver, and hepatic lipid content failed to explainthese results. About 97% of hepatic ¹⁴C was hexane extractable.HPLC of this extract indicated [¹⁴C]TCDD was the only significantnonpolar form of radiolabel in liver. In mice pretreated with511% TCDD/kg and subsequently dosed with 5 µg [¹⁴C]TCDD/kg,a more marked pretreatment disposition response was observed.These results are consistent with a predominant role for aninducible, high affinity, low capacity system in whole animalpharmacokinetics of TCDD.     

Disposition of 2,3,7,8-tetrabromodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat: biliary excretion and induction of cytochromes CYP1A1 and CYP1A2

The biologic activity and pharmacokinetic properties of 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) are similar to those of the chlorinated congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Metabolism of both compounds appears to be rate-limiting for excretion, which is primarily via the feces. Therefore, the biliary elimination of TBDD and TCDD was examined as an indirect assessment of metabolism. Male F344 rats were anesthetized with pentobarbital, and 1 nmol/kg [3H]TBDD or [3H]TCDD was administered iv. Bile was collected for up to 8 hr while rats were maintained under anesthesia. The rate of biliary excretion of radioactivity was slightly greater for TCDD than TBDD (10% vs 7% in 5 hr). All biliary radioactivity was attributable to metabolites. High pressure liquid chromatographic (HPLC) profiles of biliary radioactivity were similar for [3H]TBDD and [3H]TCDD. To determine if pretreatment altered elimination kinetics, a single dose of 100 nmol/kg TBDD or TCDD was administered to rats by oral gavage 3 days prior to iv injection of 1 nmol/kg [3H]TBDD or [3H]TCDD, respectively. Biliary excretion of the radiolabeled dose was quantitatively and qualitatively unaffected by pretreatment despite a twofold increase in hepatic levels of radiolabel in the pretreated animals. Therefore, under these experimental conditions, autoinduction of TCDD and TBDD metabolism did not occur in the rat in vivo at doses which elicited enhanced hepatic uptake. In a second set of studies, the dose-response profiles for induction of cytochromes CYP1A1 and CYP1A2 by TBDD were characterized. The ED50 value for CYP1A1 induction (measured by ethoxyresorufin O-deethylase activity and radioimmunoassay (RIA) was estimated to be 0.8-1.0 nmol/kg, similar to what has been reported for TCDD. Induction of CYP1A2 (RIA) by TBDD appeared to be a more sensitive response over the dose range studied. Finally, comparison of hepatic CYP1A2 induction vs hepatic concentrations of TBDD 3 days following treatment with 10 vs 1 nmol/kg TBDD suggested that induction of CYP1A2 alone may not account for nonlinearities in dioxin disposition exemplified by dose-related increases in the ratio of dioxin concentrations in liver and adipose tissue.     

Subcellular Localization of TCDD Differs between the Liver, Lungs, and Kidneys after Acute and Subchronic Exposure: Species/Dose Comparisons and Possible Mechanism

Subcellular localization of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) and related compounds has been examined only in the liver.The objective of this study was (1) to examine and compare thesubcellular distribution of TCDD within hepatic and nonhepatic(lungs/kidneys) tissues of female Sprague-Dawley rats acutelyexposed to TCDD, (2) to analyze species comparisons in the subcellularlocalization of TCDD in multiple tissues, (3) to investigatethe effect of dose on subcellular distribution of TCDD, (4)to analyze the effect of subchronic exposure on the subcellulardistribution of TCDD, and (5) to examine one possible mechanismfor subcellular localization of TCDD. Female Sprague-Dawleyrats and B6C3F1 mice received a single oral dose of 0.1, 1.0,or 10 µg [³H]TCDD/kg body weight and subcellular fractionsof the liver, lungs, and kidneys were prepared by differentialcentrifugation 3 days after exposure. Analysis of the rat subcellularfractions revealed that TCDD was equally distributed betweenthe hepatic P9 (mitochondrial, lysosomal, and nuclear) and S9(cytosol and microsomal) fractions at all doses tested. In contrast,TCDD was concentrated in the P9 of rat nonhepatic tissues atall doses studied. Differential centrifugation of the hepaticS9 showed that TCDD was localized within the hepatic P100 (microsomal)fraction at all doses tested. In contrast, TCDD localized inpulmonary and renal S100 (cytosolic) fractions at all doses.The subcellular distribution of TCDD in the liver and lungsof acutely exposed B6C3F1 mice was similar to that observedin the rats. Although TCDD was concentrated within the renalP9, the remainder of TCDD in the S9 was evenly distributed betweenthe S100 and the P100 fractions of acutely exposed B6C3F1 mice.Subchronic exposure of B6C3F1 mice to 1.5 or 150 µg [³H]TCDD/kg/dayrevealed that increasing dose resulted in equal distributionof TCDD between the hepatic S9 and P9 versus concentration inthe renal P9. In addition, a dose-dependent increase in accumulationof TCDD in the hepatic P100 was accompanied by a dose-dependentincrease in TCDD localization in the renal S100 of mice subchronicallyexposed to TCDD. TCDD exposure in rats resulted in a dose-dependentincrease in the induction of CYP1A1 protein and associated enzymeactivity in hepatic, pulmonary, and renal microsomes. TCDD-inducedCYP1A2 protein levels and associated enzymatic activity wereonly present in hepatic microsomes. This is the first reportto suggest that subcellular distribution of TCDD differs betweenhepatic and nonhepatic tissues and demonstrate that the liver-specificmicrosomal localization of TCDD in female Sprague-Dawley ratsalso occurs in the liver of female B6C3F1 mice acutely or subchronicallyexposed to TCDD. In addition, these data are consistent withthe hypothesis that the hepatic sequestration of TCDD is dueto an interaction with CYP1A2. Furthermore, the lack of pulmonary/renalsequestration coupled with the lack of localization of TCDDin pulmonary/renal microsomes also supports the role of CYP1A2as a hepatic microsomal binding protein involved in TCDD sequestration.     

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.     

Hepatic Uptake and Metabolism of 2,3,7,8-Tetrachlorodibenzo-p-dioxin and 2,3,7,8-Tetrachlorodibenzofuran

The pharmacokinetics of TCDD and related compounds is congener,dose, and species specific, with urinary and biliary excretionbeing dependent on the metabolism of these compounds. Isolatedhepatocytes and liver slices in suspension culture and hepaticmicrosomes were used as in vitro models to assess the hepaticuptake and metabolism of [³H]- and [¹⁴C]- TCDD and [³H]TCDF(0.01–1.0 µM) in control and induced (5 µgTCDD/kg, 3 days earlier) male Sprague-Dawley rats. TCDD pretreatment,with an increase in cytochromes P450 1A1 and 1A2 (CYP1 Al, CYP1A2),produced an increase in the hepatic uptake of TCDD, while noincrease in the hepatic uptake of TCDF was observed. The resultsare consistent with CYP1A2 serving as a hepatic binding proteinfor TCDD but not for TCDF. The rates of metabolism of TCDD andTCDF were directly proportional to their concentrations, indicatingthat the reaction follows first order kinetics at concentrationsfrom 0.01 to 1.0 µM. Very limited metabolism of TCDD andTCDF was observed in control rat liver (0.45 and 3.2 pmol/hr/ghepatocyte wet wt at 0.1 µm, respectively). TCDD inducedits own rate of metabolism about two- to fivefold at 1.0 µMbut no induction was observed at 0.01 and 0.1 µM. In contrast,TCDD markedly induced the rate of TCDF metabolism at all substrateconcentrations. While the results support the role of rat CYP1A1in TCDF metabolism, the data suggest that CYP1 Al or CYP1A2may not metabolize TCDD. These results also support the hypothesisthat the more rapid metabolism and excretion of TCDF accountsfor the relative resistance of the rat to the acute toxicityof TCDF. Comparative studies in rat and human liver microsomesfound that TCDF metabolism exhibited first order kinetics inboth species. Furthermore, the rate of TCDF metabo-lism in humanliver microsomes was similar to that of control rat liver microsomes.Together the results suggest that TCDF will be far more persistentin rats, and possibly humans, following exposure at low doseswhich do not significantly induce cytochrome P450 1A1 and/or1A2.     

Assessment of the Developmental Toxicity, Metabolism, and Placental Transfer of N,N-Dimethylformamide Administered to Pregnant Rats

This study evaluates the developmental toxicity and placentaland milk transfer of N,N-dimethylformamide (DMF) in rats. Sprague-Dawleyrats were given 0, 50, 100, 200, and 300 mg DMF/kg/day, by gavage,on Gestational Days (GD) 6 through 20. Maternal toxicity wasindicated by depressions in weight gain and food consumptionat doses 100 mg/kg. Fetal toxicity was indicated by decreasedfetal body weight at doses 100 mg/kg, and by increased incidencesof two skeletal variations (absent or poorly ossified supraoccipitaland sternebrae) at 200 and 300 mg/kg. Thus, the maternal anddevelopmental no-observed-adverse-effect level was 50 mg/kg/day.The time course disposition of [¹⁴C]DMF was examined over a48-hr period in GD12- and GD18-pregnant rats after a singleoral dose of 100 mg [¹⁴C]DMF/kg Peak concentrations of radiocarbonoccurred within 1 hr after dosing. Embryonic (GD 12) and fetal(GD18) tissues accounted for 0.15 and 6% of the administereddose, respectively. Levels of radiocarbon in embryonic and fetaltissues were equal or slightly less than in maternal plasmaup to 8 and 24 hr, respectively, and higher thereafter. HPLCanalysis performed at intervals from 1 to 8 hr on GD12 and 1–24hr on GD18 indicated that unchanged DMF and metabolites werereadily transferred to the embryonic and fetal tissues, wheretheir levels were generally equal to those in maternal plasma.The parent compound accounted for most of the radioactivityuntil 4–8 hr and then decreased. N-Hydroxymethyl-N-methylformamide(HMMF) and N-methylformamide (NMF) were the predominent metabolitesand increased with time. Much lower concentrations were foundfor formamide and N-acetyl-S-(N-methylcarbamoyl)cysteine. Transferof radioactivity into milk was studied in dams given a singleoral administration of 100 mg [¹⁴C]DMF on Lactation Day 14.DMF, HMMF, and NMF were found in the milk at concentrationsequal to those in plasma.     

Chlordecone Pretreatment Alters [14C]Chlordecone and [14C]Cholesterol Transport Kinetics in the Perfused Rat Liver

Previous work demonstrated that pretreatment of mice with lowdoses of the organochlorine insecticide chlordecone (CD) alteredthe tissue disposition of a subsequent [¹⁴C]CD or [¹⁴C]- cholesterolchallenge dose. The profile of these changes was consistentwith the induction of a protein integral to hepatic CD/cholesterolturnover. The present study was undertaken to confirm similarin vivo effects in the rat and to analyze potential CD-inducedchanges in hepatic transport kinetics in the per fused rat liver.For in vivo experiments, male, Sprague-Dawley rats were treatedwith CD (5, 15, or 40 mg/kg) and challenged 3 or 7 days laterwith a 5 mg/kg [¹⁴C]CD tracer dose. Rats challenged 3 days aftertreatment and evaluated 16 hr later showed a dose-dependentdecrease in hepatic [¹⁴C]CD relative to controls. This decreasecould not be attributed to alterations in liver mass or totalliver lipid. For kinetics studies, rats received 15 mg/kg CDand livers were perfused 3 days later. Following a brief (5–7min) single-pass perfusion, the perfusate was replaced withrecirculating buffer containing albumin-bound [³H]oleic acidor high-density lipoprotein-bound [¹⁴C]CD or [¹⁴C]cholesterolLivers from pretreated animals had significantly decreased ratesof [¹⁴C]CD and [¹⁴C]cholesterol uptake. Efflux of [¹⁴C]CD andbiliary excretion of [¹⁴C] were increased. No changes were observedin uptake or biliary excretion of [³H]oleic acid. SDS-PAGE ofhepatic cytosol revealed an enhanced band in tensity correspondingto a M_r of 25,600 in livers from pretreated rats. These resultsare supportive of a competitive interaction between cholesteroland CD for proteins associated with hepa tocellular transportand excretion and suggest that CD pretreatment may have an inductiveeffect on these proteins.     

Biliary Excretion Appears Rate Limiting for Hepatic Elimination of Benzo[a]pyrene by Temperature-Acclimated Rainbow Trout

Biliary Excretion Appears Rate Limiting for Hepatic EliminationOf Benzo[a]pyrene by Temperature-Acclimated Rainbow Trout. CURTIS,L. R., FREDERICKSON, L. K., AND CARPENTER, H. M. (1990) Fundam.Appl. Toxicol. 15, 420–428. Previous work demonstratedthat mixed function oxidase activities of hepatic microsomesfrom cold- and warm-acclimated rainbow trout were similar whenassayed at temperatures to which fish were acclimated. This"ideal temperature compensation" was partially explained byconstitutive differences in microsomes. In the work reportedhere, rainbow trout were acclimated at 10 or 18°C for 4weeks and then ip injected with 10 µmol [³H] or [¹⁴C]benzo[a]pyrene(BP)/kg in one of two temperature regimens. First, fish wereacclimated and exposed at the same temperature and killed after4, 24, or 48 hr. Concentrations of [³H]BP equivalents in liver,bile, and fat but not in plasma, muscle, intestine, gill, orkidney increased with time. There were no differences in hexaneor ethyl acetate extract-able [³H] or [¹⁴C]BP tissue concentrationsin 10 and 18°C-acclimated fish exposed at their acclimationtemperatures. At 24 hr after injection, biliary excretion of[³H]BP equivalents was about twofold higher at 18°C thanat 10°C. Therefore, warmer temperature stimulated biliaryexcretion without a marked effect on in vivo BP metabolism.In the second regimen, 10 and 18°C-accli-matedfish wereshifted to 14°C, injected with [³H] or [¹⁴C]BP 1 hr later,and killed after an additional 24 hr. There were no differencesin tissue concentrations of total [³H]BP equivalents betweenacclimation groups at 14°C. However, the biliary concentrationof [¹⁴C]BP not extracted by ethyl acetate was significantlyhigher in bile from 10°C-acclimated fish than from 18°C-acclimatedfish when both groups were exposed at 14°C. In this case,in vivo BP metabolism was altered without coincident effectson biliary excretion. Aryl hydrocarbon hydroxylase assayed at14°C was about threefold higher in hepatic microsomes from10°C-acclimated fish than from 18°C-acclimated fish.Exposure temperature selectively modulated BP metabolism andbiliary excretion in temperature-acclimated rainbow trout.     

Adduction of Hemoglobin and Albumin in Vivo by Metabolites of Trichloroethylene, Trichloroacetate, and Dichloroacetate in Rats and Mice

Adducts to macromolecules from trichloroethylene formed by invivo and in vitro metabolism have been reported by many investigators.We examined the in vivo adduction of the blood proteins hemoglobin(Hb) and albumin in rats and mice dosed orally with [¹⁴C]trichloroethylene([¹⁴C]TRI) to explore the development of a protein adduct biomarkerof TRI exposure. We also examined the adduction of these twoproteins from doses of [¹⁴C]trichloroacetate (TCA) and [¹⁴C]dichloroacetate(DCA), two metabolites of TRI. Association of label with albuminpeaked at 4–8 hr in the rat (2480 nmol eq TRI/mg protein)and 2–4 hr in the mouse (1580 nmol eq TRI/mg protein).The decay was exponential with a half-life consistent with thatof rat or mouse albumin (approx 24 hr). The time course of labelwith Hb was characterized by an early plateau at 8 hr in rat(28 nmol eq TRI/ mg protein), 4 hr in mouse (7 nmol eq TRI/mgprotein), and followed by a slow steady increase, peaking at120 hr (54 nmol eq TRI/mg protein, rat; 38 nmol eq TRI/mg protein,mouse). This apparent binding was linear with dose in the rat,but was convex in the mouse albumin (mouse Hb label was belowdetection at low dose). We also found that a portion of theirreversibly associated label, referred to by previous investigatorsas "binding," could be accounted for as metabolic incorporationof label into glycine and serine. The fraction accounted forby metabolic incorporation was constant in albumin (approximately), while in Hb, this portion was time dependent, approximately30% at the early sampling time, 75% at the late time, implyingthe observed late increase could be accounted for by metabolicincorporation. TCA and DCA also formed Hb and albumin adducts.Portions of this binding was also due to metabolic incorporation.The pattern of the binding from TCA in albumin was differentfrom that of TRI, implying a route to adduct from TRI whichdoes not proceed through TCA.     

Synergistic interactions of 2,3,7,8-TCDD and 2,2',4,4',5,5'-hexachlorobiphenyl in C57BL/6J and DBA/2J mice: role of the Ah receptor

Treatment of C57BL/6J mice with 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP, 500 mumol/kg) elevated hepatic cytosolic Ah receptor levels 82-107% for up to 14 days. Scatchard analysis of the [3H]2,3,7,8-TCDD (TCDD)-Ah receptor saturation binding curves from corn oil and HCBP treated rats gave KD values of 0.80 and 0.90 nM, respectively and confirmed that treatment with HCBP did not significantly alter receptor-radioligand affinities. Administration of HCBP to DBA/2J mice did not result in detectable hepatic cytosolic Ah receptor levels. Cotreatment of C57BL/6J mice with HCBP (500 mumol/kg) at a dose level of TCDD (1 nmol/kg) which elicited less than 10% of the maximum induction response resulted in significant synergistic induction of hepatic EROD and AHH [compared to animals treated only with TCDD (1 nmol/kg)]. In contrast, cotreatment of C57BL/6J mice with HCBP (500 mumol/kg) and maximally inducing dose levels of TCDD (100 or 500 nmol/kg) resulted in either a slight or no difference in the induction of AHH or EROD compared to the induction responses observed in mice treated only with TCDD. In contrast, cotreatment of DBA/2J mice with TCDD and HCBP (500 mumol/kg) resulted in significant synergistic induction of AHH and EROD at both submaximal (10-500 nmol/kg) and maximal (5000 nmol/kg) induction levels of TCDD. The only significant interactive effect of HCBP (500 mumol/kg) on the toxicity of TCDD in C57BL/6J and DBA/2J was protection from body weight loss observed after cotreatment of HCBP and TCDD in DBA/2J mice.     

Induction of Cytochrome P450 Isoenzymes after Toxicokinetic Interactions between 2,3,7,8-Tetrachlorodibenzo-p-dioxin and 2,2',4,4',5,5'-Hexachlorobiphenyl in the Liver of the Mouse

One group of male C57BL/6J mice received a single oral doseof 1 nmol 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg. Sixother groups received single oral doses of 100, 300, or 1000µmol2,2',4,4',5,5'-hexachlorobiphenyl (HxCB)/kg, alone or in combinationwith 1 nmol/kg TCDD. Liver deposition of both compounds wasstudied at Day 3 after dosage. Hepatic CYP1A1 and CYP1A2 proteinlevels and related 7-ethoxyres-orufin-O-deethylation (EROD)and acetanilide 4-hydroxylation (ACOH) activities were alsostudied. A significant increase in the hepatic deposition ofTCDD was observed in all three mixed dose groups but TCDD didnot influence hepatic HxCB deposition. TCDD did increase bothCYP1A1 and CYP1A2 protein levels. In the HxCB-treated groups,CYP1A2 levels were also increased in a dose-dependent way butCYP1A1 levels were not increased. CYP1A2 activities (ACOH),but not protein levels, in the TCDD groups cotreated with HxCBwere higher than those in the group treated with TCDD alone.CYP1A1-dependent EROD activity and CYPlA2-dependent ACOH activitywere induced in all treated dose groups. It is concluded thatthe present results do not confirm a direct role of CYP1A2 inductionin the increase of hepatic TCDD levels by HxCB cotreatment inthe mixed HxCB/TCDD dose groups. However, in this aspect, thediscrepancy between CYP1A2 activities and protein levels remainsto be explained.     

Use of Model-Based Compartmental Analysis to Study Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Vitamin A Kinetics in Rats

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic,widespread environmental contaminant that has dramatic adverseeffects on the metabolism of vitamin A. We used model-basedcompartmental analysis to investigate sites and quantitativeimpacts of TCDD on vitamin A kinetics in rats given an oralloading dose of TCDD in oil (3.5 µg/kg) followed by weeklymaintenance doses (0.7 µg/kg) or oil only. [³H]retinylin its plasma transport complex (experiment 1) or lymph containingchylomicrons labeled mainly with [³H]retinyl esters (experiment2) were administered iv, and tracer kinetics in plasma, liver,carcass, urine, and feces were measured for up to 42 days. TCDDtreatment caused significant reductions in liver vitamin A levelsand significant changes in tracer kinetics and tracer excretion.A four-compartment model was used to fit tracer data for experiment1; for experiment 2, compartments were added to describe themetabolism of newly absorbed vitamin A. The compartmental modelspredict that TCDD caused a slight delay in plasma clearance(via an increased recycling to plasma), and in liver processing,of chylomicronderived vitamin A. Models for both experimentspredict that TCDD exposure did not affect the fractional uptakeof plasma retinol from the rapidly turning-over extravascularpool, but it doubled the fractional transfer of recycled retholfrom slowly turning-over pools of vitamin A to plasma The residencetime for vitamin A was reduced by 70% in TCDD-treated rats,transfer into urine and feces was tripled, and vitamin A utilizationrates were significantly increased. Since our results do notindicate that retino1 esterification is inhibited, we hypothesizethat some of the significant effects of TCDD on vitamin A metabolismresult from increased catabolism and mobilization of vitaminA from slowly turning-over pools (especially the liver).     

Pharmacokinetics, Tissue Distribution, and Placental Permeability of All-trans- and 13-cis-N-Ethyl Retinamides in Pregnant Hamsters

Pharmacokinetics, Tissue Distribution, and Placental Permeabilityof All-trans- and 13-cis-N-Ethyl Retinamides in Pregnant Hamsters.HOWARD, W. B., WILLHITE, C. C., OMAYE, S. T., AND SHARMA, R.P. (1989). Fundam. Appl. Toxicol. 12, 621–627. Retinamideshave clinical applications in therapy of dermatologic disease,have cancer chemopreventive/chemotherapeutic activities, andpossess larger therapeutic ratios than their acidic congeners.The N-ethyl-all-trans-retinamide (NERA) and its 13-cis congener(CNERA) failed to induce terata in hamsters, but an equivalentoral dose of all -trans- or 13-cis-retinoic acid was associatedwith a significant teratogenic response. Following intubationof 11.4 mg/kg of [³H]NERA or[³H]CNERA to pregnant hamsters duringa sensitive stage of development, radioactivity accumulatedin maternal bladder and liver. Although plasma concentrationsof the parent retinamides declined to nondetectable levels within12 hr of dosing, near-peak concentrations of retinamide metabolitesper sisted in maternal plasma until termination of the study(96 hr). Cis/trans isomerization of each retinamide at Cl3 occurred,but only 15–20% of the total dose could be accounted foras parent retinamide and its Cl3 isomer. The retinamides werenot metabolized to detectable concentrations of circulatingall-trans- or 13-cis-retinoic acid. Although the label associatedwith the retinamides and their biotransformation products crossedthe placenta, there was no evidence for preferential accumulationin embryonic or fetal tissues. The results presented here showthat the reduced teratogenic potency of retinamides comparedto acidic retinoids cannot be ascribed to reduced placentaltransfer.     

Metabolism of [14C]Acetylisoniazid and [14C]Acetylhydrazine by the Rat and Rabbit

Metabolism of [¹⁴C]Acetylisoniazid and [¹⁴C]Acctylhydrazineby the Rat and Rabbit Thomas, B. H., WHITEHOUSE, L. W., andZEITZ, W. (1984). Fundam. Appl. Toxicol. 4, 646–653. Malerats and rabbits were singly dosed with either l-[¹⁴C]acetylisoniazid (acetylisonicotinoylhydrazine, acctyl-INH, 200 mg/kgpo) or 1-[^l4C]acetylhydrazine (50 or 100 mg/kg ip). Urine andexpired ¹⁴CO₂ were collected, and after 6 hr the animals werekilled for the analysis of tissue ¹⁴C concentrations and covalentbinding of ¹⁴C to hepatic protein. Rats excreted proportionatelymore ¹⁴C in urine and had lower ¹⁴C levels in their tissuescompared to rabbits. When acetyl-INH was administered, covalenthepatic protein binding of the acetyl moiety was greater inthe rabbit than the rat, but the opposite was observed whenacetylhydrazine was administered. Analysis of blood and urineby TLC revealed that the rabbit more rapidly metabolized bothacetyl-INH to acetylhydrazine, and acetylhydrazine to diacetylhydrazinethan did the rat. These observations suggest that higher amidaseactivity in the rabbit compared to the rat leads to faster conversionof acetyl-INH to acetylhydrazine which in turn leads to greatercovalent binding and hepatotoxicity.     

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.     

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.     

Disposition of the Aromatase Inhibitor LY56110 and Associated Induction and Inhibition Studies in Rats, Dogs, and Monkeys

Disposition of the Aromatase Inhibitor LY56110 and AssociatedInduction and Inhibition Studies in Rats, Dogs, and Monkeys.LINDSTROM, T. D., AND WHITAKER, G. W. (1987). Fundam. Appl.Toxicol. 8, 595–604. Compound LY56110 was well absorbedbut slowly excreted in the rat, dog, and monkey. Oral administrationof 5 mg/kg of [¹⁴C]LY56110 (5-bis(4-chlorophe-nyl)methylpyrimidine)to the rat, monkey, and dog resulted in a total excretion of68, 65, and 30% of the radioactivity within 5 days, respectively.Very low urinary excretion was observed in the rat and dog (2%),with fecal excretion being the predominant mode of eliminationin all three species. The plasma radioactivity half-life was49, 41, and greater than 100 hr in the rat, monkey, and dog,respectively. The plasma half-life of parent compound was 18hr in the rat and 10 hr in the dog. LY56110 accounted for only25, 12, and 1% of the plasma radioactivity area under the curvein the rat, dog, and monkey, respectively. High levels of radioactivitywere observed in the target tissues of fat, adrenals, and ovariesof rats. LY56110 induced hepatic cytochromes b₅ and P-450 andcytochrome c reductase in rats after 14 days of oral dosingat 10 mg/kg but not in monkeys after 10 days of oral dosingat 10 mg/kg. The compound was more potent than aminoglutethimideor cimetidine in inhibiting hepatic ethylmorphine and p-nitro-anisoledemethylase activity in vitro. LY56110 also inhibited ethinamate-inducedsleeping time in rats in vivo. The compound induced a reversetype I binding spectrum with rat ovarian microsomes.     

4,4'-Methylenebis(2-chloroaniline) (MOCA): The Effect of Multiple Oral Administration, Route, and Phenobarbital Induction on Macromolecular Adduct Formation in the Rat

The effect of multiple oral administration of MOCA, a suspecthuman carcinogen, was studied in the adult male rat. As manyas 28 consecutive daily doses of [¹⁴C]MOCA at 28.1µmol/kgbody wt (5 µC1/day) were administered and rats were euthanizedat weekly intervals for 7 weeks. MOCA adduct formation for globinand serum albumin was evaluated by determination of [¹⁴C]MOCAcovalent binding. The covalent binding associated with globinshowed a linear increase over the 28-day exposure period with342 fmol/mg globin 24 hr after the final dose. More extensivecovalent binding was detected for albumin with 443 fmol/mg albuminafter the final dose, but increases were not linear. After cessationof dosing, the albumin adduct levels decreased rapidly (t _1/2=4.6 days) in relation to globin adduct levels (t _1/2 =16.1days). The MOCA-globin adduct t _1/2 is consistent with thatdetermined after a single 281 µmol/kg oral dose of MOCA.Significant differences related to route of administration weredetected for 24-hr globin covalent binding with ip > po >dermal. Distribution of undifferentiated [¹⁴C]MOCA was highestin the liver at 24 hr with tissue levels for liver > kidney> lung > spleen > testes > urinary bladder. Inductionof cytochrome P450 enzymes by administration of phenobarbital(100 mg/kg/day/3 days) resulted in a significant (p < 0.05)increase in MOCA-globin adduct formation detected with 33.5pmol/ mg globin for induced rats versus 13.6 pmol/mg globinfor control rats. Although MOCA-globin and albumin adducts showdiffering stability, quantification of such MOCA adducts maybe useful for long-term industrial biomonitoring of MOCA.     

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.