TOXICOLOGY

Tissue

AND

APPLIED

Distribution

PHARMACOLOGY

32,330-338

and Excretion in the

(1975)

of Octachlorodibenzo-p-Dioxin Rat’

D. H. NORBACK, J. F. ENGBLOM, AND J. R. ALLEN Department ofPathology, University of Wisconsin Medical School, and Regional Primate Research Center, University of Wisconsin, Madison, Wisconsin 53706 Received Ja!y 30,1974; accepted November l&l974

Tissue Distribution and Excretion of Octachlorodibenzo-p-Dioxin in the Rat. NORBACK, D. H., ENGBLOM, J. F. AND ALLEN, J. R. (1975).Toxicol. Appl. Pharmacol. 32, 330-338. The contamination of several commercial compounds by chlorinated dibenzo-p-dioxins and the health hazards presentedby certain dioxins have beenwell documented.In this study the relative absenceof toxicity following high dosagesof octachlorodibenzop-dioxin (OCDD) in rats wasconfirmed. A moderateproliferation of the hepatic endoplasmicreticulum (ER) and hepatomegalyoccurred. Daily administration of a radioactive analogof OCDD to rats by gastricintubation for 21 daysresultedin the recovery of over 90% of the total dosein the fecesas unabsorbedmaterial. A smalllipid-solublefraction wasexcreted in the urine. After 21 days of administration, lessthan 1y0 of the total dosewaspresentin the rat tissues.The liver containedthe highestconcentration with approximately 50% of the body load found within this organ. The radioactivity in the adiposetissuewasapproximately 25% that in the liver. Significant levels of radioactivity were found in the kidneys, heart, testes, skeletal muscle, skin, and serum. The greatest reservoirs of the material were liver, skin, and adipose tissue. Over 95% of the radioactivity within the liver was associatedwith the microsomesand was equally distributed within the rough and smooth fractions. Distribution of the detectable radioactivity within rats that subsequentlyreceived a control diet for 6 wk wasconfined to the liver, adiposetissue,and skin at levels approximately 20-25x that found immediately after the 21-day period of administration. The route of excretion wasthrough the urinary systemand the rate correspondedto a biological half-life of approximately 3 wk. Toxicity, hepaticalterations,percentageof absorption,body distribution, and biologicalhalf-life of OCDD are comparedto thoseparametersof the extremely toxic tetrachlorodibenzo-p-dioxin (TCDD). Microsomal localization of the compoundsispostulatedto result in sequestrationof the material. The development of hydropericardium, hydrothorax, and ascitesin poultry following ingestion of feed supplemented with certain industrially contaminated fats occurred in late 1957 (Schmittle et al., 1958). Advancements in the isolation and identification of the fat soluble toxic component were made by Harman et al. (1960) and Wootton and Courchene (1964), and the chlorinated dibenzo-p-dioxin (CDD) structure waslater 1Supportedinpart by U.S.PublicHealthServiceGrantsES-00472 andRR-00167 fromtheNational Institutesof Health.PrimateCenterpublicationno. 1@18. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

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demonstrated by Cantrell et al. (1969). The CDDs are formed by condensation of chlorophenols (Higginbotham et al., 1968; Kimmig and Schulz, 1957), and contamination apparently occurred through the use of chlorophenols as animal hide preservatives with addition of recovered tallows to animal feeds (Firestone, 1973). The most toxic isomer of the CDD group, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), can apparently be formed during the synthesis and remains as a contaminant of the widely used herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (Kearney et al., 1973). Evidence for the effects of TCDD on humans resulted from the development of chloracne and porphyria cutanea tarda in factory workers exposed to 2,4,5-T (Bleiberg et al., 1964). The etiology of the porphyria cutanea tarda was related to TCDD contamination of the 2,4,5-T by Poland et al. (1971). The relationship of chloracne to CDDs had previously been made by Kimmig and Schulz (1957). Toxic levels and numerous alterations have been documented in experimental animals following TCDD ingestion. The oral LD50 values were shown to be 23 and 45 pg/kg for male and female rats, respectively, and for male guinea pigs 0.6 ,ug/kg (Sparschu et al., 1971). Administration of TCDD to subhuman primates caused gastric hyperplasia and ulceration, decreased hematopoiesis, hydropericardium, ascites, reduced spermatogenesis, focal liver necrosis, and eventual mortality (Allen and Carstens, 1967). In rats, teratogenic effects and fetal mortality have been demonstrated (Sparschu et al., 1971). Focal necrosis of the liver, epicardial hemorrhage, and lymphocytic infiltration of the cardiac muscle occurred in chickens (Sanger et al., 1958’). Hepatic &aminolevulinic acid synthetase was induced in chick embryos (Poland and Glover, 1973). Within the hepatic parenchymal cells there was a marked proliferation of the endoplasmic reticulum (ER) and formation of concentric membrane arrays (CMAs) (Norback and Allen, 1969). Associated with the proliferated ER was an increased hepatic microsomal enzymatic activity with a several fold increase in glucuronal UDP transferase activity (Lucier et al., 1973). Octachlorodibenzo-p-dioxin (OCDD) has been detected in commercial samples of tetrachlorophenol and in pentachlorophenol, a commercial wood preservative, at concentrations up to 2500 ppm (Johnson et al., 1973; Firestone et al., 1972). Toxicity studies of OCDD have not demonstrated lethality at g/kg dosages in rats or mice, acnegenic activity, production of chick edema, nor teratogenicity in rats given 100 or 500 mg/kg/day on days 6-15 of gestation. OCDD was embryotoxic at the dosage of 500 mg/kg/day (Schwetz et al., 1973). This study further describes the effects of OCDD in comparison with TCDD. Hepatic alterations induced by OCDD, efficiency of absorption, percentage and routes of excretion, and organ distribution with hepatocyte organelle association immediately following and 6 wk after a 21-day period of administration by gastric intubation of a radioactive analog of OCDD are reported. Evidence demonstrating hepatic localization and association of the hepatic fraction with the ER is also presented. METHODS Male Sprague-Dawley rats, initially weighing 100 g, were individually housed in cages and given free access to food and water. Six rats received 100 pg OCDD in 1 ml corn oil daily for 21 days; a control group of 6 rats received 1 ml corn oil daily for 21

332

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days. After the 21-day period of administration the rats were deprived of food for 24 hr and subsequently killed by exsanguination. Liver tissue was obtained for light and electron microscopy. Eleven rats received a radioactive CDDZ which was prepared by subjecting OCDD to direct neutron bombardment in a nuclear reactor with the resultant product of OCDD containing [35S]thioheptachlorodibenzo-p-dioxin (SCDD) (tllz equal to 86.7 days) through the reaction of 35Cl (n, p) 35S. A second reaction yielding 36Cl theoretically occurred at a much lesser frequency; however, the quantity of radiation emitted from 36Cl decay was not significant in the prepared material. One hundred micrograms of OCDD suspended in 1 ml corn oil and initially containing 12.6 pg SCDD (66,000 dpm) were administered daily by gastric intubation. Total urine and total feces from five rats were collected daily. Cages were rinsed with water and the washings were added to the urinary fraction. Following 21 days of administration (total dose of 2.1 mg OCDD containing SCDD), six rats were starved for 24 hr and sacrificed by decapitation and exsanguination. Five rats which also received the radioactive dioxin by daily intubation for 21 days were allowed to remain in the metabolism cages on a control diet. Total feces and urine were collected from this group for an additional 6 wk. These rats were also starved for 24 hr and sacrificed in a similar manner. At autopsy, blood was collected, portions of the liver were prepared for light and electron microscopic examination, and the remaining liver was homogenized for preparation of nuclear, mitochondrial, lysomal, and microsomal fractions. The brain, testes, kidneys, adrenals, lungs, spleen, heart, and skin were removed and weighed. In addition, portions of skeletal muscle and adipose tissue were also removed. Subcellular fractions of the liver from the experimental and control groups were prepared from a 25 % homogenate of liver in 0.25 M sucrose. One preparation of each of the hepatic subcellular fractions was examined with the electron microscope. Nuclear, mitochondrial, and lysosomal fractions were prepared by differential centrifugation at 600g for 10 min, 5000g for 10 min, and 15,000g for 10 min, respectively. Each fraction collected above was resuspended in distilled water equal to the original quantity of liver homogenate. The lipid layer collecting at the top of the tube during the initial centrifugation was removed, dried, and weighed. The postlysosomal supernatant was separated into total microsomes and into rough and smooth microsomes, after adjustment to 15 mM KC1 with the addition of 1 M KCl, by a modification of the procedure reported by Glaumann and Dallner (1968). Twenty-five milliliters of the 0.25 M sucrose-15 mM KC1 containing the microsomal fraction was layered over 12 ml of 1.3 1 M sucrose-l 5 mM KC1 and recentrifuged in an SW-27 rotor (13 l,OOOg,,,,X)for 8 hr. The double layer at the gradient boundary was collected with a pipette and designated the smooth microsomal fraction. The pellet and lower particulate portion that traversed the 1.31 M sucrose-15 mM KC1 was designated the rough microsomal fraction. The smooth fraction, rough fraction, and 10 ml of the original postlysosomal supernatant (total microsomes) were centrifuged in a fixed angle 40 rotor (144,88Og,,,) for 60 min. The pellets were washed with distilled water, recentrifuged, and resuspended in an amount of distilled water equal to the original liver homogenate (four times the liver weight). The measured representative portions of body tissues, urine, and feces were prepared ’ Procured from Drs. I. Solomon and M. King, Illinois Institute of Technology, Chicago, Illinois.

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333

for liquid scintillation counting3 and evaluated on a Packard Tri-Carb liquid scintillation counter. One hundred milligram tissue samples or 0.5 ml serum were directly solubilized in the medium for direct radioactivity analysis. Larger samples of tissue or excrement (l-5 g, as were available) from each animal were repeatedly extracted with Ccl, and the extract was dried and prepared for liquid scintillation counting. The urine samples were separated into water and lipid soluble fractions by repeated extractions with Ccl, and the two samples separately analyzed. Two-milliliter aliquots each of nuclear, mitochondrial, lysosomal, total microsomal, smooth and rough microsomal, and an accurately weighed portion of the lipid fractions obtained from the liver homogenates were added to the scintillation vials. Water was allowed to evaporate until the samples were near dryness and fractions were then prepared for radioactivity measurement using the materials described above. Two-milliliter aliquots of the microsomal fractions were also repeatedly extracted with Ccl,; the Ccl, phase was placed in a scintillation vial, and after evaporation of the solvent, the residue was measured for radioactivity. All radioactivity analyses from the group killed on the 22nd day of the experiment were performed on day 23. Each rat at this time had received a total dosage of 2.1 mg OCDD which on day 23 corresponded to a total activity of 1.16 x IO6 dpm and 221 pg SCDDs after correction for the spontaneous decay occurring during the initial 23 days of the experiment. All radioactivity analyses of the tissues from the group which was sacrificed after the 6-wk recovery period were done on day 65 of the experiment. Since day 23 the radioactive decay of the SCDDs accounted for a loss of 28.4 ?< of the radioactivity. The loss of microsomes that occurred during the differential centrifugations prior to separation of microsomes was estimated by the comparison of the activity of glucose6-phosphatase, an enzyme located exclusively in the microsomes, of the total liver homogenate to the activity present in the microsomal fraction. Glucose-6-phosphatase activity was determined by incubation at 37°C of the liver material with 40 mM glucose6-phosphate at pH 6.5 and measurement of inorganic phosphate (Baginski et al., 1967) formed by the reaction. The smooth and rough microsomal fractions of the two groups obtained from the rats receiving radioactive material were analyzed for phospholipid content. Lipid was extracted (Folch et al., 1957), and phospholipid concentration was determined by digestion of the lipid extract and determination of inorganic phosphate (Baginski et al., 1967).

The radioactivity level of each tissue was determined from the analysis of the Ccl, extract or from analysis of the direct tissue and expressed as dpm/g tissue or dpm/ml serum. The radioactivity excreted in the urine or feces was expressed as dpm/animal for each 24-hr period. The radioactivity level of the hepatic microsomal fraction was corrected for this loss which occurred during the centrifugation procedures and expressed as dpm/total microsomes from 1 g liver. The radioactivity of the hepatic free lipid was presented as dpm/lipid isolated from 1 g liver. The radioactivity and the phospholipid concentrations of the microsomal subfractions were determined on each preparation; in order to present radioactivity per quantity of membrane, dpms were presented per quantity of phospholipid. In each of the above categories there were 3 Unisol-Complement

or Scintisol-Complete,

Isolab, Elkhart, Indiana.

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NORBACK,

ENGBLOM

AND

ALLEN

five or six values, each representing one experimental animal; were determined for each tissue or hepatic subcellular fraction.

means and SD

RESULTS

OCDD at the daily dosage of 100 jig/rat for 21 days (appox 12.4 mg/kg administrated over 21 days) produced little morbidity in the rats. The animals weighed 243 + (SD) 9 g while the control group weighed 235 + 10 g. The relative liver weight was 3.80 + 0.20 g/100 g body wt (vs 2.85 + 0.05 g for the controls). The animals continued to eat well throughout the experiment and they were normal in appearance and activity. Ultrastructural changes were confined to a moderate increase of hepatic smooth ER. The quantity and the morphologic appearance of lipid droplets, mitochondria and lysosomes were unaltered. The light microscopic appearance of the experimental livers was indistinguishable from the controls. Gross appearance, relative liver weight, and hepatic cytologic changes present in animals receiving OCDD with SCDDs were indistinguishable from the effects induced by OCDD alone. Ninety-three + 6% of the total ingested radioactive dose over the 21-day period (after correction for spontaneous decay) was recovered from the feces. Daily excretion levels within the feces were constant from day 2 throughout the 21-day feeding period. After discontinuation of SCDDs in the diet, daily excretion levels decreased on day 1 to 20 % of the level during the feeding period, 4 % on day 2, and 1% on day 3, and after day 4 no radioactivity was present within the feces. Over the 21-day period of ingestion of SCDDs, the urinary levels of radioactivity were constant and the total radioactivity found in the urine accounted for a total of 5.2 f 0.8 % of the ingested dosage. A portion of the radioactivity of the urine, collected when the radioactivity of the feces was high, may represent contamination from the radioactivity of the feces. Over the 6-wk recovery period, urinary values were nearly constant for 4 wk and decreased more rapidly over the fifth and sixth weeks. The total amount excreted during the recovery period was 0.78 % of the total dosage. Since radioactivity was not present in the feces after day 4 of the recovery period, this fraction likely represents a true urinary component. All radioactivity was found within the CCI, extracted portion of the urine with no radioactivity being present within the water soluble portion After 21 days of daily administration of radioactive SCDDs followed by 1 day of starvation, the total radioactivity detected in the body tissue analyzed, with omission of that in the gastrointestinal tract, was 0.56 % of the total dosage. The distribution of the material within the tissues is presented in Table 1. Radioactivity concentrations per gram of tissue and per total organ were greatest within the liver and approximately 50% of all body radioactivity was found within this organ. The radioactivity in the retroperitoneal adipose tissue was approximately 25 % that of the liver. Additional reservoirs of the compound were the skeletal musculature and skin. Following discontinuation of the 21-day radioactive SCDD diet and resumption of a control diet for 6 wk, disappearance of 28.4 % of the radioactivity occurred due to the spontaneous decay of the isotope. Corrections were made for the spontaneous destruction of the material, and the compound was determined to be distributed

ABSORPTION AND EXCRETION OF DIOXINS

3.35

TABLE 1 DISTRIBUTION OF RADIOACTIVITY WITHIN RAT TISSUES AFTER INGESTION OF [35SlT~~~~~~~~~~~~~~~~~~~~o-~-~~~~~~~ (SCDDs)

Organ Liverd Hepatic Hepatic Hepatic Hepatic Adipose Heartd Kidneyd Serumh Skind Lungsd Testesd Muscled

microsomese free lipidf smooth microsomesB rough microsomesg tissued

After 3-wk SCDD administrationb 382 If: 31 367 t- 11 29 of:7 9.0 +- 1.4 11.1 4 2.4 107 + 19 103 4 27 97& 10 57 * 10 4ot- 10 39 + 33 24 + 7 15*5

After 3-wk SCDD administration followed by 6-wk recovery period’ 52 f so* 3+2 2.3 f. 2.4 + 19f8 0 0 0 7t3 0 0 0

24 12 1.2 1.5

* 5.24 x lo3 dpm/pg. b Data represent mean + SD of six values from six rats. c Data represent mean It SD of five values from five rats. d &m/g. e dpm/total microsomes from 1 g liver. s dpm/lipid isolated from 1 g liver. g dpm/mg microsomal phospholipid. h dpm/ml. within the liver, adipose tissue, and skin at levels approximately 20-25 % that found immediately after the 21-day dietary period. This rate of disappearance corresponds to a biological half-life of approximately 3 wk. Examination of the hepatic subfractions with the electron microscope demonstrated the theoretically anticipated contamination of the rapidly sedimentingparticles with the slowly sedimenting particles; microsomes were in the nuclear, mitochondrial. and lysosomal fractions. The microsomal preparations were free of the early sedimenting particles. The smooth and rough microsomal preparations showed a small proportion of cross contamination. Forty-five f 5.4% of the total hepatic microsomal material was recovered within the microsomal preparations as was determined by the activity of glucose-6-phosphatase in the microsomal and total liver preparations. After correction for the percentage of microsomes lost in the initial centrifugations, 96.3 f 8.2 7: of the hepatic radioactivity was determined to be contained in the microsomal fraction. The combined radioactivity of the nuclear, mitochondrial, and lysosomal fractions per gram of liver was approximately 50 % of that found within the liver and presumably represents radioactivity from microsomal contamination of these fractions. Free lipid contained 7.6 + 2.1% of the hepatic radioactivity present. The radioactivity per quantity of phospholipid (Table 1) of smooth microsomes was not significantly different

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from that found in the rough microsomes. Following discontinuation of the dietary SCDDs, the greatest percentage of the intrahepatic radioactivity was also located in the microsomal fraction (Table 1). DISCUSSION

The OCDD compound, which consists of the dibenzo-p-dioxin structure completely substituted with chlorine, is relatively nontoxic compared to the symmetrically substituted TCDD compound, which contains four chlorine atoms. Alterations of the OCDD-fed animals were confined to a moderate hepatomegaly and increase of hepatic smooth ER while the liver changes induced by TCDD included organ hypertrophy, ER proliferation, CMA formation (Norback and Allen, 1969), and degeneration of the hepatocytes with eventual death of the rat (Gupta et al., 1973). Relative to TCDD, small percentages of OCDD or SCDD are absorbed from the gastrointestinal tract. In this experiment, approximately 1% of the given SCDDs was present in the body tissues after 21 days of continuous administration. Other workers reported approximately 1% or less of OCDD (approx dosage of 150 pg/kg body wt or 800 pg/kg body wt) recoverable from body tissues (Williams et al., 1972). Approximately 70 % of TCDD administered in corn oil was absorbed from the gastrointestinal tract and recovered from the body tissues (Piper et al,, 1973). Preferential localization of TCDD and OCDD within the liver and within the adipose tissue at lower concentrations was demonstrated. The biologic half-life of 17.4 + 5.6 days reported for TCDD (Piper et al., 1973) was similar to the approximate 21 days estimated for the OCDD derivative. Metabolic stability of the OCDD derivative is indicated by the ability to extract hepatic and urinary fractions with hydrophobic solvents. The disappearance of radioactivity from the feces with the cessation of administration of the compounds further indicates lack of hepatic metabolism and lack of or only minute biliary excretion. TCDD is also metabolically stable within the mouse, rat, or rabbit (Vinopal and Casida, 1973); however, the major route of TCDD excretion in the rat was via the feces (Piper et al., 1973). The hepatic localization of OCDD and TCDD is likely due to association with the intracellular membranes. After administration of SCDDs approximately 95% of the hepatic radioactivity was localized to the microsomes with equal distribution between the rough and smooth fractions. Concentration of TCDD within the hepatic microsomes has also been described (Piper et al., 1973). TCDD, OCDD, and the SCDDs are extremely hydrophobic and location within the microsomal fraction is presumed dependent on the hydrophobic association of the material within the lipids of the membrane. Current molecular models of the membrane allow for lipid regions where hydrophobic fatty acids of phospholipids are sandwiched between the protein layers. These hydrophobic fluid areas could provide regions for the SCDD localization. Extraction of over 90% of the microsomal radioactivity with carbon tetrachloride in this experiment indicates hydrophobic association as the predominant interaction of the radioactive compound with the membranes. The sequela of association of hydrophobic compounds with the microsomes is likely contingent on the capacity of the microsomal enzyme to metabolize the compounds and the biological activity of the metabolites.

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REFERENCES ALLEN, J. R. AND CARSTENS, L. A. (1967). Light and electron microscopic observations in Macaca mulatta monkeys fed toxic fat. Amer. J. Vet. Res.28,1513-l 516. BAGINSKI, E. S., FOA, P. P. AND ZAK, B. (1967). Microdetermination of inorganic phosphate, phospholipids, and total phosphate in biologic materials. Clin. Chem.13, 326-332, BLEIBERG, J., WALLEN, M., BRODKIN, R. AND APPELBAUM, I. L. (1964). Industrially acquired porphyria. Arch. Dermatol. 89, 793-797. CANTRELL, J. S., WEBB, N. C. AND MABIS, A. J. (1969). The identification and crystal structure of a hydropericardium producing factor: 1,2,3,7,8,9-hexachlorodibenzo-p-dioxin. Acta Cryst. B25,150-155. FIRESTONE, D., RESS, J., BROWN, N. L., BARRON, R. P. AND DAMICO, J. N. (1972). Determination of polychlorodibenzo-p-dioxins and related compounds in commercial chlorophenols.

J. Ass. Ofic, Anal. Chem.55, 85-92. FIRESTONE, D. (1973). Etiology of chick edemadisease.Environ. Health Persp.5, 59-66. FOLCH,J., LEES,M. ANDSLOANESTANLEY,G. H. (1957).A simplemethod for the isolation and purification of total lipids from animal tissue.J. Biol. Chem.226,497-509. GLAUMANN,H. AND DALLNER,G. (1968). Lipid composition and turnover of rough and smooth microsomal membranes in rat liver. J. Lipid Res.9, 720-729. GUPTA, B. N., Vos, J. G., MOORE, J. A., ZINKL, J. G. AND BULLOCK, B. C. (1973). Pathologic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals. Environ. Health Perspect.5, 111-118. HARMAN, R. E., DAVIS, G. E., OTT, W. H., BRINK, N. G. AND KUEHL, F. A. (1960). The

isolation and characterization of the chick edemafactor. J. Amer. Chem.Sot. 82, 20782079. HIGGINBOTHAM, G. R., HUANG, A., FIRESTONE, D., VERRET~, J., REES, J. AND CAMPBELL, A. D. (1968). Chemical

and toxicological

evaluations

of isolated

and synthetic

chloro

derivatives of dibenzo-p-dioxin. Nature (London)220, 702-703. JOHNSON, R. L., GEHRING, P. J. AND KOCIBA, R. J. (1973).Chlorinated dibenzo-dioxinsand pentachlorophenol.Envinon.HealthPerspect.5,171-175. KEARNEY, P. C., WOOLSON, E. A., ISENSEE, A. R. AND HELLING, C. S. (1973). Tetrachlorodibenzodioxin in the environment: sources,fate, and decontamination.Environ. Health Perspect.5, 273-277. KIMMIG,

J. AND SCHULZ, K. H. (1957). Berufliche

Akne (sog. Chloracne)

durch chlorierte

aromatischezyklische Jther. Dermatologica115, 540. LUCIER, G. W., MCDANIEL, 0. S., FOWLER, B. A., FAEDER, E., HOOK,G. AND SONAWANE, B. R. (1973). Studies on TCDD-induced changes in rat liver microsomal and mitochondrial enzymes. Environ. Health Perspect.5, 199-209. NORBACK, D. H. AND ALLEN, J. R. (1969). Morphogenesis of toxic fat-induced concentric

membranearrays in rat hepatocytes.Lab. Invest. 20, 338-346. PIPER, W. N., ROSE, J. Q. AND GEHRING, P. J. (1973). Excretion and tissue distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Environ. Health Perspect.5, 241-244. POLAND, A. AND GLOVER, E. (1973). 2,3,7,8-Tetrachlorodibenzo-p-dioxin: a potent inducer of &aminolevulinic acid synthetase.Science179,476-477. POLAND, A. P., SMITH, D., METTER, G. AND POSSICK, P. (1971).A health survey of workers

in a 2,4-D and 2,4,5-T plant. Arch. Environ. Health 22, 316-327. SANGER, V. L., SCOTT, L., HAMDY, A., GALE, C. AND POUNDEN, W. D. (1958). Alimentary toxemia in chickens.J. Amer. Vet. Med. Ass. 133, 172-176. SCHMI~~LE, S. C., EDWARDS, H. M. AND MORRIS, D. (1958).A disorder of chickensprobably due to a toxic feed-Preliminary report. J. Amer. Vet. Med. Ass. 143, 216219. SCHWETZ, B. A., NORRIS, J. M., SPARSCHU, G. L., ROWE, V. K., GEHRING, P. J., EMERSON, J. L. AND GERBIG, C. G. (1973). Toxicology of chlorinated dibenzo-p-dioxin%Environ.

Health Perspect.5, 87-99. SPARSCHU, G. L., DUNN, F. L. AND Row, V. K. (1971). Study of the teratogenicity tetrachlorodibenzo-p-dioxin in the rat. Food Cosmet.Toxicol. 9, 405412.

of 2,3,7,8-

338

NORBACK,

ENGBLOM

AND

ALLEN

J. H. AND CASIDA, J. E. (1973). Metabolic stability of 2,3,7,8-tetrachlorodibenzo-pdioxin in mammalian liver microsomal systems and in living mice. Arch. Environ. Contum. Toxicol. 1, 122-132. WILLIAMS, D. T., CUNNINGHAM, H. M. AND BLANCHFIELD, B. J. (1972). Distribution and excretion studiesof octachlorodibenzo-p-dioxinin the rat. Bull. Environ. Contam.Toxicol. VINOPAL,

7, 57-62.

J. C. AND COURCHENE, W. L. (1964). A contribution to the knowledgeof the structure of two hydropericardium-producingfactors from a toxic fat. J. Agric. Food Chem.

WOOTTON,

12,94-98.