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Distribution and biliary excretion of polychlorinated biphenyls in rats
JOURNAL
OF TOXICOLOGY
Distribution
AND
APPLIED
and
PHARMACOLOGY
38,609~619 (1976)
Biliary Excretion of Polychlorinated Biphenyls in Rats’
RICHARDE. PETERSON, JONL. SEYMOUR, AND JAMES R. ALLEN School of Pharmacy, Department of Pathology, School of Medicine, and Regional Primate Research Center, University of Wisconsin, Madison, Wisconsin 53706 Received May 18,1976; acceptedJuIy 29,1976
Distribution and Biliary Excretion of PolychlorinatedBiphenylsin Rats. PETERSON, R. E., SEYMOUR, J. L., AND ALLEN, J. R. (1976). Toxicol. Appl. Pharmacol. 38, 609-619. Absorption, distribution, and excretion of two 3H-labeled
polychlorinated
biphenyls
(PCB),
2,4,5,2’,4’,5’,-hexachloro-
biphenyl (HCB) and 2,5,2’,5’-tetrachlorobiphenyl(TCB), were studiedin surgically prepared male and female rats. Approximately 3-5 hr after surgery, HCB or TCB (50 mg/kg) was administeredinto the stomach. Bile, urine, and feceswerecollectedfor 24 hr after which the animalswere sacrificedand tissuestaken for determination of 3H content. The distribution of 3H remainingin the rats, expressedas percentageof dose,was highestin skeletal muscle, skin, liver, and small intestine for both isomers. The major differenceobservedbetweenthe PCBswasin biliary excretion. For HCB, 0.5 f 0.2 % (males, mean + SE) and I .l + 0.3 % (females) of the dose were excreted in bile in 24 hr; for TCB, 42.2 & 8.5% (males)and 25.7 + 7.8% (females)were excreted by the sameroute. The lower biliary excretion of HCB than of TCB cannot be accountedfor by a differencein absorption from the gastrointestinaltract and is thought to be due to a slower rate of HCB metabolism.More explicitly, the chlorinesin the 4,4 positionsof HCB appearto preventrapid biliary excretion of the compound by eliminating adjacent unsubstitutedcarbons which are necessaryfor rapid metabolismto occur. Urinary excretion of HCB and TCB was of minor importance compared to biliary excretion. Generally, absorption, distribution. and excretion of the PCBswere similar in malesand females. Polychlorinated bjphenyls (PCBs) have been widely used in industry over the past 40 years. Becauseof their chemical stability, detectable levels have built up in many sectors of the environment. The presence of PCBs in the environment was first reported by Jensen(1966). Since then, PCBs have been detected in food destined for human consumption (Kolbye, 1972) and in human adipose tissue (Yobs, 1972). The PCBs have been implicated in the reproductive failure of severalanimal speciessuch asbirds (Risebrough et al., 1968), mink (Ringer et al., 1972), and rhesus monkeys (Barsotti et al., 1976). Although PCBs have been shown to be toxic and harmful environmental contaminants, information on the absorption, distribution, and excretion of individual PCBs 1 A preliminary
report of this work was presented at the Society of Toxicology Meeting, Atlanta,
Georgia,March 1976.Supportedby USPHSGrantsNo. ES01332,No. ES.00958, andNo. RRO0167. Copyright Q 1976 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
609
610
PETERSON,
SEYMOUR
AND
ALLEN
has been investigated only recently. Matthews and Anderson (1975a, b) reported on the distribution and excretion of four PCBs in male rats. Their results show that greater than 90% of an iv dose of mono-, di-, tri-, and pentachlorobiphenyl was eliminated from the animals over a period of 42 days. Analysis of the elimination of 2,4,5,2’,4’,5’hexachlorobiphenyl (HCB), however, indicated that less than 20 % of the dose would ever be eliminated. Interestingly, the monochlorobiphenyl was excreted equally in urine and feces, but the higher chlorinated compounds were mainly eliminated in the feces. Matthews and Anderson (1975b) suggested that the differences observed in the rate of elimination of these five PCBs were due to differences in their rate of metabolism. In the present study more information about the distribution and excretion of individual PCB isomers in rats was obtained by studying the biliary excretion of these compounds. The only other in viva study of excretion into bile of an individual PCB was in anesthetized male rats (Matthews and Anderson, 1975a). In their study approximately 23 “/, of an iv dose of pentachlorobiphenyl was excreted in bile in 8 hr. In the present report biliary excretion of TCB and HCB was determined in unanesthetized male and female rats for 24 hr after oral administration of the compounds. Urinary excretion was also determined as was the tissue distribution of the compounds. To our knowledge the present paper contains the first report on the distribution and elimination of individual PCBs in female rats. METHODS
Chemicals [3H]-2,5,2’,5’-tetrachlorobiphenyl (TCB) and [3H]-2,4,5,2’,4’,5’-hexachlorobiphenyl (HCB) (Fig. 1) were prepared by the method of Hutzinger and Safe (1972). The compounds were shown to be greater than 99.5% pure by gas-liquid chromatography. Specific activity after dilution with nonradioactive TCB or HCB was 1.0 and 2.0
2
il FIG.
biphenyl,
1. Chemical TCB (2).
structure
of 2,4,5,2’,4’,5’-hexachlorobiphenyl,
61 HCB
(l),
and 2,5,2’,5’-tetrachloro-
BILIARY
EXCRETION
OF PCBS
611
&i/mg, respectively. The compounds were dissolved in corn oil as final preparation for dosage. Animal Treatment and Surgical Preparation
Male and female Sprague-Dawley rats (95-105 g) were fasted for 24 hr prior to surgery with water available ad libitum. The animals were anesthetized with pentobarbital sodium (35 mg/kg, ip) and the common bile duct, the avascular part of the stomach, and the duodenum near the papilla of Vater, were cannulated with the appropriate size polyethylene tubing. After surgery the rats were placed in restraining cages (Stoelting Co., Chicago, Ill.) for 3-5 hr to recover from anesthesia. During recovery and throughout the 24-hr duration of the experiment, the duodenal cannula was connected to an infusion pump (Model 940, Harvard Apparatus Co., Inc., Dover, Mass.). Bile collected 24 hr earlier from donor rats and stored in the dark under nitrogen at -70°C until use was infused (5.1 pljmin) into the intestine. After recovering from anesthesia, a Thermistor probe was inserted into the rectum and the temperature was determined with a Telethermometer (Yellow Springs Instrument Co., Yellow Springs, Ohio). When rectal temperature remained constant at 37 ? 1°C for approximately 30 min, 50 mg/kg of TCB or HCB in 0.5 ml of corn oil was administered into the stomach through the stomach cannula and washed in with 0.3 ml of corn oil. Bile was collected at 6-hr intervals and urine and feces for 24 hr. At the end of 24 hr the animals were sacrificed and tissues were taken for determination of 3H content. Analytical Procedures
The weight of bile in each sample was determined gravimetrically. To estimate the amount of 3H in tissues and excreta, 75-125 mg of material, when available, were oxidized in a sample oxidizer (Model 306, Packard Instrument Co., Downers Grove, Ill.), collected in Monophase 40 scintillation medium (Packard Instrument Co.), and counted in a liquid scintillation counter (Isocap 300, Searle Analytic, Inc., Des Plaines, Ill.). All samples were run in triplicate and quenching was estimated by the sample channels ratio method. To determine the percentage of jH in bile due to excretion of parent compound and unconjugated metabolites, radioactivity was extracted from bile samples with hexane followed by extraction with diethyl ether. Bile collected from O-6 and 6-12 hr was combined to give a O-12 hr sample. Similarly, 12-18 and 18-24 hr collections were combined to give a 12-24 hr sample. These combinations were done separately for all male and female rats for each PCB isomer. The combined samples were extracted three times with 2-3 volumes of hexane to remove unmetabolized PCB and monohydroxy metabolites (Van Miller et al., 1975). The radioactivity which remained in bile was then extracted three times with 2-3 volumes of diethyl ether to remove more polar metabolites. The percentage radioactivity which remained after diethyl ether extraction is designated as “aqueous” and it represents the most polar metabolites and conjugates of TCB and HCB that were found in bile. This radioactivity in the aqueous fraction was not reduced by freeze-drying and therefore was not due to [3H]water. [3H]PCB recovered from the gastrointestinal tract contents and feces was minced with a Polytron tissue homogenizer (Kinematica, Luzern, Switzerland) in 5 volumes (v/w) of hexane several times until the last hexane extract was near background. The
612
PETERSON, SEYMOUR AND ALLEN
hexane extract wasthen analyzed for total 3H and the remaining fecesand contents were dried, weighed, and oxidized to determine the 3H remaining. Gas-liquid chromatography was carried out on the hexane extracts (Hewlett-Packard 7620A, 3 % SE-30 on Gaschrom Q, 2OO”C,5 ft. x l/8 in. id, 35 ml/min, Argon-CH, (95/5)) utilizing an electron capture detector.
Statistical Analysis The Student’s t test (Snedecor and Cochran, 1967)wasusedto compare bile flow and excretion of TCB- and HCB-derived radioactivity in bile and urine. Significance was set at p 6 0.05. RESULTS
Table 1 shows the tissue distribution of radioactivity 24 hr after TCB and HCB administration. Overall the distribution tended to follow a similar pattern for both compounds in female and male animals. Tissues which consistently contained the greatest percentage of the administered dosewere skeletal muscle, skin, liver, and small TABLE
1
PERCENTAGE 3H DOSE IN TISSUES OF BILE DUCT-CANNULATED
RATS 24 HR AFTER [3H]PCB ADMINISTRATION INTO THE STOMACH”
Females 2,5,2’,5’-TCB
Skeletalmuscle Skin Liver Small intestine Blood Stomach Large intestine Testes Uterus Ovaries Brain Adrenal glands Lungs Heart Kidneys Spleen Pancreas Thymus Urinary bladder Submaxillary glands
7.4 5.5 3.8 13.6
* 2.0 f 2.2 r!~2.7 f 8.0
(5)b (5) (5) (5)
Males
2,4,5,2’,4’,5’-HCB
12.4 7.4 7.8 6.3
+ + -t *
5.0 3.9 5.7 5.7
(4) (4) (4) (4)
14.7 8.2 5.4 6.6
* + + +
1.9 1.3 0.7 3.6
(5) (5) (5) (5)
1.4 * 0.4 (4) 1.4 + 0.5 (5) 1.2 + 0.3 (5)
0.8 f 0.3 (3) 0.8 L- 0.2 (4) 1.0 f 0.6 (4)
2.4 1.2 1 .o 0.6
+ f f f.
1.4 0.4 0.3 0.1
(3) (4) (4) (4)
1.3 0.8 1.4 1.2
+ 2 + +
0.7 0.2 0.3 0.2
(4) (5) (5) (5)
0.2 0.2 0.3 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.04
0.06 0.05 0.3 0.1 0.2 0.1 0.3 0.02 0.3 0.1 0.01
0.7 0.3 0.3 0.2 0.3 0.1 0.1 0.1 0.1
& 0.3 (4) & 0.1 (3) + 0.04 (4) + 0.03 (3) * 0.04 (4) * 0.04 (3) * 0.01 (3) + 0.04 (3) + 0.06 (3)
1.5 0.8 0.9 0.6 1.3 0.4 0.8 0.4 0.1
+ 0.3 (5) + 0.1 (5) +_ 0.2 (5) k 0.2 (5) + 0.2 (5) * 0.1 (5) + 0.3 (3) + 0.2 (5) f 0.03 (4)
0.02 f 0.02 (3)
f. f f +
f + f f + 2 + + * f f
4.7 2.6 6.5 0.5
2,4,5,2’,4’,5’-HCB
(4) (4) (4) (4)
+ 0.04 (3) f 0.1 (3) + 0.1 (5) & 0.03 (3) + 0.05 (5) + 0.02 (3) + 0.04 (4) + 0.02 (3) f 0.07 (3) + 0.03 (3) If: 0.03 (3)
5.3 3.0 23.1 1.7
2,5,2’,5’-TCB
0.03 (3) 0.03 (3) 0.1 (3) 0.01 (3) 0.02 (3) 0.02 (3) 0.1 (3) 0.01 (3) 0.1 (3) 0.02 (3) 0.01 (3)
0.1 * 0.02 (3)
0.1 -t 0.03 (3)
0.8 f 0.4 (5)
o [3H]-2,5,2’,5’-Tetrachlorobiphenyl (TCB) or [3H]-2,4,5,2’,4’,5’-Hexachlorobiphenyl (HCB). b Mean f SE of three to five rats. The figures in parentheses indicate number of animals used.
BILIARYEXCRETION
613
OFPCBS
intestine. All other tissues contained less than 2.5% of the administered dose. An unexpected finding was the greater percentage dose of radioactivity in the livers of female than of male rats given HCB. Table 2 shows the distribution of radioactivity in gastrointestinal tract contents, feces, urine, and bile. In considering these results, it should be recalled that TCB and HCB were administered into the stomach of bile duct-cannulated animals. Since bile was collected continuously and not returned to the gastrointestinal tract, the total percentage dose of radioactivity recovered from gastrointestinal tract contents and feces together represents unabsorbed TCB and HCB. This was verified by hexane extraction and gas-liquid chromatography (glc) where greater than 99 % of the 3H in TABLE 2 PERCENTAGE 3H DOSEIN GASTROINTESTINALTRACT CONTENTSAND EXCRETAOFBILE CANNULATED RATS 24 HRAFTER [3H]PCB ADMINISTRATIONINTOTHESTOMACH~
Females
Males
2,5,2’,5’-TCB 2,4,5,2’,4’,5’-HCB 2,5,2’,5’-TCB Stomachcontents 6.7 f 4.2 (5)b Smallintestine contents 11.1 f 4.5 (5) Large intestine 11.3 + 4.2 (5) contents Feces
11.6+
Total of gut contentsand feces
40.6 f 1.1 (5)
Bile Urine
5.0(5)
25.7 f 7.8 (5)d
1.7+ 1.0(5)
DUCT-
2,4,5,2’,4’,5’-HCB
5.3 f 2.9 (4)
0.05 L- 0.05 (4)
1.1 f 1.0(5)
19.8 f 6.4 (4)
2.6 f 1.4 (4)
7.2 + 4.5 (5)
10.2 f 2.1 (4) 17.8 + 4.2 (4)
13.1 If: 7.3 (4) 9.9 -t 3.7 (4)
20.7 + 2.1 (5) 10.8 + 4.1 (5)
55.1 * 7.4 (4)
25.6
44.9
1.1 + 0.3 (4) 0.03 * 0.03 (4)
f 4.4 (4)’
42.2 + 8.5 (4)d 1.7 f 0.6 (4)d
+ 8.2 (5)
0.5 + 0.2 (5) 0.02 f 0.02 (5)
’ [3H]-2,5,2’,5’-Tetrachlorobiphenyl (TCB) or [3H]-2,4,5,2’,4’,5’-Hexachlorobiphenyl (HCB). b Mean + SE of four to five. The figures in parentheses indicate number of animals used. c TCB vs HCB within sexes p c 0.05. d TCB vs HCB within sexes p < 0.01.
gastrointestinal contents and feceswas extracted by hexane, and the extracts showed no marked abnormalities on glc tracings when compared to unmetabolized synthetic PCB. In examining the total percentage dose of 3H recovered from gut contents and fecesin Table 2, it is seenthat from 25.6-55.1x of the dose wasnot absorbed. TCB was excreted into bile and urine to a greater extent than HCB in animals of both sexes (p < 0.01). Also, there was a distinct preference for biliary rather than urinary excretion of the PCBs. This preference for biliary excretion was more obvious for TCB than for HCB. The data presented in Tables 1 and 2 are summarized in Fig. 2 to illustrate major differences between TCB and HCB. The full height of each bar is for recovery of the dose of radioactivity in all body tissues and excreta analyzed. These results show, depending upon the compound and the sex of the animal, that 93 (mean) to 109% of
614
PETERSON,
SEYMOUR
AND
ALLEN
the doses were recovered. With respect to absorption, TCB tended to be absorbed to a greater extent than HCB. This is shown in Fig. 2 by the percentage dose of radioactivity recovered from gastrointestinal tract contents and feces tending to be lower in rats administered TCB. This tendency for greater absorption of TCB than of HCB was significant only in male rats (p < 0.05). Interestingly the total percentage dose of radioactivity that was recovered from all of the tissues analyzed, was similar for TCB and HCB, varying from 36 % (mean) of the TCB dose in female rats to 47 % of the HCB dose in males. m
URINE
0
BILE
m
OTHER
m
SKELETAL
MUSCLE,
‘3. TRACT
CONTENTS,
TISSUES SKIN,
LIVER.
SMALL
INTESTINE
FECES
60 3
' l-
60
E B P
40 20 n ”
TCB
HCB FEMALES
TCB
HCB MALES
FIG. 2. Major tissues and excreta involved in the distribution and elimination of TCB- and HCBderived radioactivity in rats. The full height of each bar and vertical line next to it represents the percentage dose recovered from the entire animal (mean + SE of three to five rats). The subdivisions within each bar show the percentage dose recovered from different tissues and excreta.
The most obvious difference between TCB and HCB shown in Fig. 2 involves elimination of the dose in bile and urine. Excretion in bile and urine together accounted for approximately 27 and 44% of the TCB dose in female and male rats, respectively. On the other hand, less than 2 % of the HCB dose was excreted in animals of either sex by these routes. The difference in gut absorption between TCB and HCB is not of sufficient magnitude to account for the far greater excretion of TCB- than of HCBderived radioactivity. One result, however, where a difference in gut absorption may have been responsible for a difference in biliary excretion in seen in the data for TCB. Figure 2 shows that female rats excreted a lower percentage of the TCB dose in bile (25.7 +_7.8 %, mean f SE) than males (42.2 f 8.5 %). Figure 3 (top panel) shows the time course for biliary excretion of radioactivity during the 24-hr period after TCB and HCB administration. For female and male rats
BILIARY
EXCRETION
615
OF PCBS
administered TCB the time course for appearance of radioactivity in bile was similar. Biliary excretion was lowest at O-6 hr, highest at 6-12 and 12-18 hr, and then decreased 18-24 hr after administration. In the case of HCB, the appearance of radioactivity in bile followed a different time course, being nonexistent between 0 and 6 hr and then remaining relatively constant for the 6-12, 12-18, and 18-24 hr collections. At all collection periods, except 18-24 hr for female rats, the biliary excretion of radioactivity was significantly greater for TCB than for HCB (p < 0.05). The bottom panel of Fig. 3 shows that bile flow was similar in rats given TCB and HCB. Thus, the difference in biliary excretion between the two compounds was not caused by a difference in bile MALES
FEMALES 20 3H-2,5,2:5’-TCB
,5
;
3H-2,4,5,2:4:5'-HCB
I
IOk
O-6
6-Q
12-18
16-24
HOURS
AFTER
O-6 3wPCB
6-12
12-m
la-24
ADMINISTRATION
FIG. 3. Biliary excretion of TCB- and HCB-derived radioactivity in male and female rats (top panel) and bile flow (bottom panel). Each value is the mean rt SE of three to five rats. The asterisk indicates a significant difference @ < 0.05) between rats of the same sex administered TCB and HCB.
Table 3 provides preliminary information on the disposition of TCB- and HCBderived radioactivity in bile. As was mentioned previously, the radioactivity extracted into hexane represents the parent compound and monohydroxy metabolites of this compound (Van Miller et al., 1975). Radioactivity extracted into diethyl ether is predominantly due to metabolites that are more polar than the monohydroxy metabolites. The most polar metabolites of TCB and HCB were not significantly extracted into hexane or ether and are designated in Table 3 as “aqueous.” Approximately 90 % of the 3H remaining in all aqueous fractions (male-female, HCB-TCB) could be extracted with diethyl ether after a 24-hr incubation at 37°C with sulfatase (containing &glucuronidase) (Sigma Chemical Co., St. Louis, MO.) with 50 Sigma units/ml in 0.2 M sodium acetate buffer, pH 5.0. For rats treated with TCB, greater than 78 % of the radioactivity in bile was present in the aqueous phase, and represented polar metabolites of TCB. Also, when the smaller percentage of radioactivity present in the hexane extract of bile
616
PETERSON,
SEYMOUR
AND
ALLEN
was purified by thin-layer chromatography and analyzed by gas-liquid chromatography no unmetabolized TCB could be found. Thus, for rats administered TCB, all of the radioactivity in bile was due to metabolites. The distribution of radioactivity in hexane and ether extracts of bile and the aqueous phase was different for rats given HCB. For HCB, between 30 and 40 % of the radioactivity in bile was extracted into hexane as compared to between 2 and 17% for TCB. Thus, for HCB a greater percentage of the radioactivity in bile was due to nonpolar material. Gas-liquid chromatography of the hexane extract indicated that the extractable 3H portion is at least 95 % parent compound. TABLE PERCENTAGE
DISTRIBUTION
OF 3H IN HEXANE
3
AND ETHER
EXTRACTS
OF POOLED
BILE
SAMPLES
FROM RATS ADMINISTERED t3H]PCB
Females O-12hr” 12-24 hrb bile bile
Males O-12 hr
12-24 hr
bile
bile .-
13H]-2,5,2’,5’-TCB
Hexaneextract Ether extract Aqueous’
3.2 91.8
2.8 0.8 96.4
17.1 4.5 78.4
8.8 0.9 90.3
30.2 10.3 59.5
40.1 9.4 50.5
40.1 7.8 52.1
40.0 4.5 55.5
5.0
[3H]-2,4,5,2’,4’,5’-HCB
Hexaneextract Ether extract Aqueous
LISamples of O-6 and &12-hr bile from four to five rats were pooled prior to extraction. * Samples of 12-18- and 18-24-hr bile from four to five rats were pooled prior to extraction. c 3H in bile not extracted into hexane or ether. DISCUSSION
Generally speaking, absorption, distribution, and excretion of radioactivity after TCB or HCB administration were similar in female and male animals for each isomer. In both sexes skeletal muscle, skin, liver, and small intestine were the major tissues involved in the localization of radioactivity, and bile was the main route of excretion. Greater biliary excretion of TCB in males than in females was caused by greater absorption of the compound in males(Table 2, Fig. 2) and is not due to a sex difference in hepatic excretory function. Recovery of HCB in the liver was higher in female than in male animals. This difference (23.1 f 6.5 %, mean f SE, femalesversus 5.4 + 0.7 %, males)cannot be accounted for by differencesin absorption or biliary excretion of HCB. Although absorption, distribution, and excretion of each PCB were similar overall in female and male rats, chronic feeding studies of PCBs in other specieshave shown that females develop signs of PCB toxicity at lower levels of PCB intake. This has been
BILIARY
EXCRETION
OF PCBS
617
demonstrated for rhesus monkeys (Barsotti et al., 1976) and mink (Platonow and Karstad, 1973). With respect to PCB absorption from the gastrointestinal tract, our results in restrained rats differ from those of other investigators where rats that were not exposed to anesthesia, surgery, and body restraint were used. Van Miller et al. (1975) and Matthews and Anderson (1975b) recovered as unmetabolized material less than 5% of an oral dose of TCB and HCB from gut contents and feces of rats 24 hr after administration. In the present study, depending on the PCB and sex of the animal, between 25 and 55 % of the dose was not absorbed. The cause of the incomplete absorption was not related to an absence of bile from the gastrointestinal tract because bile was continuously infused into the duodenum. In preliminary experiments where bile was not infused, only 5-10 % of the PCB dose was absorbed in 24 hr as compared to 45-75 “, when bile was infused. Thus, the presence of bile in the intestine was necessary for significant absorption of PCBs to occur, and lack of bile was not the reason for the slower absorption. Also hypothermia cannot be invoked as an explanation because body temperature was maintained near 37°C. The most feasible explanation is that stress associated with anesthesia, surgery, and body restraint in the present study, but not in those of Van Miller et al. (1975) and of Matthews and Anderson (1975a, b), was involved in producing the effect. Among the various tissues analyzed for distribution of TCB and HCB, the greatest percentage of the dose was located in skeletal muscle, skin, liver, and small intestine. The high recovery from skeletal muscle was expected since this tissue constitutes approximately 50 % of the body weight. Also Matthews and Anderson (1975a, b) have shown in male rats that skeletal muscle is a major depot for iv-administered PCBs. The skin and liver which represent a lower percentage of the total body weight, 16 and 5 2;, respectively, were also involved to a major extent in the tissue distribution of radioactivity. Van Miller et al. (1975) and Matthews and Anderson (1975a, b) have also demonstrated a greater involvement of skin and liver in the distribution of TCB and HCB in non-bile duct-cannulated male rats. In the present study the small intestine was also included as a major tissue depot for radioactivity. This was probably caused by ongoing absorption of TCB and HCB through the tissue of the small intestine at the time the animals were sacrificed. This suggestion is supported by data in Table 2 which show that a significant amount of unabsorbed TCB and HCB was present in the small intestinal contents at this time. In distribution studies of PCBs in rats, adipose tissue is usually a major storage depot (Matthews and Anderson, 1975a, b; Van Miller et al., 1975). This was not the case in the present study, because very little adipose tissue was present in the animals (95-105 g) to begin with and virtually none was present at the time of sacrifice. With respect to metabolism and elimination, a major finding of this study was the 25- to 40-fold greater biliary excretion of TCB than of HCB. This difference in excretion cannot be adequately explained by a difference in absorption of the compounds and appears to relate instead to the more rapid rate of TCB than HCB metabolism (Van Miller et al., 1975; Matthews and Anderson, 397513). Ghiasuddin et al. (1976) have also shown in a liver microsomal system prepared from phenobarbital-treated rats that the lower chlorinated isomers are metabolized to a greater extent than the higher chlorinated ones. This difference in rate of metabolism was reflected in the present study by 21
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differences in the disposition of these compounds in bile. For rats treated with TCB, 100% of the radioactivity in bile was from metabolites. On the other hand, for rats given HCB, 3@-40% of the radioactivity in bile was extracted into hexane, of which greater than 95 % was from the parent compound. Recently, Mehendale (1976) has obtained biliary excretion results for individual PCBs, using an isolated perfused rat liver preparation, that are in agreement with the greater biliary excretion of TCB (26 % females, 42 % males) than of HCB (1.1% females, 0.5 % males) reported here in intact animals. In his investigation, Mehendale (1976) found that 48,30,20, and 1% of the dose of mono-, di-, penta-, and hexachlorobiphenyl were excreted by the isolated perfused rat liver in 4 hr. Thus, biliary excretion was inversely related to the degree of chlorination. In the present study the chlorines in the 4,4’ positions of HCB (Fig. 1) appear to prevent the rapid biliary excretion of HCB by eliminating adjacent unsubstituted carbons which are necessary for rapid metabolism to occur. Jondorf et al. (1955) first demonstrated that two adjacent unsubstituted carbon atoms, which are present in TCB but not in HCB, were necessary for an appreciable rate of metabolism of the trichlorobenzenes. The possibility that a similar requirement pertains to the rate of metabolism of individual PCBs has been suggested by Schulte and Acker (1974) and Matthews and Anderson (1975b). Interestingly, a preliminary study by Matthews and Tuey (1976) has shown that 3,5,3’,5’-tetrachlorobiphenyl, which is like HCB in the sense that it lacks two adjacent unsubstituted carbon atoms, is excreted in the feces of rats after iv administration at a significantly greater rate than HCB. Thus, the presence of two adjacent unsubstituted carbon atoms in TCB may partially explain the greater biliary excretion of TCB than of HCB in the present study, but it is probably not the only factor involved. ACKNOWLEDGMENTS We wish to thank Miss Patricia Hack, Mr. Steven Schmidt, and Mr. King Yan Lee for their expert technical assistance. REFERENCES
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S. M., MENZER, R. E., AND NELSON, J. 0. (1976).Metabolismof 2,5,2’-trichloro-, 2,5,2’,5’-tetrachloro-, and 2,4,5,2’,5’-pentachlorobiphenyl in rat hepatic microsomal systems.Toxicol. Appl. Pharmacol. 36, 187-194. HUTZINGER, 0. AND SAFE, S. (1972).A generalmethod for the preparation of tritiated polychlorobiphenylsof high specificactivity: 2,2’,5,5’-Tetrachlorobiphenyl-[3H]and 2,2’,4,4’,5, 5’-hexachlorobiphenyl-[3H].Bull. Environ. Contam. Toxicol. 7, 374-375. JENSEN, S. (1966).Report on a newchemicalhazard. New Sci. 32,612. JONDORF, W. F., PARKE, D. V., AND WILLIAMS, R. T. (1955).Detoxication LXVI. The metabolism of halogenobenzenes 1,2,3-,1,3,5-trichlorobenzenes.Biochem. J. 61, 512-521. KOLBYE, A. C. (1972).Food exposureto polychlorinated biphenyls.Environ. Health Perspec. GHIASUDDIN,
1,85-88.
H. B.,ANDANDERSON, M. W. (1975a)The distribution and excretion of 2,4,5,2’,5’pentachlorobiphenylin the rat. Drug Metab. Dispos. 3,211-219. MATTHEWS, H. B., AND ANDERSON, M. W. (1975b).Effect of chlorination on the distribution and excretion of polychlorinated biphenyls. Drug. Metab. Dispos. 3, 371-380. MATTHEWS,
BILIARY
EXCRETION
OF PCBS
619
MATTHEWS, H. B., AND TUEY, D. B. (1976).The distribution and excretion of 3,5,3’,5’-tetra-
chlorobiphenyl in the malerat. Abstract No. 133,Society of Toxicology Meeting, Atlanta, Georgia, March 14-18, Toxicol. Appl. Pharmacol. 37, 147. MEHENDALE, H. M. (1976).Uptake and dispositionof chlorinated biphenylsby isolatedperfusedrat liver. Drag. Metab. Dispos. 4, 124-132. PLATONOW, N. S., AND KARSTAD, L. H. (1973). Dietary effectsof polychlorinated biphenyls on mink. Canad. J. Comp. Med. 37,391-400. RINGER, R. K., AULERICH, R. J., AND ZABIK, M. (1972). Effect of dietary polychlorinated biphenylson growth and reproduction of mink. Proc. Amer. Chem. Sot. 12,149. RISEBROUGH, R. W., HERMAN, S. G., PEAKALL, D. B., AND KIRVEN, M. N. (1968). Polychlorinated biphenylsin the global ecosystem.Nature (London) 220, 1098-l 102. SCHULTE, E., AND ACKER, L. (1974). Identification and metabolizability of polychlorinated biphenyls.Naturwisserzschaften 61, 79-90. SNEDECOR, G. W., AND COCHRAN, W. G. (1967). Statistical Methods. Iowa State University Press,Ames,Iowa. VAN MILLER, J. P., Hsu, 1. C., AND ALLEN, J. R. (1975). Distribution and metabolismof 3H-2,5,2’,5’-tetrachlorobiphenylin rats. Proc. Sot. Exp. Biol. Med. 148, 682-687. Yoes, A. R. (1972). Levels of polychlorinated biphenyls in adiposetissueof the general population of the nation. Environ. Health Perspec. 1, 79-8 1.
OF TOXICOLOGY
Distribution
AND
APPLIED
and
PHARMACOLOGY
38,609~619 (1976)
Biliary Excretion of Polychlorinated Biphenyls in Rats’
RICHARDE. PETERSON, JONL. SEYMOUR, AND JAMES R. ALLEN School of Pharmacy, Department of Pathology, School of Medicine, and Regional Primate Research Center, University of Wisconsin, Madison, Wisconsin 53706 Received May 18,1976; acceptedJuIy 29,1976
Distribution and Biliary Excretion of PolychlorinatedBiphenylsin Rats. PETERSON, R. E., SEYMOUR, J. L., AND ALLEN, J. R. (1976). Toxicol. Appl. Pharmacol. 38, 609-619. Absorption, distribution, and excretion of two 3H-labeled
polychlorinated
biphenyls
(PCB),
2,4,5,2’,4’,5’,-hexachloro-
biphenyl (HCB) and 2,5,2’,5’-tetrachlorobiphenyl(TCB), were studiedin surgically prepared male and female rats. Approximately 3-5 hr after surgery, HCB or TCB (50 mg/kg) was administeredinto the stomach. Bile, urine, and feceswerecollectedfor 24 hr after which the animalswere sacrificedand tissuestaken for determination of 3H content. The distribution of 3H remainingin the rats, expressedas percentageof dose,was highestin skeletal muscle, skin, liver, and small intestine for both isomers. The major differenceobservedbetweenthe PCBswasin biliary excretion. For HCB, 0.5 f 0.2 % (males, mean + SE) and I .l + 0.3 % (females) of the dose were excreted in bile in 24 hr; for TCB, 42.2 & 8.5% (males)and 25.7 + 7.8% (females)were excreted by the sameroute. The lower biliary excretion of HCB than of TCB cannot be accountedfor by a differencein absorption from the gastrointestinaltract and is thought to be due to a slower rate of HCB metabolism.More explicitly, the chlorinesin the 4,4 positionsof HCB appearto preventrapid biliary excretion of the compound by eliminating adjacent unsubstitutedcarbons which are necessaryfor rapid metabolismto occur. Urinary excretion of HCB and TCB was of minor importance compared to biliary excretion. Generally, absorption, distribution. and excretion of the PCBswere similar in malesand females. Polychlorinated bjphenyls (PCBs) have been widely used in industry over the past 40 years. Becauseof their chemical stability, detectable levels have built up in many sectors of the environment. The presence of PCBs in the environment was first reported by Jensen(1966). Since then, PCBs have been detected in food destined for human consumption (Kolbye, 1972) and in human adipose tissue (Yobs, 1972). The PCBs have been implicated in the reproductive failure of severalanimal speciessuch asbirds (Risebrough et al., 1968), mink (Ringer et al., 1972), and rhesus monkeys (Barsotti et al., 1976). Although PCBs have been shown to be toxic and harmful environmental contaminants, information on the absorption, distribution, and excretion of individual PCBs 1 A preliminary
report of this work was presented at the Society of Toxicology Meeting, Atlanta,
Georgia,March 1976.Supportedby USPHSGrantsNo. ES01332,No. ES.00958, andNo. RRO0167. Copyright Q 1976 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
609
610
PETERSON,
SEYMOUR
AND
ALLEN
has been investigated only recently. Matthews and Anderson (1975a, b) reported on the distribution and excretion of four PCBs in male rats. Their results show that greater than 90% of an iv dose of mono-, di-, tri-, and pentachlorobiphenyl was eliminated from the animals over a period of 42 days. Analysis of the elimination of 2,4,5,2’,4’,5’hexachlorobiphenyl (HCB), however, indicated that less than 20 % of the dose would ever be eliminated. Interestingly, the monochlorobiphenyl was excreted equally in urine and feces, but the higher chlorinated compounds were mainly eliminated in the feces. Matthews and Anderson (1975b) suggested that the differences observed in the rate of elimination of these five PCBs were due to differences in their rate of metabolism. In the present study more information about the distribution and excretion of individual PCB isomers in rats was obtained by studying the biliary excretion of these compounds. The only other in viva study of excretion into bile of an individual PCB was in anesthetized male rats (Matthews and Anderson, 1975a). In their study approximately 23 “/, of an iv dose of pentachlorobiphenyl was excreted in bile in 8 hr. In the present report biliary excretion of TCB and HCB was determined in unanesthetized male and female rats for 24 hr after oral administration of the compounds. Urinary excretion was also determined as was the tissue distribution of the compounds. To our knowledge the present paper contains the first report on the distribution and elimination of individual PCBs in female rats. METHODS
Chemicals [3H]-2,5,2’,5’-tetrachlorobiphenyl (TCB) and [3H]-2,4,5,2’,4’,5’-hexachlorobiphenyl (HCB) (Fig. 1) were prepared by the method of Hutzinger and Safe (1972). The compounds were shown to be greater than 99.5% pure by gas-liquid chromatography. Specific activity after dilution with nonradioactive TCB or HCB was 1.0 and 2.0
2
il FIG.
biphenyl,
1. Chemical TCB (2).
structure
of 2,4,5,2’,4’,5’-hexachlorobiphenyl,
61 HCB
(l),
and 2,5,2’,5’-tetrachloro-
BILIARY
EXCRETION
OF PCBS
611
&i/mg, respectively. The compounds were dissolved in corn oil as final preparation for dosage. Animal Treatment and Surgical Preparation
Male and female Sprague-Dawley rats (95-105 g) were fasted for 24 hr prior to surgery with water available ad libitum. The animals were anesthetized with pentobarbital sodium (35 mg/kg, ip) and the common bile duct, the avascular part of the stomach, and the duodenum near the papilla of Vater, were cannulated with the appropriate size polyethylene tubing. After surgery the rats were placed in restraining cages (Stoelting Co., Chicago, Ill.) for 3-5 hr to recover from anesthesia. During recovery and throughout the 24-hr duration of the experiment, the duodenal cannula was connected to an infusion pump (Model 940, Harvard Apparatus Co., Inc., Dover, Mass.). Bile collected 24 hr earlier from donor rats and stored in the dark under nitrogen at -70°C until use was infused (5.1 pljmin) into the intestine. After recovering from anesthesia, a Thermistor probe was inserted into the rectum and the temperature was determined with a Telethermometer (Yellow Springs Instrument Co., Yellow Springs, Ohio). When rectal temperature remained constant at 37 ? 1°C for approximately 30 min, 50 mg/kg of TCB or HCB in 0.5 ml of corn oil was administered into the stomach through the stomach cannula and washed in with 0.3 ml of corn oil. Bile was collected at 6-hr intervals and urine and feces for 24 hr. At the end of 24 hr the animals were sacrificed and tissues were taken for determination of 3H content. Analytical Procedures
The weight of bile in each sample was determined gravimetrically. To estimate the amount of 3H in tissues and excreta, 75-125 mg of material, when available, were oxidized in a sample oxidizer (Model 306, Packard Instrument Co., Downers Grove, Ill.), collected in Monophase 40 scintillation medium (Packard Instrument Co.), and counted in a liquid scintillation counter (Isocap 300, Searle Analytic, Inc., Des Plaines, Ill.). All samples were run in triplicate and quenching was estimated by the sample channels ratio method. To determine the percentage of jH in bile due to excretion of parent compound and unconjugated metabolites, radioactivity was extracted from bile samples with hexane followed by extraction with diethyl ether. Bile collected from O-6 and 6-12 hr was combined to give a O-12 hr sample. Similarly, 12-18 and 18-24 hr collections were combined to give a 12-24 hr sample. These combinations were done separately for all male and female rats for each PCB isomer. The combined samples were extracted three times with 2-3 volumes of hexane to remove unmetabolized PCB and monohydroxy metabolites (Van Miller et al., 1975). The radioactivity which remained in bile was then extracted three times with 2-3 volumes of diethyl ether to remove more polar metabolites. The percentage radioactivity which remained after diethyl ether extraction is designated as “aqueous” and it represents the most polar metabolites and conjugates of TCB and HCB that were found in bile. This radioactivity in the aqueous fraction was not reduced by freeze-drying and therefore was not due to [3H]water. [3H]PCB recovered from the gastrointestinal tract contents and feces was minced with a Polytron tissue homogenizer (Kinematica, Luzern, Switzerland) in 5 volumes (v/w) of hexane several times until the last hexane extract was near background. The
612
PETERSON, SEYMOUR AND ALLEN
hexane extract wasthen analyzed for total 3H and the remaining fecesand contents were dried, weighed, and oxidized to determine the 3H remaining. Gas-liquid chromatography was carried out on the hexane extracts (Hewlett-Packard 7620A, 3 % SE-30 on Gaschrom Q, 2OO”C,5 ft. x l/8 in. id, 35 ml/min, Argon-CH, (95/5)) utilizing an electron capture detector.
Statistical Analysis The Student’s t test (Snedecor and Cochran, 1967)wasusedto compare bile flow and excretion of TCB- and HCB-derived radioactivity in bile and urine. Significance was set at p 6 0.05. RESULTS
Table 1 shows the tissue distribution of radioactivity 24 hr after TCB and HCB administration. Overall the distribution tended to follow a similar pattern for both compounds in female and male animals. Tissues which consistently contained the greatest percentage of the administered dosewere skeletal muscle, skin, liver, and small TABLE
1
PERCENTAGE 3H DOSE IN TISSUES OF BILE DUCT-CANNULATED
RATS 24 HR AFTER [3H]PCB ADMINISTRATION INTO THE STOMACH”
Females 2,5,2’,5’-TCB
Skeletalmuscle Skin Liver Small intestine Blood Stomach Large intestine Testes Uterus Ovaries Brain Adrenal glands Lungs Heart Kidneys Spleen Pancreas Thymus Urinary bladder Submaxillary glands
7.4 5.5 3.8 13.6
* 2.0 f 2.2 r!~2.7 f 8.0
(5)b (5) (5) (5)
Males
2,4,5,2’,4’,5’-HCB
12.4 7.4 7.8 6.3
+ + -t *
5.0 3.9 5.7 5.7
(4) (4) (4) (4)
14.7 8.2 5.4 6.6
* + + +
1.9 1.3 0.7 3.6
(5) (5) (5) (5)
1.4 * 0.4 (4) 1.4 + 0.5 (5) 1.2 + 0.3 (5)
0.8 f 0.3 (3) 0.8 L- 0.2 (4) 1.0 f 0.6 (4)
2.4 1.2 1 .o 0.6
+ f f f.
1.4 0.4 0.3 0.1
(3) (4) (4) (4)
1.3 0.8 1.4 1.2
+ 2 + +
0.7 0.2 0.3 0.2
(4) (5) (5) (5)
0.2 0.2 0.3 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.04
0.06 0.05 0.3 0.1 0.2 0.1 0.3 0.02 0.3 0.1 0.01
0.7 0.3 0.3 0.2 0.3 0.1 0.1 0.1 0.1
& 0.3 (4) & 0.1 (3) + 0.04 (4) + 0.03 (3) * 0.04 (4) * 0.04 (3) * 0.01 (3) + 0.04 (3) + 0.06 (3)
1.5 0.8 0.9 0.6 1.3 0.4 0.8 0.4 0.1
+ 0.3 (5) + 0.1 (5) +_ 0.2 (5) k 0.2 (5) + 0.2 (5) * 0.1 (5) + 0.3 (3) + 0.2 (5) f 0.03 (4)
0.02 f 0.02 (3)
f. f f +
f + f f + 2 + + * f f
4.7 2.6 6.5 0.5
2,4,5,2’,4’,5’-HCB
(4) (4) (4) (4)
+ 0.04 (3) f 0.1 (3) + 0.1 (5) & 0.03 (3) + 0.05 (5) + 0.02 (3) + 0.04 (4) + 0.02 (3) f 0.07 (3) + 0.03 (3) If: 0.03 (3)
5.3 3.0 23.1 1.7
2,5,2’,5’-TCB
0.03 (3) 0.03 (3) 0.1 (3) 0.01 (3) 0.02 (3) 0.02 (3) 0.1 (3) 0.01 (3) 0.1 (3) 0.02 (3) 0.01 (3)
0.1 * 0.02 (3)
0.1 -t 0.03 (3)
0.8 f 0.4 (5)
o [3H]-2,5,2’,5’-Tetrachlorobiphenyl (TCB) or [3H]-2,4,5,2’,4’,5’-Hexachlorobiphenyl (HCB). b Mean f SE of three to five rats. The figures in parentheses indicate number of animals used.
BILIARYEXCRETION
613
OFPCBS
intestine. All other tissues contained less than 2.5% of the administered dose. An unexpected finding was the greater percentage dose of radioactivity in the livers of female than of male rats given HCB. Table 2 shows the distribution of radioactivity in gastrointestinal tract contents, feces, urine, and bile. In considering these results, it should be recalled that TCB and HCB were administered into the stomach of bile duct-cannulated animals. Since bile was collected continuously and not returned to the gastrointestinal tract, the total percentage dose of radioactivity recovered from gastrointestinal tract contents and feces together represents unabsorbed TCB and HCB. This was verified by hexane extraction and gas-liquid chromatography (glc) where greater than 99 % of the 3H in TABLE 2 PERCENTAGE 3H DOSEIN GASTROINTESTINALTRACT CONTENTSAND EXCRETAOFBILE CANNULATED RATS 24 HRAFTER [3H]PCB ADMINISTRATIONINTOTHESTOMACH~
Females
Males
2,5,2’,5’-TCB 2,4,5,2’,4’,5’-HCB 2,5,2’,5’-TCB Stomachcontents 6.7 f 4.2 (5)b Smallintestine contents 11.1 f 4.5 (5) Large intestine 11.3 + 4.2 (5) contents Feces
11.6+
Total of gut contentsand feces
40.6 f 1.1 (5)
Bile Urine
5.0(5)
25.7 f 7.8 (5)d
1.7+ 1.0(5)
DUCT-
2,4,5,2’,4’,5’-HCB
5.3 f 2.9 (4)
0.05 L- 0.05 (4)
1.1 f 1.0(5)
19.8 f 6.4 (4)
2.6 f 1.4 (4)
7.2 + 4.5 (5)
10.2 f 2.1 (4) 17.8 + 4.2 (4)
13.1 If: 7.3 (4) 9.9 -t 3.7 (4)
20.7 + 2.1 (5) 10.8 + 4.1 (5)
55.1 * 7.4 (4)
25.6
44.9
1.1 + 0.3 (4) 0.03 * 0.03 (4)
f 4.4 (4)’
42.2 + 8.5 (4)d 1.7 f 0.6 (4)d
+ 8.2 (5)
0.5 + 0.2 (5) 0.02 f 0.02 (5)
’ [3H]-2,5,2’,5’-Tetrachlorobiphenyl (TCB) or [3H]-2,4,5,2’,4’,5’-Hexachlorobiphenyl (HCB). b Mean + SE of four to five. The figures in parentheses indicate number of animals used. c TCB vs HCB within sexes p c 0.05. d TCB vs HCB within sexes p < 0.01.
gastrointestinal contents and feceswas extracted by hexane, and the extracts showed no marked abnormalities on glc tracings when compared to unmetabolized synthetic PCB. In examining the total percentage dose of 3H recovered from gut contents and fecesin Table 2, it is seenthat from 25.6-55.1x of the dose wasnot absorbed. TCB was excreted into bile and urine to a greater extent than HCB in animals of both sexes (p < 0.01). Also, there was a distinct preference for biliary rather than urinary excretion of the PCBs. This preference for biliary excretion was more obvious for TCB than for HCB. The data presented in Tables 1 and 2 are summarized in Fig. 2 to illustrate major differences between TCB and HCB. The full height of each bar is for recovery of the dose of radioactivity in all body tissues and excreta analyzed. These results show, depending upon the compound and the sex of the animal, that 93 (mean) to 109% of
614
PETERSON,
SEYMOUR
AND
ALLEN
the doses were recovered. With respect to absorption, TCB tended to be absorbed to a greater extent than HCB. This is shown in Fig. 2 by the percentage dose of radioactivity recovered from gastrointestinal tract contents and feces tending to be lower in rats administered TCB. This tendency for greater absorption of TCB than of HCB was significant only in male rats (p < 0.05). Interestingly the total percentage dose of radioactivity that was recovered from all of the tissues analyzed, was similar for TCB and HCB, varying from 36 % (mean) of the TCB dose in female rats to 47 % of the HCB dose in males. m
URINE
0
BILE
m
OTHER
m
SKELETAL
MUSCLE,
‘3. TRACT
CONTENTS,
TISSUES SKIN,
LIVER.
SMALL
INTESTINE
FECES
60 3
' l-
60
E B P
40 20 n ”
TCB
HCB FEMALES
TCB
HCB MALES
FIG. 2. Major tissues and excreta involved in the distribution and elimination of TCB- and HCBderived radioactivity in rats. The full height of each bar and vertical line next to it represents the percentage dose recovered from the entire animal (mean + SE of three to five rats). The subdivisions within each bar show the percentage dose recovered from different tissues and excreta.
The most obvious difference between TCB and HCB shown in Fig. 2 involves elimination of the dose in bile and urine. Excretion in bile and urine together accounted for approximately 27 and 44% of the TCB dose in female and male rats, respectively. On the other hand, less than 2 % of the HCB dose was excreted in animals of either sex by these routes. The difference in gut absorption between TCB and HCB is not of sufficient magnitude to account for the far greater excretion of TCB- than of HCBderived radioactivity. One result, however, where a difference in gut absorption may have been responsible for a difference in biliary excretion in seen in the data for TCB. Figure 2 shows that female rats excreted a lower percentage of the TCB dose in bile (25.7 +_7.8 %, mean f SE) than males (42.2 f 8.5 %). Figure 3 (top panel) shows the time course for biliary excretion of radioactivity during the 24-hr period after TCB and HCB administration. For female and male rats
BILIARY
EXCRETION
615
OF PCBS
administered TCB the time course for appearance of radioactivity in bile was similar. Biliary excretion was lowest at O-6 hr, highest at 6-12 and 12-18 hr, and then decreased 18-24 hr after administration. In the case of HCB, the appearance of radioactivity in bile followed a different time course, being nonexistent between 0 and 6 hr and then remaining relatively constant for the 6-12, 12-18, and 18-24 hr collections. At all collection periods, except 18-24 hr for female rats, the biliary excretion of radioactivity was significantly greater for TCB than for HCB (p < 0.05). The bottom panel of Fig. 3 shows that bile flow was similar in rats given TCB and HCB. Thus, the difference in biliary excretion between the two compounds was not caused by a difference in bile MALES
FEMALES 20 3H-2,5,2:5’-TCB
,5
;
3H-2,4,5,2:4:5'-HCB
I
IOk
O-6
6-Q
12-18
16-24
HOURS
AFTER
O-6 3wPCB
6-12
12-m
la-24
ADMINISTRATION
FIG. 3. Biliary excretion of TCB- and HCB-derived radioactivity in male and female rats (top panel) and bile flow (bottom panel). Each value is the mean rt SE of three to five rats. The asterisk indicates a significant difference @ < 0.05) between rats of the same sex administered TCB and HCB.
Table 3 provides preliminary information on the disposition of TCB- and HCBderived radioactivity in bile. As was mentioned previously, the radioactivity extracted into hexane represents the parent compound and monohydroxy metabolites of this compound (Van Miller et al., 1975). Radioactivity extracted into diethyl ether is predominantly due to metabolites that are more polar than the monohydroxy metabolites. The most polar metabolites of TCB and HCB were not significantly extracted into hexane or ether and are designated in Table 3 as “aqueous.” Approximately 90 % of the 3H remaining in all aqueous fractions (male-female, HCB-TCB) could be extracted with diethyl ether after a 24-hr incubation at 37°C with sulfatase (containing &glucuronidase) (Sigma Chemical Co., St. Louis, MO.) with 50 Sigma units/ml in 0.2 M sodium acetate buffer, pH 5.0. For rats treated with TCB, greater than 78 % of the radioactivity in bile was present in the aqueous phase, and represented polar metabolites of TCB. Also, when the smaller percentage of radioactivity present in the hexane extract of bile
616
PETERSON,
SEYMOUR
AND
ALLEN
was purified by thin-layer chromatography and analyzed by gas-liquid chromatography no unmetabolized TCB could be found. Thus, for rats administered TCB, all of the radioactivity in bile was due to metabolites. The distribution of radioactivity in hexane and ether extracts of bile and the aqueous phase was different for rats given HCB. For HCB, between 30 and 40 % of the radioactivity in bile was extracted into hexane as compared to between 2 and 17% for TCB. Thus, for HCB a greater percentage of the radioactivity in bile was due to nonpolar material. Gas-liquid chromatography of the hexane extract indicated that the extractable 3H portion is at least 95 % parent compound. TABLE PERCENTAGE
DISTRIBUTION
OF 3H IN HEXANE
3
AND ETHER
EXTRACTS
OF POOLED
BILE
SAMPLES
FROM RATS ADMINISTERED t3H]PCB
Females O-12hr” 12-24 hrb bile bile
Males O-12 hr
12-24 hr
bile
bile .-
13H]-2,5,2’,5’-TCB
Hexaneextract Ether extract Aqueous’
3.2 91.8
2.8 0.8 96.4
17.1 4.5 78.4
8.8 0.9 90.3
30.2 10.3 59.5
40.1 9.4 50.5
40.1 7.8 52.1
40.0 4.5 55.5
5.0
[3H]-2,4,5,2’,4’,5’-HCB
Hexaneextract Ether extract Aqueous
LISamples of O-6 and &12-hr bile from four to five rats were pooled prior to extraction. * Samples of 12-18- and 18-24-hr bile from four to five rats were pooled prior to extraction. c 3H in bile not extracted into hexane or ether. DISCUSSION
Generally speaking, absorption, distribution, and excretion of radioactivity after TCB or HCB administration were similar in female and male animals for each isomer. In both sexes skeletal muscle, skin, liver, and small intestine were the major tissues involved in the localization of radioactivity, and bile was the main route of excretion. Greater biliary excretion of TCB in males than in females was caused by greater absorption of the compound in males(Table 2, Fig. 2) and is not due to a sex difference in hepatic excretory function. Recovery of HCB in the liver was higher in female than in male animals. This difference (23.1 f 6.5 %, mean f SE, femalesversus 5.4 + 0.7 %, males)cannot be accounted for by differencesin absorption or biliary excretion of HCB. Although absorption, distribution, and excretion of each PCB were similar overall in female and male rats, chronic feeding studies of PCBs in other specieshave shown that females develop signs of PCB toxicity at lower levels of PCB intake. This has been
BILIARY
EXCRETION
OF PCBS
617
demonstrated for rhesus monkeys (Barsotti et al., 1976) and mink (Platonow and Karstad, 1973). With respect to PCB absorption from the gastrointestinal tract, our results in restrained rats differ from those of other investigators where rats that were not exposed to anesthesia, surgery, and body restraint were used. Van Miller et al. (1975) and Matthews and Anderson (1975b) recovered as unmetabolized material less than 5% of an oral dose of TCB and HCB from gut contents and feces of rats 24 hr after administration. In the present study, depending on the PCB and sex of the animal, between 25 and 55 % of the dose was not absorbed. The cause of the incomplete absorption was not related to an absence of bile from the gastrointestinal tract because bile was continuously infused into the duodenum. In preliminary experiments where bile was not infused, only 5-10 % of the PCB dose was absorbed in 24 hr as compared to 45-75 “, when bile was infused. Thus, the presence of bile in the intestine was necessary for significant absorption of PCBs to occur, and lack of bile was not the reason for the slower absorption. Also hypothermia cannot be invoked as an explanation because body temperature was maintained near 37°C. The most feasible explanation is that stress associated with anesthesia, surgery, and body restraint in the present study, but not in those of Van Miller et al. (1975) and of Matthews and Anderson (1975a, b), was involved in producing the effect. Among the various tissues analyzed for distribution of TCB and HCB, the greatest percentage of the dose was located in skeletal muscle, skin, liver, and small intestine. The high recovery from skeletal muscle was expected since this tissue constitutes approximately 50 % of the body weight. Also Matthews and Anderson (1975a, b) have shown in male rats that skeletal muscle is a major depot for iv-administered PCBs. The skin and liver which represent a lower percentage of the total body weight, 16 and 5 2;, respectively, were also involved to a major extent in the tissue distribution of radioactivity. Van Miller et al. (1975) and Matthews and Anderson (1975a, b) have also demonstrated a greater involvement of skin and liver in the distribution of TCB and HCB in non-bile duct-cannulated male rats. In the present study the small intestine was also included as a major tissue depot for radioactivity. This was probably caused by ongoing absorption of TCB and HCB through the tissue of the small intestine at the time the animals were sacrificed. This suggestion is supported by data in Table 2 which show that a significant amount of unabsorbed TCB and HCB was present in the small intestinal contents at this time. In distribution studies of PCBs in rats, adipose tissue is usually a major storage depot (Matthews and Anderson, 1975a, b; Van Miller et al., 1975). This was not the case in the present study, because very little adipose tissue was present in the animals (95-105 g) to begin with and virtually none was present at the time of sacrifice. With respect to metabolism and elimination, a major finding of this study was the 25- to 40-fold greater biliary excretion of TCB than of HCB. This difference in excretion cannot be adequately explained by a difference in absorption of the compounds and appears to relate instead to the more rapid rate of TCB than HCB metabolism (Van Miller et al., 1975; Matthews and Anderson, 397513). Ghiasuddin et al. (1976) have also shown in a liver microsomal system prepared from phenobarbital-treated rats that the lower chlorinated isomers are metabolized to a greater extent than the higher chlorinated ones. This difference in rate of metabolism was reflected in the present study by 21
618
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AND
ALLEN
differences in the disposition of these compounds in bile. For rats treated with TCB, 100% of the radioactivity in bile was from metabolites. On the other hand, for rats given HCB, 3@-40% of the radioactivity in bile was extracted into hexane, of which greater than 95 % was from the parent compound. Recently, Mehendale (1976) has obtained biliary excretion results for individual PCBs, using an isolated perfused rat liver preparation, that are in agreement with the greater biliary excretion of TCB (26 % females, 42 % males) than of HCB (1.1% females, 0.5 % males) reported here in intact animals. In his investigation, Mehendale (1976) found that 48,30,20, and 1% of the dose of mono-, di-, penta-, and hexachlorobiphenyl were excreted by the isolated perfused rat liver in 4 hr. Thus, biliary excretion was inversely related to the degree of chlorination. In the present study the chlorines in the 4,4’ positions of HCB (Fig. 1) appear to prevent the rapid biliary excretion of HCB by eliminating adjacent unsubstituted carbons which are necessary for rapid metabolism to occur. Jondorf et al. (1955) first demonstrated that two adjacent unsubstituted carbon atoms, which are present in TCB but not in HCB, were necessary for an appreciable rate of metabolism of the trichlorobenzenes. The possibility that a similar requirement pertains to the rate of metabolism of individual PCBs has been suggested by Schulte and Acker (1974) and Matthews and Anderson (1975b). Interestingly, a preliminary study by Matthews and Tuey (1976) has shown that 3,5,3’,5’-tetrachlorobiphenyl, which is like HCB in the sense that it lacks two adjacent unsubstituted carbon atoms, is excreted in the feces of rats after iv administration at a significantly greater rate than HCB. Thus, the presence of two adjacent unsubstituted carbon atoms in TCB may partially explain the greater biliary excretion of TCB than of HCB in the present study, but it is probably not the only factor involved. ACKNOWLEDGMENTS We wish to thank Miss Patricia Hack, Mr. Steven Schmidt, and Mr. King Yan Lee for their expert technical assistance. REFERENCES
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