1OXICOLOGY

AND

Distribution after

APPLIED

37,415-423

PHARMACOLOGY

and Excretion Their Prolonged

of [‘4C]Polychlorinated Administration to Male

Biphenyls Rats

K. HASHIMOTO,’S. AKASAKA, Y. TAKAGI, M. KATAOKA, T. OTAKE, Y. MURATA, S. ABURADA, T. KITAURA, AND H. UDA Osaka

Prefectural

Received

Institute

October

of Public

22, 1975;

Health,

accepted

April

Osaka,

Japan

IS, 1976

Distribution kmd Excretion of [WlPolychlorinated Biphenyls after Their ProlongedAdministration to Male Rats. HASHIMOTO, K., AKASAKA, S., TAKAGI, Y., KATAOKA, M., OTAKE, T., MURATA, Y., ABURADA, S., KITAURA, T., AND UDA, H. (1976). Toxicol. Appl. Pharmacol. 37,415-423. After repeatedadministration of [14C]PCBto malerats, the concentration distribution and the amountsexcreted were studied. The PCB contained mostly either four or six chlorine substituents(Cl-4 or Cl-6), andthe longest dosingperiod was 1 year. After Cl-4 administration,between1.9 and 4.9 % of the dose was excreted in the urine, the amounts being significantly higher in thosegroups given PCB for longer periods.After Cl-6 administration, only 0.3y0 of the dosewasexcreted in the urine. Larger amounts of both Cl-4 and Cl-6 were excreted in feces,being47 to 68% of the dose with no significant differencesbetweenthe two compounds.The concentration of PCB washighestin the adiposetissues,intermediatein the skin, adrenal gland, aorta, and sciatic nerve, and lowestin the blood and other tissues.The concentrationsdecreasedwith time, most rapidly in the blood and more slowly in adiposetissue.The rate of decreasewas lower after longer periods of administration. Most of the PCB were presentin the lipid fraction of each tissue,and the concentration values differed with eachlipid fraction. PCB seemto haveno specificaffinity for any component of lipid. Polychlorinated biphenyls (PCB) have prcduced a widespread contamination of the environment (Jensen, 1966; Widmark, 1967; Risebrough et al., 1968). Although many toxicity studieshave been cor,ducted with PCB, little is known about their pharmacodynamic behavior. The present report describescomparative studieson the distribution and excretion of PCB of two different types after repeated administration to male rats. METHODS Animals. Male albino rats of JCL-S.D. strain, 6 weeks of age and 188 g (f9.7) at the beginning of the experiments, were randomly distributed into six groups of 12 animals per group (one control and five treated groups, G-l-G-S). They were housed in stainless 1 Present affiliation : Department of Hygiene, Japan. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

School 415

of Medicine,

Kanazawa

University,

Kanazawa,

416

HASHIMOTO

ET AL.

steel cages in groups of two animals per cage and were fed normal chow and water confined to metabolic cages with a wire floor. [14C]PCB. Two PCB compounds, [U-14C]KC-400 and [U-i4C]KC-600, having specific radioactivities of 12.6 &i/mg and 4.7 ,uCi/mg, respectively, were obtained from the Japan Atomic Energy Research Institute. The distribution patterns due to mass signals on the gas chromatograms of both [14C]PCB are almost identical to those of the corresponding commercial PCB, Kanechlor-400 and Kanechlor-600, in which percentages of fixed chlorine were 2;1%, 3;22, 4;59, 5;12, 6;6, 7;0.5, and 2;tr, 3; 2%, 4;5,5;5,6;38,7;39,8 ; 11, respectively. On the radiogas chromatograms the radioactivity is distributed among all components in proportion to the mass peaks. Gas chromatography and mass spectrometry (JOEL GC- 1100) of nonradioactive KC-400 and KC-600 synthesized under the same conditions as the radioactive PCB revealed that there were resemblances between the signal from the total-ion monitor of the mass spectrometer and the signal from the FID, used for the mass assay in the radiogas chromatography (Hoizumi and Moriya, 1974). Hereafter, the two radioactive PCBs will be referred to as Cl-4 and Cl-6, respectively. Administration of PCB. The PCBs were dissolved in olive oil, in appropriate concentrations, and were administered orally with blunt-tip metal intubation needles. Cl-4 was administered to four groups of animals once a week for periods of 5, 10,30, and 50 weeks, respectively. Cl-6 was administered to a fifth group for a period of 5 weeks. The control animals received a comparable volume of pure olive oil. Experimental conditions, including number of rats, body weight at the beginning and the end of the experiments, and dosing schedules are summarized in Table 1. Assay of radioactivity in urine andfeces. A 0.5-ml urine sample was put into a counting vial and was counted with 10 ml of dioxane scintillation fluid. After being dried at room temperature, feces were ground, weighed in small pieces of blotting paper in approximately 50-mg amounts, oxidized with an automatic sample oxidizer (Packard Model 305), and finally counted with a liquid scintillation counter (Packard Model 3380) using the external-standard method. Assa-v of radioactivity in tissues. After the administration periods, four rats each of the control and treated groups were sacrificed on Days 3, 10, and 30 following the final administration. Blood samples were withdrawn from the heart under ether anesthesia, and whole body transfusion was conducted with physiological saline. Thirty-six organ and tissue samples were dissected from each rat. After the surfaces of the organs were washed with saline, they were blotted and weighed. Aliquots of these tissues where then weighed either in counting vials in approximately 200 mg of amounts or on small pieces of blotting paper in approximately 50-mg quantities. The samples in the vials were dissolved with Packard Soluene 350 solubilizer, and the samples on blotting paper were burned with the sample oxidizer. The radioactivity in these samples was counted with the liquid scintillation counter. 14C0, in expiration. Expired CO2 was absorbed in a solution of ethanolamine in ethylene glycol monomethyl ether (1 :2) (Jeffay, 1961) in two absorption tubes (each contained 50 ml of the solution) connected serially. Extraction and chromatography of the lipid fraction of tissues. According to Folch’s method (Folch et al., 1957), lipid in tissues was extracted three times with 20 volumes of ad libitum. During collection of urine and feces, animals were individually

-

189 +- 9.5* -

0

12 -

186 + 13 377 k 18 (372 k 23) 1.33 7.14

12 Cl-4” 5

G-l

1

188 + 8.0 493 f 43 (460 f 45) 0.72 7.17

12 Cl-4 10

G-2

CONDITIONS

(679 f 82) 0.16 7.85

(611 + 63) 0.24 7.64

Cl-4 50

Cl-4 30

750 194 f+ 98 9.2

12

12

658 191 f* 49 9.1

G-4

G-3

(372 + 23) 1.33 6.35

341 188 2+ 8.6 12

Cl-6” 5

12

G-5

n Cl-4 and Cl-6 denote that the PCB contained mostly either 4 or 6 chlorine atoms. The PCBs were dissolved in olive oil and given orally once a week. * Mean i SD.

No. of rats (male) PCB Period of dosing (weeks) Mean body weight (g) Original Final (Control) Single dose (mg/kg) Total dose (mg/kg)

Control

TABLE EXPERIMENTAL

9 52 $ ti

2 2 F g

t; j;l 5 2

0

418

HASHIMOTO

ET AL.

chloroform-methanol solution (2 : 1). The lipid fraction being extracted was chromatographed with a silicic acid column of 180 x 10 cm using chloroform, chloroformmethanol (I : 4), and methanol as stepwise solvents. Statistical analysis. Student’s t test was employed in the statistical analysis of differences between means. RESULTS

Body weight. No significant differences in growth were seen between groups, and no particular abnormality in behavior or general appearance was detected in any group. Liver/body weight ratio. No significant differences were seen in the liver/body weight ratios between the control and treated groups at any stages of the experiments. Radioactivity in urine,feces, and expiration. 14C in urine was mostly excreted by Day 2 after each administration in any group. The percentage of Cl-4 excreted in 7 days after final doses of 5, 10,30, and 50 were 1.9,2.4,4.4, and 4.9 %, respectively, and that of Cl-6 after five doses was 0.56 %. Significant differences in the total excretion rates were seen among groups, as shown in Table 2. 14C in feces was mostly excreted by Day 2 after

CUMULATIVE

EXCRETION

Group

OF

TABLE 2 PCB IN URINE 7 DAYS AFTER ADMINISTRATION OF PCBs TO RATS

PCB

Dose period (weeks)

Cl-4” Cl-4 Cl-4 Cl-4 Cl-6“

5 10 30 50 5

Percentage excretion of the last dose

_____ G-l G-2 G-3 G-4 G-5

1.9 2.4 4.4 4.9 0.56

+ 0.96’ _+ 0.36’ k 0.96d + lSd + 0.15d

0 Cl-4 and Cl-6 denote the PCB contained mostly either 4 or 6 chlorines. They were dissolved in olive oil and given orally. b Values are mean f SD. c p c 0.05 significantly different from G-l. d p < 0.001 significantly different from G-l.

administration. The total percentages excreted in each group were 67.6 + 6.6(G-l), 60.4 + 5,O(G-2), 46.7 k 13.8(G-3), and 53.6 -t 5.4(G-4), and 57.5 + 7&G-5) of the Cl-6 group. These values did not differ significantly from each other. Only a trace amount of radioactivity was detected in the expiration of rats during the 24 hr after PCB administration. Radioactivity in tissues.Figures l-3 show time course of PCB concentration in certain tissues after the administration of either Cl-4 or Cl-6 for different periods. In all groups, regardless of dosing schedule, the highest concentration of PCB (5 x 103-2 x lo3 ppb) was detected in adipose tissues such as the subcutaneous, perirenal, brown, and mesenteric fat. Intermediate concentrations (1 x 103-3 x 10’ ppb) were detected in the skin, adrenal gland, aorta, and sciatic nerve, in which the proximal parts contained a higher

DISTRIBUTION OF [14C]PCB IN RATS

419

,content of PCB than the central parts. Concentrations in the lung, liver, kidney, pancreas, spleen, esophagus, stomach, small intestine, colon, thyroid gland, thymus, genital organs, urinary bladder, heart, skeletal muscle, diaphragm, nervous tissues, eye, bone, hair, nail, and blood were the lowest (less than 3 x lo2 ppb). Concentrations in alimentary canal showed a wide range of values, presumably because of contamination by fat

FIG. 1. Concentration of PCB in tissues. Groups 1 to 4 received Cl-4 PCB in olive oil and group 5 received Cl-6 orally once a week. Animals were sacrificed 3,10, and 30 days following the final administration of each group. WI

10’ : s

3

2

I,-3 i

3

10

V&Y 30 T,ME&FTER THELASTDOSE

2. Concentration

of PCB in tissues. See Fig. 1.

Fig. 3. Concentration

of PCB in tissues. See Fig. 1.

FIG.

420

HASHIMOTO

ET AL.

in the samples. In the genital organs, the epidydimis showed comparatively higher content of PCB than the testis. In the nervous tissues, the gray and white matters of the cerebrum, cerebellum, spinal cord, and spinal ganglions showed about the same values. More than 90 % of the PCB concentration in the blood was detected in the serum fraction. As illustrated by the figures, the concentration of PCB in most tissues tended to show less value as the periods of administration prolonged. After five doses of Cl-6, tissue concentrations were generally higher than those after the same doses of Cl-4. The concentration in tissues decreased with time, the most rapid decrease occurring in the blood. The rate seems to be more rapid in groups to which PCB were administered for shorter periods of time. In the group which was given Cl-6, the concentration of PCB in the adipose tissues was about twice as great as that in the group which was given Cl-4 for the same period. As the rate of concentration decrease was smaller than in the Cl-4 group, the concentration on Day 30 was also much higher than in the latter. Distribution of radioactive 14C in lipid andprotein fraction of tissues. Table 3 shows the radioactivity detected in lipid and protein fractions of the liver, brain, and subTABLE 3 RADIOACTIVITY

IN THE LIPID AND PROTEIN AFTER ADMINISTRATION

FRACTIONS OF TISSUES OF RATS OF [‘*C]PCB

dpm/g tissue

-

PCB

Dose period (weeks)

Fraction

Liver

Brain

Subcutaneous adipose tissue

.- -__ (x-4” (J-6”

5 5

Lipid Protein Lipid Protein

1500 + 260'

850 + 104

Trace 1470 * 510

0

120 -t 23

640 _+170 89 + 9.0

22,800 + 3360 0 44,500 + 4400

Trace

a Cl-4 and Cl-6 denote the PCB contained mostly either 4 or 6 chlorines. They were dissolved in olive oil and given orally once a week. Lipid in tissues was extracted three times with 20 volumes of chloroform-methanol solution (2:l). b Values are mean k SD.

cutaneous adipose tissue. As seen in this table, most radioactivity due to PCB was recovered from the lipid fraction of every tissue in the Cl-4 group, while about 10 % of the total radioactivity was found in the protein fraction of the Cl-6 group in the liver and brain. Silicic acid chromatography of the lipidfraction. Since most of the PCB in tissues were present in the lipid fraction, the lipid was subfractionated by silicic acid chromatography to see the affinity of PCB for the three main components of lipid. The elution peak of the PCB was not the same as that of any subfraction, although cholesterol and neutral fat were eluted near the PCB as shown in Fig. 4. Concentration of PCB in tissue lipid. The concentration of PCB in tissue lipid on Day 3 after the last of five doses differs in each tissue, being highest in the serum,

DISTRIBUTION OF [i4C]PCB

IN RATS

421

CPM p\

105 I b- PCB I I

PHOSPHOLIPID

0

10 TUBE

20 NO,

FIG. 4. Fractionation of lipids from subcutaneous adipose tissue. Lipids were extracted with Folch’s method from tissues of animals which received Cl-4 five times and chromatographed with a silicic acid column as described in the Methods section.

followed by that in the subcutaneous adipose tissue. The concentrations decreased with time, most quickly in the serum, followed by the heart, nervous tissues, kidney, liver, and subcutaneous adipose tissue. DISCUSSION

Many authors have reported the distribution and excretion of PCB based on investigations with both animals and humans (Goto and Higuchi, 1969; Grant et al., 1971; Hansen et al., 1971; Yoshimura and Oshima, 1971; Platonow et al., 1971). In some animal experiments, labeled compounds were used (Yoshimura et al., 1971; Yoshida et al., 1973). These studies in particular have shown that PCB have a strong affinity for every adipose tissue in the body and that PCB are excreted mostly in the feces and only slightiy in urine. The present study has been carried out with two PCB compounds to compare their distribution in the body and their excretion in feces and urine. Prolonged administration of 100 ppm PCB has been reported to inhibit the growth rate of dogs (Keplinger et al., 1971). In the present study, however, no inhibition of growth was seen. Inagami et al. (1969) have reported increased body weight in hairless mice after PCB administration and have interpreted this as being due to an increased liver weight, which has generally been observed in connection with PCB (Nishizumi, 1970; Kimbrough et al., 1972; Bruchner et al., 1973). In our experiment, no increase of liver weight was seen in any group probably because of the small doses of PCB. Urinary excretion of PCB was not high, totaling less than 5 % of each Cl-4 dose and 0.56 % after five doses of Cl-6 during 7 days after administration. The difference between the two figures is probably due to differences in water solubility and the tendencies to be metabolized into more soluble compounds. In the groups which were administered Cl-4, the urinary excretion increased with longer periods of administration, presumably due to increased metabolism of PCB in the body with time. In this study, the total dose

422

HASHIMOTO

ET AL.

of PCB was the same in all animals regardless of the periods of administration; i.e., each dose of PCB was smaller in groups with longer administration periods. The increased percentage of urinary excretion, therefore, might also be relevant to this factor. Excretion of PCB in feces was much higher than that in urine, totaling from 47 to 68 % in 7 days after each dose, there being no significant difference between the Cl-4 and Cl-6 groups. Radioactivity was detected in the expired air in only trace amounts, indicating that PCBs were neither expired nor metabolized into CO,. Concentration of PCB in adipose tissue was highest of all tissues examined after both Cl-4 and Cl-6 administration. Tissues which showed intermediate concentrations were the skin, adrenal gland, aorta, and sciatic nerve, presumably because of high contents of lipids in these tissues. These results are consistent with those obtained from pregnant rats (Takagi et al., 1976). The concentrations in tissues after five doses of Cl-6 were generally higher than those after the same doses of Cl-4, even though the total amount of PCB administered was smaller in the first case. It is not clear in the present experiments whether the difference is due to preferential absorption from gastrointestinal tract or to preferential elimination of the two compounds. As the period of administration of Cl-4 prolonged, the concentration of PCB in tissues tended to be lower and the elimination from tissues became slower. It is possible that PCB isomers of high chlorine contents, which are more difficult to be metabolized as indicated by Hutzinger et al. (1972), remained preferentially in tissues after a prolonged administration. After five doses of Cl-6, the elimination rate from tissues was also slower than that after the same doses of Cl-4. Burse ef al. (1974) have obtained similar results from a comparative study of Arochlor 1016 and 1242. Since the results in this study were mostly presented in terms of total radioactivity, the relationship between the high concentration of PCB in some tissues and the toxic mechanism of action of the compounds could not be directly inferred from the present animal experiments. Further studies should also be carried out with individual isomers in order to understand the whole picture of the pharmacodynamic behavior of PCB. The concentrations of PCB in the lipid of each tissue were not identical. This difference may be due to factors such as the permeability of tissue vascular systems to PCB, solubility of the compounds and the structure of lipid in each tissue. From the results of fractionation of tissue lipid with silicic acid column chromatography, PCBs are unlikely to have specific affinity for any component of lipid such as have been shown in DDT (Tinseley et al., 1971).

REFERENCES J. V., KHANNA, K. L., AND CORNISH, H. H. (1973). Biological responsesof the rat to polychlorinated biphenyls. Toxicol. Appl. Pharmacol.24,434-448.

BRUCKNER,

BURSE, V. W., KIMBROUGH, R. D., VILLANUEVA, E. C., JENNINGS, R. W., LINDER, R. E., AND SOVOCOOL, G. W. (1974).Polychlorinated biphenyls. Storage,distribution, excretion and

recovery: Liver morphology after prolonged dietary ingestion.Arch. Environ. Health 29, 301-307. FOLCH, J., LEE, M., AND STANLEY, S. G. H. (1957). A simplemethod for the isolation and purification of total lipids from animal tissues.J. BioE.Chem.226,497-507. GOTO, M., AND HIGUCHI, K. (1969). The symptomatology of Yusho (chlorobiphenyls poisoning)in dermatology. Fukuoka Acta Med. 60,409-431.

DISTRIBUTION

OF

[14C]PCB

IN RATS

423

D. L., PHILLIPS, W. E. J., AND VILLENEUVE, D. C. (1971). Metabolism of a polychlorinated biphenyl (Arochlor 1254)mixture in the rat. Bull. Environ. Contamin.Toxicol. 6, 102-l 12. HANSEN, D. J., PARRISH, P. R., ROWE, J. I., AND WILSON, A. J. (1971).Chronic toxicity, uptake, and retention of Arochlor 1254in two estuarinefishes.Bull. Environ. Contamin.Toxicol. 6, 113-119. HOIZUMI, K., AND MORIYA, T. (1974). Synthesis of carbon-14 labeled polychlorinated biphenyls.J. LabelledCompd.10, 499-508. HUTZINGER, O., NASH, D. M., SAFE, S., DEFREITAS, A. S. W., NORSTROM, R. J., WILDISH, D. J., AND ZITKO, V. (1972). Polychlorinated biphenyls: Metabolic behavior of pure isomersin pigeons,rats, and brook trout. Science178, 3 12-3 14. INAGAMI, K., KOGA, T., KIKUCHI, M., HASHIMOTO, M., TAKAHASHI, H., AND WADA, K. (1969). Experimental study of hairlessmice following administration of rice oil usedby a “Yusho” patient. FukuokaActa Med. 60,548-553. JEFFAY, H. (1961). Counting PO, with liquid scintillation counter. In Advancesin Tracer Methodology (S. Rothchild, ed.), Vol. 1, pp. 113-l 14.PlenumPress,New York. JENSEN, S. (1966). Report of a new chemical hazard. New Scientist32, 612. KEPLINGER, M. L., FANCHER, 0. E., AND CALANDRA, J. C. (1971).Toxicologic studies with polychlorinated biphenyls.Toxicol. Appl. Pharmacol.19,402. KIMBROUGH, R. D., LINDER, R. E., AND GAINES, T. B. (1972).Morphological changes in livers of rats fed polychlorinated biphenyls.Arch. Environ. Health 25, 354-364. NISHIZUMI, M. (1970).Light and electron microscopestudy of chlorobiphenyl poisoning: In mouseand monkey liver. Arch. Environ. Health 21, 620-632. PLATONOW, N. S., LIPTRAP, R. M., AND GEISSINGER, H. D. (1971). The distribution and excretion of polychlorinated biphenyls(Arochlor 1254)and their effect on urinary gonadal steroidlevelsin the boar. Bull. Environ. Contamin.Toxicol. 6, 358-368. RISEBROUGH, R. W., RIECH, P., PEAKALL, D. B., HERMAN, S. G., AND KIRVEN, M. N. (1968). Polychlorinated biphenylsin the global ecosystem.Nature (London)220, 1098-l 102. TAKAGI, Y., OTAKE, T., KATAOKA, M., MURATA, Y., ABURADA, S., AKASAKA, S., HASHIMOTO, K., UDA, H., AND KITAURA, T. (1976). Studies on the transfer and distribution of 14Cpolychlorinated biphenylsfrom maternalto fetal andsucklingrats. Toxicol. Appl. Pharmacol. to appear. TINSELEY, I. J., HAQUE, R., AND SCHMEDDING, D. (1971).Binding of DDT to lecithin. Science GRANT,

174, 145-147. WIDMARK,

G. (1967).Possibleinterferencechlorinated biphenyls.J. Assoc.Off. Anal. Chem.

50, 1069. T., TAKASHIMA, F., AND WATANABE, T. (1973). Distribution of [14C]PCBin carp. AMBZO 2, 111-113. YOSHIMURA, H., AND OSHIMA, M. (1971).Studieson the tissuedistribution and elimination of severalcompoundsof KC-400 (chlorobiphenyls)in mice.Fukuoka Acta Med. 62,5-l 1. YOSHIMURA, H., YAMAMOTO, H., NAGAI, J., YAE, Y., UZAWA, H., ITO, Y., NOTOMI, A., MINAKAMI, S., ITO, A., KATO, K., AND TSUJI, H. (1971). Studieson the tissuedistribution and the urinary and fecal excretion of 3H-Kanechlor(chlorobiphenyls)in rats. Fukuoka Acta Med. 60, 12-19. YOSHIDA,