Pulmonary Bioavailability and Fine Particle Enrichment of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in Respirable Soil Particles

The pulmonary bioavailability of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) and the enrichment of polychlorinated dioxins (PCDDs)and furans (PCDFs) in fine particles were evaluated to assessthe implications that these factors have on risk and exposureassessments. Respirable subfractions of PCDD-contaminated soilfrom a former 2,4,5-trichlorophenoxyacetic acid manufacturingsite were isolated by chemical dispersion and gravity sedimentation.Analysis of the subfractions revealed that there was a size-dependentenrichment of PCDDs and PCDFs, with smaller particles more highlycontaminated. TCDD was enriched up to 33-fold as compared tounfractionated soil. Soil and laboratory-recontaminated galliumoxide, which served as the positive control, were administeredby intratracheal instillation to female Sprague-Dawley rats.Animals were terminated up to 28 days following treatment andpulmonary bioavailability of TCDD was assessed by hepatic enzymeinduction and TCDD concentration. Enzyme induction was dependenton the duration of exposure with up to 56 and 918% increasesin cytochrome P450 and aryl hydrocarbon hydroxylase (AHH) activity,respectively, following exposure to PCDD-contaminated soil.There was no significant difference in AHH induction betweenanimals which received contaminated soil and those treated withthe positive control. Hepatic concentration of TCDD in soil-exposedrats was 115, 101, and 179% of positive controls at 1, 7, and28 days post-treatment, suggesting that the soil or co-contaminantsinfluenced retention of TCDD in the liver. These data indicatethat the relative pulmonary bioavailability of TCDD on respirablesoil particles is 100% as compared to laboratory-recontaminatedgallium oxide and that PCDDs and PCDFs are highly enriched onrespirable particles. Utilization of these results will reducethe uncertainty and improve the accuracy of envi ronmental riskassessments of PCDDs and PCDFs.     

Ingestion of Soil Contaminated with 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Alters Hepatic Enzyme Activities in Rats

Ingestion of Soil Contaminated with 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) Alters Hepatic Enzyme Activities in Rats. LUCIER, G.W., RUMBAUGH, R. C., MCCOY, Z., HASS, R., HARVAN, D., AND ALBRO,P. (1986). Fundam. Appl. Toxicol. 6, 36.4–371. Femalerats were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)in either corn oil or contaminated soil from the Minker sitein Missouri. Eight doses ranging from 0.015 to 5 µg TCDD/kgwere used in the corn oil group; the range was 0.015 to 5.5TCDD/kg in the TCDD-contaminated soil group. Rats in a thirdgroup were given equal amounts of soil uncontaminated with TCDD.No acute toxicity or effects on body weight gain were observedat these doses. In general, equivalent doses of TCDD in cornoil or TCDD in soil produced similar increases in hepatic arylhydrocarbon hydroxylase activity (AHH) and UDP glucuronyltransferaseactivity although effects were slightly greater in the TCDD–cornoil groups. In the corn oil groups, the induction of AHH rangedfrom about 30-fold at the highest dose to twofold at the lowestdose studied. TCDD also caused an increase in cytochrome P-450concentration 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 ppbin the TCDD-contaminated soil group. Our results suggest thatthe bioavailability of TCDD in soil in rats is approximately50%. Therefore, ingestional exposure to TCDD-contaminated soilmay constitute a significant health hazard in view of its extremelyhigh toxicity and relatively high bioavailability.     

Interactive effects of estradiol and 2,3,7,8-tetrachlorodibenzo-p-dioxin on hepatic cytochrome P-450 and mouse uterus

Weanling C57B/6 female mice treated with 6 micrograms/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 3 times a week for one month (total dose 72 micrograms/kg) were observed to have greatly reduced relative uterine weights and histopathological changes in the uterus. Weanling CD-1 female mice were then treated with estradiol (E2) subcutaneously daily for 2 weeks. Half the mice also received 10 micrograms/kg TCDD in corn oil: acetone (9:1) by gavage 4 times during the second week. Control mice received either no E2 or no TCDD. Mice were killed on day 15 and autopsied. Relative uterine weights increased with increasing E2 doses; however, TCDD decreased this effect of E2 markedly. Liver microsomes from these animals showed that cytochrome P1-450 and P3-450 and, aryl hydrocarbon hydroxylase (AHH) induction by TCDD were independent of E2 dosage. Epoxide hydrolase was induced in TCDD treated animals. Gels showed an E2 dose dependent decrease in a protein migrating near epoxide hydrolase and 'P-450a' in animals receiving both E2 and TCDD. These results suggest that: E2 may act at the TCDD receptor; the TCDD receptor may be related to the estrogen receptor; the anti-estrogenic effects of TCDD are possibly independent of the Ah locus and AHH induction, and in TCDD-treated mice a protein migrating near epoxide hydrolase and 'P-450a' may be controlled by estrogen levels.     

Bioavailability of Soil-Bound TCDD: Oral Bioavailability in the Rat

Bioavailability of Soil-Bound TCDD: Oral Bioavailability inthe Rat. SHU, H., PAUSTENBACH, D., MURRAY, F. J., MARPLE, L.,BRUNCK, B., DEI ROSSI, D., TEITELBAUM, P. (1988). Fundam ApplToxicol. 10, 648–654. The implications to the public healthof trace amounts of 2,3,7,8-TCDD in the environment are underevaluation by regulatory agencies in the United States and WesternEurope. One major consideration in such evaluations is the contributionto human exposure via ingestion of TCDD-contaminated soil. An80% figure is under consideration by some regulators for estimatedhuman exposure. A contractor for one agency has, in fact, useda value of 1007% bioavailability for estimating human bioavailability.Several studies have investigated the oral bioavailability ofTCDD from contaminated soil in animals. Most have reported estimatesof 25–50%, although one has reported <0.5 and 85%,depending on the source of the contaminated soil. This paperreports an oral bioavailability of approximately 43% in therat dosed with three environmentally contaminated soil samplesfrom Times Beach, Missouri. This figure did not change significantlyover a 500-fold dose range of 2 to 1450 ng TCDD/kg of body weightfor soil contaminated with approximately 2, 30, or 600 ppb ofTCDD. The relevance of animal oral bioavailability data forthe human remains to be evaluated. However, since regulatoryagencies use animal data for extrapolating to humans, the 43%or 25–50% figure would be more accurate than the 80 or100% estimates.     

Induction of cytochrome P450IA1 in mouse hepatoma cells by several chemicals. Phenobarbital and TCDD induce the same form of cytochrome P450

The mouse hepatoma cell line Hepa-1 was studied for aryl hydrocarbon hydroxylase (AHH) inducibility by sixteen compounds known to be inducers of cytochrome P450 of different "classes". Both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and sodium phenobarbital induced AHH activity. A cytochrome P450IA1-specific (P1-450) mouse cDNA probe was used to quantitate mRNA induction. There was a good correlation between the amount of cytochrome P450IA1 mRNA induced and AHH activity. Immunoblots with monoclonal antibody 1-7-1, which recognizes rat liver P450IA1 and P450IA2 (P450c and P450d, respectively), showed that both phenobarbital and TCDD increase the amount of a P450 isozyme immunorelated to P450IA1 in this cell line. Hepa-1 mutants with no AHH inducibility (no functional P450IA1 structural gene; no Ah receptor; no nuclear translocation of the inducer-receptor complex; and presence of dominant repressor) did not respond to phenobarbital. The cytosolic receptor for TCDD (Ah receptor) was characterized to see if phenobarbital induced cytochrome P450IA1 mRNA and the hydroxylase enzyme through the same mechanism as TCDD. 20 mM Phenobarbital almost completely abolished the binding of 3H-TCDD to the cytosolic receptor. These data indicate that phenobarbital can be a weak ligand for the Ah receptor and thus induce cytochrome P450IA1 and AHH activity. The observation increases the list of different P450 forms inducible by phenobarbital.     

Bioavailability of Soil-Bound TCDD: Dermal Bioavailability in the Rat

Bioavailability of Soil-Bound TCDD: Dermal Bioavailability inthe Rat. SHU, H., TEITELBAUM, T., WEBB, A. S., MARPLE, L., BRUNCK,B. DEI ROSSI, D., MURRAY, J., AND PAUSTENBACH, D. (1988). Fundam.Appl. Toxicol. 10, 335-343. 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD), an unwanted by-product formed during the manufactureof hexachlorophene and phenoxyherbicides, has been found asan environmental contaminant in many U.S. and Western Europeansites. This study examines in the rat the degree of dermal absorptionof TCDD bound to soil. Such information would assist regulatoryagencies in evaluating the degree of exposure of humans whocome in contact with TCDD-contaminated soil. Several parameterswhich may influence dermal absorption were studied, includingTCDD dose, duration of contact, presence of crankcase oil asa co-contaminant, and environmentally contaminated vs laboratory-preparedsoil. The dermal penetration of TCDD following 4 hr of contactwith skin was approximately 60% of that following 24 hr of contact(P 0.05). Following 24 hr of contact with the skin, the degreeof dermal uptake of TCDD contaminated soil was approximately1% of the administered dose. Under the conditions of the presentstudy, the degree of uptake does not appear to be influencedto any significant extent by the concentration of TCDD on soil,the presence of crankcase oil as co-contaminants, or by environmentallyvs laboratory-contaminated soil. Although a number of parametersexamined in this study did not significantly influence the degreeof dermal absorption of TCDD in the rat following 24 hr of contactwith the contaminated soil, the unqualified use of the 1% valueto estimate human exposure would overestimate human exposure,since there is general agreement among researchers that ratskin tends to be more permeable than human skin to highly lipid-solublecompounds such as TCDD.     

Changes in Hepatocyte Ploidy in Response to Chromium, Analyzed by Computer-Assisted Microscopy

Changes in Hepatocyte Ploidy in Response to Chromium, Analyzedby Computer-Assisted Microscopy. GARRISON, J.C., BISEL, T. U.,PETERSON, P., AND UYEKJ, E. M. (1990). Fundam. Appl. Toxicol.14, 346–355. BDF1 mice were given single injections ofsodium dichromate (25 mg/kg) on an acute (6 hr to 7 days) orintermediate (2-4 weeks) basis, or multiple injections (12.5mg/kg) on a chronic (4.5 months) basis. Observed hepatic changesincluded programmed cell death (apoptosis) in the periportalregion with acute exposure and fusion of liver lobes with chronicexposure. Response to chromate exposure was measured by changein hepatocyte nuclear ploidy state (e.g., the proportion ofdiploid, tetraploid, and octaploid nuclei) based on computer-assistedimaging from histological sections. The computer-assisted imagingsystem used in this study was superior to traditional methodsbecause it (1) allows rapid ploidy determinations from histologicalmaterial and (2) can be used to collect regional information.Regional differences in ploidy were seen to occur in a consistentfashion among both control and treated animals. Nuclei adjacentto the portal triad had the lowest ploidy value (highest proportionof diploid nuclei), an intermediate value was found adjacentto the central vein, and the highest ploidy was found in themidzone. These three ploidy-based zones roughly correspond tothe three functional zones of A. M. Rappaport (1973, Microvasc.Res. 6, 212–228) and W. H. Lam-ers el al. W. H. Lamers,A. Hilberts, E. Furt, J. Smith, G. N. Jonges, J. F. Van Noorden,J. W. G. Janzen, R. Charles, and A. F. M. Moorman, 1989, Hepatology,10, 72–76. Temporal changes in ploidy were seen amongcontrol animals (all zones), with young animals (56 days) displayingrelatively low ploidy values compared to older animals (184days). Chromate exposure caused increased ploidy (all zones)among animals treated on an acute basis (the youngest animals).Chromate had no apparent effect on ploidy among animals treatedfor longer periods of time, probably because of age-relatedfactors.     

Age-Related Changes in Dermal Absorption of 2,3,7,8-Tetrachlorodibenzo-p-dioxin and 2,3,4,7,8-Pentachlorodibenzofuran

Age-Related Changes in Dermal Absorption of 2,3,7,8-Tetrachlorodibenzo-p-dioxinand 2,3,4,7,8-Pentachlorodibenzofuran. BANKS, Y. B., BREWSTER,D. W., AND BIRNBAUM, L. S. (1990). Fundam. Appl. Toxicol. 15,163–173. Changes in the structure and function of agedskin may alter percutaneous absorption of environmental compoundssuch as the halogenated aromatic hydrocarbons. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)and 2,3,4,7,8-pentachlorodibenzofuran (4PeCDF) were previouslyfound to be poorly absorbed in 3 month male Fischer 344 ratswithin 3 days after dermal administration. In order to examineage-related changes in dermal absorption and changes in potentialfor systemic exposure, the absorption, distribution, and eliminationof TCDD and 4PeCDF were examined in male Fischer 344 rats ofvarious ages (TCDD: 10, 36, and 96 week; 4PeCDF: 10, 36, 64,96, and 120 week). Each compound was applied at a dose of 0.1µmol/kg and/or 0.04 µmol in 60 µ1 acetoneto a preclipped region of the back and covered with a stainless-steelperforated cap. Rats were housed in individual metabolism cagesfor 3 days. Dermal absorption of both compounds was decreasedin older age groups compared to 10 week rats. The major tissuedepots for both compounds were liver, adipose, skin, and muscleand in comparable age groups, the liver.fat ratio was greaterin 4PeCDF-treated animals. Age-related changes in the distributionof the administered dose and the absorbed dose varied with thecompound as well as the depot. Elimination of TCDD and 4PeCDFwas limited at all ages. Results indicate that percutaneousabsorption of these compounds is decreased in older animals,suggesting that systemic bioavailability may be decreased inolder organisms following dermal exposure to TCDD or 4PeCDF.     

Aryl Hydrocarbon Hydroxylase Induction in Adult Rat Hepatocytes in Primary Culture by Several Chlorinated Aromatic Hydrocarbons Including 2,3,7,8-Tetrachlorodibenzo-p-dioxin

Aryl Hydrocarbon Hydroxylase Induction in Adult Rat Hepatocytesin Primary Culture by Several Chlorinated Aromatic HydrocarbonsIncluding 2,3,7,8-Tetrachlorodibenzo-p-dioxin. JANSING, R. L.,AND SHAIN, W. (1985). Fundam. Appl. Toxicol. 5, 713–720.The induction of aryl hydrocarbon hydroxylase (AHH) by Aroclor1254, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other polychlorinatedorganic compounds was examined in primary cultures of adultrat hepatocytes isolated by a collagenase perfusion technique.Following exposure of fresh hepatocyte cultures to 1 µg/mlAroclor 1254, AHH induction was undetected for 48 hr and thenincreased dramatically up to 96 hr. Cultures maintained in controlmedium for either 24, 48, or 72 hr prior to a 24-hr exposureto Aroclor 1254 displayed significant inducible AHH which wassustained to 96 hr. AHH induction was extremely sensitive totwo planar polyaromatic hydrocarbons, 2,3,7,8-TCDD and 2,3,7,8-tetrachlorodibenzofuran,and the PCB congener 3,4,3',4'-tetrachlorobiphenyl, but insensitiveto 2,6-dichlorodibenzofuran, 2,5,2',5'-tetrachlorobiphenyl,2,4,5,2',4',5'-hexachlorobiphenyl, and hexachlorobenzene. Theinduction of AHH activity in primary cultures of adult rat hepatocytesmay represent a useful bioassay for screening extracts of foodstuffs,biological fluids, or environmental samples for dioxin-likeactivity     

Acute Inhalation Toxicity of Soman and Sarin in Baboons

Acute Inhalation Toxicity of Soman and Sarin in Baboons. ANZUETO,A., DELEMOS, R. A., SEIDENFELD, J., MOORE, C, HAMIL, H., JOHNSON,D., AND JENKJNSON, S. G. (1990). Fundam. Appl. Toxicol. 14,676–687. Adult baboons (Papio sp.; 8–12 kg) wereanesthetized with sodium pentobarbital (20 mg/kg iv). The animalswere instrumented for measurement of mean blood pressure (MBP),pulmonary artery pressure (PAP), ECG, arterial and mixed venousblood gases, lung volumes, lung pressures, and efferent phrenicnerve activity. Bronchoalveolar lavage (BAL) was performed.Studies were done prior to exposure, at intervals during thefirst 4 hr postexpo-sure, and at 4 and 28 days after exposure.Control animals received a sham exposure to 2-propanol (N =5). Soman (pinacolyl methylphosphonofluoridate) at 13.14 µg/kg(2 x LD50) was vaporized into the upper airway in a second groupof animals (N = 5), and sarin (isopropyl methylphosphonofluoride)30 µg/kg (2 x LD50) was vaporized into a third group ofanimals (N = 4). Controls showed no change in any parametereither immediately after diluent exposure or during the monitoringperiod. Soman and sarin produced a decline in MBP and bradyarrhyth-miasthat were reversed with atropine. Apnea occurred in all soman-and sarin-exposed animals within 5 min postexposure, and wasassociated with absence of phrenic nerve signal. Ventilationwas mechanically supported until the animal could maintain normalarterial blood gases during spontaneous breathing. BAL studiesrevealed an increase in total white cell population and neutrophilsat 4 hr in all three groups. There were signs of impaired hemodynamicsand persistent lung injury for 4 days that resolved by 28 daysafter exposure. In conclusion, inhalation of soman and sarinin the baboon is associated with cardiac arrhythmias, developmentof apnea, and a significant decrease in MBP. Inhalation exposurealso resulted in a persistent influx of neutrophils and hypoxemia.     

Pharmacological Studies on the in vivo Cataractogenicity of Acetaminophen in Mice and Rabbits

Pharmacological Studies on the in Vivo Cataractogenicity ofAcetaminophen in Mice and Rabbits. LUBEK, B. M., AVARIA, M.,BASU, P. K., AND WELLS, P. G. (1988). Fundam Appl Toxicol 10,596–606. Acetaminophen can be enzymatically bioactivated,which may play a role in cataractogenesis. This study evaluatedthe relation of dose, sex, plasma drug concentration, cytochromesP-450 (P-450 and P448) induction, and hepatocellular toxicityto cataract-ogenic susceptibility in inbred mice and rabbits.C57BL/6 or DBA/2 mice, which respectively are genetically responsiveand nonresponsive to P448 induction, were treated with acetaminophen,300 to 1000 mg/kg intrapentoneally (ip), following pretreatmentwith the P448 inducer 3-methylcholanthrene (3-MC). Bilateralcataracts developed, independent of sex, in 83% of C57BL/6 micewithin 4 hr of acetaminophen administration, compared with 7%of DBA/2 mice. A dose-response relation for cataractogenesiswas evident in C57BL/6 mice using doses of 300 and 400 mg/kg,with the higher dose producing similar plasma acetaminophenconcentrations but twofold higher glucuronide concentrations.Both strains had increased plasma concentrations of glutamic-pyruvictransaminase (GPT). New Zealand white or Chinchilla pigmentedrabbits were treated with single or multiple doses of acetaminophen,500 to 1500 mg/kg/day ip, following pretreatment with a cytochromesP-450 inducer: pheno-barbital, 3-MC, or ß3-naphthoflavone.Acetaminophen given chronically caused lenticular opacitieswithin 1 week in 19 of 20 rabbits pretreated with P-450 inducers,regardless of pigmentation, but not in animals without priorP-450 induction. No opacities were observed after a single doseof acetaminophen, even with P-450 induction. There was no increasein plasma GPT in rabbits with any treatment. Over 85% of acetaminophenwas recovered in urine as a glucuronide conjugate, and the restas acetaminophen or conjugates with sulfate, cysteine, or N-acetylcysteine.Susceptibility to acetaminophen cataractogenesis can be geneticallypredetermined and may involve enzymatic bioactivation, possiblyindependent of hepatic biotrans-formation and toxicity.     

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.     

Induction of aryl hydrocarbon hydroxylase in rat tissue following intratracheal instillation of diesel particulate extract and benzo[a]pyrene

The potential for aryl hydrocarbon hydroxylate (AHH) induction by inhaled diesel particles was assessed by intratracheal administration. Benzo[a]pyrene, (B[a]P) a reference compound, or an extract of particles, dissolved in gelatin-saline solution was administered to Fischer 344 rats at several dose levels. Twenty-four hours after administration of B[a]P or diesel particulate extract, the AHH activity increased in a dose-response manner in the lung, but not in liver. The maximum increase in the AHH activity in the lung was observed 12 h after intratracheal instillation of B[a]P (5 mg/kg), and the levels remained elevated for seven days. The AHH activity in the liver reached the maximum 24 h after the administration, and returned rapidly to control values. In contrast, intratracheal instillation of diesel particulate extract resulted in a significant increase of AHH activity in the lungs only after doses greater than 6 mg kg-1. The activity, however, declined rapidly and returned to control values within 75 h. The liver AHH activity in this instance, remained unchanged throughout the experimental period. These data indicate that in the lung, hydrocarbons extracted from diesel particles are weak enzyme inducers and exposure to these compounds by intratracheal administration did not result in AHH induction in the liver. The results suggest that doses higher than those normally expected from occupational exposures will be required to induce AHH activity in organs examined.     

Protection by Zinc against Acetaminophen Induced Hepatotoxicity in Mice

Protection by Zinc against Acetaminophen Induced Hepatotoxicityin Mice. CHENGELIS, C. P., DODD, D.C., MEANS, J. R., AND KOTSONIS,F. N. (1986). Fundam. Appl. Toxicol. 6, 278–284. The purposeof this investigation was to assess protection by zinc againstacetaminophen induced hepatotoxicity and to evaluate possiblemechanisms of protection. Mice were treated with zinc (3 mg/kg,ip) or saline (ip) 48 and 24 hr before and sacrificed 12 hrafter acetaminophen administration (375. 500, or 750 mg/kg,po). Liver toxicity was then assessed by histological examination.The incidence of hepatotoxicity was significantly less at 375and 500 mg/kg of acetaminophen in zinc treated animals. Thesame dosage of zinc was not hepatoprotective when given 1 hrafter acetaminophen. Mice were also treated with 1 to 10 mg/kgof zinc (ip) 48 and 24 hr prior to sacrifice, and metallothionein,cytochrome P-450, glutathione, and UDP-glucuronosyl transferase(GT) were determined in the liver. Metallothionein and UDP-GTwere increased and P-450 and glutathione decreased at the higherdosages of zinc; however, only metallothionein was significantlychanged at the dosage of zinc (3 mg/kg) used in the hepatoprotectionexperiments. Further, mice were similarly treated with 3 mg/kgof zinc before administration of 375 mg/kg of [³H] acetaminophen(po) and the amount of acetaminophen and acetaminophen boundto me tallothionein were determined in the liver for 0.5 to24 hr. In addition, after 6 hr the subcellular distributionand covalent binding to protein of acetaminophen were also determined.Zinc treatment had no significant effect in any of the abovedeterminations. These results indicate that zinc protects againstacetaminophen induced hepatotoxicity and that the observed protectionis probably due to an induced biochemical change, but it isapparently not the result of any of the commonly invoked mechanisms.     

Comparative Potencies of Aroclors 1232, 1242, 1248, 1254, and 1260 in Male Wistar Rats--Assessment of the Toxic Equivalency Factor (TEF) Approach for Polychlorinated Biphenyls (PCBs)

Immature male Wistar rats were treated with several differentdoses of the commercial polychlorinated biphenyls (PCBs) Aroclors1232, 1242, 1248, 1254, and 1260 (10, 40, 160, 480, and 2000mg/kg) and the effects on body weight gain, thymic atrophy,and the induction of hepatic microsomal aryl hydrocarbon hydroxylase(AHH), ethoxyresorufin O-deethylase (EROD), and pentoxyresorufin(O-deethylase (PROD) activities were measured 14 days aftertreatment. A significant inhibition in body weight gain wasobserved only in rats treated with high doses of Aroclors 1232and 1248 and thymic atrophy was not observed for any of theAroclors. All the Aroclors caused a dose-dependent increasein hepatic microsomal AHH, EROD, and PROD activities. The correspondingED50 values for the induction of AHH-EROD activities variedfrom 51 to 678 mg/kg. Aroclor 1260 was the least active inducerof the P4501A1-mediated enzyme activities. In contrast, Aroclor1260 was a potent inducer of PROD activity (ED50=37 mg/kg),but Aroclors 1232, 1242,1248, and 1254 did not induce 50% ofthe maximal response at the highest dose used in this experiment(2000 mg/ kg). Previous studies have quantitated the levelsof those PCB congeners which induce AHH or EROD activities inAroclors 1232, 1242, 1254, and 1260 and their potencies or toxicequivalency factors (TEFs) relative to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) have also been estimated or experimentally determined.Using highly conservative TEF values it was demonstrated thatthe calculated ED50s for the Aroclors as inducers of AHH andEROD activity were significantly lower than the observed ED50values. However, if the TEFs were derived directly from therelative potencies of the PCBs (compared to 2,3,7,8-TCDD) asinducers of AHH and EROD activity, then there was a good correspondencebetween the observed and calculated ED50 Values.     

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.     

6-Methyl-1,3,8-trichlorodibenzofuran as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist: inhibition of the induction of rat cytochrome P-450 isozymes and related monooxygenase activities

In addition to being one of the most toxic chemicals known, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent inducer of rat liver microsomal cytochrome P-4501A1 (P-450c). Previous studies have demonstrated that a high affinity, low capacity cytosolic receptor (the Ah receptor) mediates the activity of TCDD to induce cytochrome P-4501A1, which catalyzes benzo[a]pyrene hydroxylation [aryl hydrocarbon hydroxylase (AHH]) and 7-ethoxyresorufin O-dealkylation (EROD). The results of the present study indicate that 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) effectively competes with [3H]TCDD for binding to the Ah receptor in rat liver cytosol. The concentration of MCDF effecting 50% displacement of [3H]TCDD was 4.9 X 10(-8) M, which is approximately 50 times greater than the EC50 for unlabeled TCDD (approximately 1 X 10(-9) M). However, in contrast to TCDD, MCDF was only a weak inducer of AHH and EROD activity in rat hepatoma H-4-II cells in culture. When co-incubated, MCDF diminished in a concentration-dependent manner the ability of TCDD to induce AHH and EROD activity in vitro. Treatment of rats with 20-200 mumol/kg MCDF in vivo had little or no effect on liver microsomal AHH and EROD activity, whereas treatment of rats with 16 nmol/kg TCDD caused a 6- and a 70-fold induction of AHH and EROD activity, respectively. When co-administered, MCDF diminished by approximately 50% the ability of TCDD to induce AHH and EROD activity in vivo. The partial antagonism produced by 50 mumol/kg MCDF could be partially overcome by doubling the dosage of TCDD from 16 to 32 nmol/kg. Immunochemical analysis of rat liver microsomes revealed that treatment of rats with 20-200 mumol/kg MCDF caused little or no induction of cytochromes P-4501A1 and P-4501A2 (P-450d), whereas these isozymes were induced 33- and 5-fold, respectively, in rats treated with 16 nmol/kg TCDD. When co-administered, MCDF diminished by approximately 50% the ability of TCDD to induce cytochrome P-4501A1 in vivo, which established that MCDF was not simply acting as an inhibitor of AHH and EROD activity. MCDF also antagonized the ability of TCDD to induce cytochrome P-4501A2, which suggests that the induction of both cytochromes P-4501A1 and P-4501A2 is regulated by the Ah receptor. These results indicate that MCDF binds with high affinity to the Ah receptor in rat liver cytosol and competitively blocks the binding of TCDD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pulmonary Toxicity of Cytostatic Drugs: Cell Kinetics

Pulmonary Toxicity of Cytostatic Drugs: Cell Kinetics. WITSCHI,H., GODFREY, G., FROME, E., and LINDENSCHMIDT, R. C. (1987).Fundam. Appl. Toxicol. 8, 253–262. Mice were treated withthree cytostatic drugs: cyclophosphamide, busulfan, or l,3-bis(2-chloroethyl)-l-nitro-sourea(BCNU). The alveolar labeling index was measured following drugadministration with a pulse of ³H-labeled thymidine and autoradiography.In cyclophosphamide-treated animals, peak alveolar cell proliferationwas seen 5 days after injection of the drug. In animals treatedwith busulfan or BCNU, proliferation was even more delayed (occurring2–3 weeks after administration). In contrast, with oleicacid, the highest alveolar cell labeling was found 2 days afterintravenous administration. In animals exposed to a cytostaticdrug, proliferation of type II alveolar cells was never a prominentfeature whereas in animals treated with oleic acid there wasan initial burst of type II cell proliferation. It is concludedthat the patterns of pulmonary repair vary between chemicalsdesigned to interfere with DNA replication as compared to agentswhich produce acute lung damage such as oleic acid.     

Cytoprotective Effects of Modulators of Oxidative Xenobiotic Metabolism in Precocene II-Induced Hepatotoxicity

Cytoprotective Effects of Modulators of Oxidative XenobioticMetabolism in Precocene 11-Induced Hepatotoxicity. DUDDY, S.K., AND HSIA, M. T. S. (1987). Fundam. Appl. Toxicol. 9, 304–313.This investigation was conducted to evaluate the effects ofmodulation of several phase I xenobiotic-metabolizing enzymeactivities on the expression of precocene II-induced hepatotoxicity.Precocene II (175–200 mg/kg) was given intraperitoneallyto male Sprague-Dawley rats that had been exposed previouslyto inducers (phenobarbital and 3-methylcholanthrene) or inhibitors(SKF 525-A and cimetidine) of oxidative xenobiotic metabolism.Hepatic damage was measured both biochemically (leakage of aspartateaminotransferase and alanine amino transferase into the serum)and histologically. Significant protection from precocene II-inducedhepatotoxicity was observed in all treated animals regardlessof whether the modulator employed was an inducer or an inhibitorof microsomal oxidative enzymes. These results indicate thatthe level of activity of various forms of cytochrome P-450 significantlyinfluences the severity of hepatic necrosis induced by precoceneII. Furthermore, these results suggest that inducible non-P-450factors, such as glutathione S-transferases, may be importantin modulating precocene II-induced hepatotoxicity.     

An Immunotoxicological Evaluation of 4, 4'-Thiobis-(6-t-butyl-m-cresol) in Female B6C3F1 Mice: 1. Body and Organ Weights, Hematology, Serum Chemistries, Bone Marrow Cellularity, and Hepatic Microsomal Parameters

An Immunotoxicological Evaluation of 4,4'-Thiobis-(6-t-butyl-m77-cresol)in Female B6C3F1 Mice. 1. Body and Organ Weights, Hematology,Serum Chemistries, Bone Marrow Cellularity, and Hepatic MicrosomalParameters. MUNSON, A. E., WHITE, K. L., JR., BARNES, D. W.,MUS-GROVE, D. L., LYSY, H. H., AND HOLSAPPLE, M. P. (1988).Fundam. Appl. Toxicol. 10, 691–700. Adult female B6C3F1mice were gavaged with 4,4'-thiobis-(6-t-butyl-m-cresol) (TBBC)in corn oil at doses of 10, 100, or 200 mg/kg daily for 14 consecutivedays. There was no overt toxicity, as manifested by grosslyobservable behavioral changes, decreased growth rate over theexposure period, or mortality. There were also no marked effectson serum chemistries or hematology, with the exception of asignificant increase (41%) in the number of leukocytes at thehighest dose. Absolute differential counts indicated that significantincreases occurred in the number of lymphocytes (31%) and neutrophils(177%). Studies with bone marrow indicated a significant 30%increase in the number of cells/femur from animals treated withthe highest dose of TBBC. The number of macrophage progenitors(CFU-M)/femur was significantly increased by 28%, while thenumber of granulocyte-monocyte progenitors (CFU-GM)/femur wasnonsig-nificantly increased by 20% in the high dose animals.The weight of both the spleen and liver was increased in a dose-relatedfashion, although the histopathology of the spleen of TBBC-treatedmice was not different from control. The livers of mice receivingthe high dose showed mild focal hydropic degeneration, mildhepatitis, and a slight increase in the number of Kupffer cells.No other organs were affected. Liver microsomal protein andcytochrome P-450 levels were increased in a dose-related fashion.Enzyme activities of aminopyrine demethylase and aniline hydroxylase,but not arylhydrocarbon hydroxylase, were also increased ina dose-related fashion.