Journal of Toxicology and Environmental Health
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Elimination of PBBs in rats. effect of mineral oil and/or feed restriction D. Polin , S. J. Bursian , M. S. Underwood , P. A. Wiggers , N. Biondo , I. Su , W. E. Braselton & J. A. Render To cite this article: D. Polin , S. J. Bursian , M. S. Underwood , P. A. Wiggers , N. Biondo , I. Su , W. E. Braselton & J. A. Render (1991) Elimination of PBBs in rats. effect of mineral oil and/or feed restriction, Journal of Toxicology and Environmental Health, 33:2, 197-212, DOI: 10.1080/15287399109531518 To link to this article: http://dx.doi.org/10.1080/15287399109531518
Published online: 19 Oct 2009.
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Date: 10 November 2015, At: 18:43
ELIMINATION OF PBBs IN RATS. EFFECT OF MINERAL OIL AND/OR FEED RESTRICTION D. Polin, S. J. Bursian, M. S. Underwood, P. A. Wiggers, N. Biondo Department of Animal Science, Michigan State University, East Lansing, Michigan
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I. Su, W. E. Braselton Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan J. A. Render Department of Pathology, Michigan State University, East Lansing, Michigan Rats were fed polybrominated biphenyls (PBBs) at 0.1 to 100.0 ppm for 14 d and then treated to hasten the removal of PBBs with 0, 5, or 10% mineral oil (MO) and/or 0, 15, 30, or 45% feed restriction (FR) for 21 d. PBB body burdens were determined at d 14 and expressed on a log-log basis by Y = 0.91X + 2.179 (r2 = 0.974), where X = log of PBB concentration in diet (ppm) and Y = log of PBB body burden (μg). After 21 d withdrawal, body burdens were expressed by the equation Y = 0.787X + 2.218 (r2 = 0.95). The most effective withdrawal treatment was 10% MO + 45% FR producing a reduction of body burdens inversely related to prior body burdens (69% at 0.1 ppm to 23% at 100 ppm). Body weights and fat content were significantly (p .05) reduced by feed restriction, with fat content only 39% of controls at 21 d off. Mortality averaged 0, 13.6, and 35.8% for rats fed 0, 5, or 10% MO, and 25, 15, 8.6, and 3.7% for rats feed restricted at 0, 15, 30, and 45%, respectively. Histopathology of the dead and moribund rats indicated that the clinical signs were not characteristic of PBB toxicity. In a second experiment, safflower oil at 3.5% or excess vitamins prevented the mortality and clinical signs associated with MO during withdrawal from 100 ppm PBBs. Based on these data and those in the literature, PBBs interfere with vitamin utilization.
INTRODUCTION The long-term retention of certain xenobiotics results in continuous exposure of target organs even after cessation of external exposure. This possibility poses a long-term risk to humans and/or food animals harboring the xenobiotics. As a result, there has been considerable attention directed toward reducing the body burdens of xenobiotics. Such studies used treatments involving charcoal, phénobarbital, lipotropic agents and Support came from the Michigan Department of Public Health and the Michigan Agricultural Experiment Station. Requests for reprints should be sent to Donald Polin, Department of Animal Science, Room 132 Anthony Hall, Michigan State University, East Lansing, Ml 48824. 197 Journal of Toxicology and Environmental Health, 33:197-212, 1991 Copyright © 1991 by Hemisphere Publishing Corporation
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bile acid binding resins, but all with uncertain and conflicting results (Wilson et al., 1968; Donaldson et al., 1971; Cregar and Kubena, 1971; Egebertson and Davison, 1971; Coon and Couch, 1973; Sell et al., 1977; Wyss et al., 1982; Polin and Leavitt, 1984). Polin and Leavitt (1984) revealed the need to determine the role of varying the dosages of these compounds and the length of treatment to determine if these treatments could be effective. In chickens almost 70% of the body burdens of polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs), and hexachlorobenzene (HCB), and 100% of the pentachlorophenol (PCP) body burdens were eliminated in 21 d (Polin et al., 1985; Polin et al., 1986). The combination of 45% feed restriction (FR) and 10% mineral oil (MO) in the diet appeared to be the best combination of several investigated (Polin et al., 1989). The purpose of this study was to determine if in the rat the MO + FR treatment would be a viable approach when used over a wide range of dietary PBBs. The effects of other dietary concentrations of MO and lesser amounts of FR were also studied. PROCEDURES AND METHODS Male Sprague-Dawley rats, 6 wk of age, were assigned to an environmentally controlled room (21°C). Rats were weighed weekly to the nearest gram. Thirty-nine rats were fed control diet, and 78 rats were fed either 0.1,1.0,10.0, or 100.0 mg PBBs/kg diet (as FireMaster; see Polin and Leavitt, 1984, for details on product). The rats were housed in groups of three per cage. The cages were plastic bases with stainless steel metal wire tops. There were no wire floors to keep the rats off the bedding (coprophagy could be practiced by the rats). The diets containing the PBBs were fed for 14 d. Diets were withdrawn from 3 control rats, and from 6 rats fed each of the levels of PBBs on the evening before d 14 so that the digestive contents would be at a minimum and not contribute PBBs to the body burden measurements. The rats were euthanized on d 14 with excess CO2. Their carcasses were frozen, and stored in a freezer at -20°C until processed for fat and PBBs determinations. On d 14 of exposure PBB feeding was discontinued. Each of the 5 groups exposed to the different dietary levels of PBBs was then treated with 0, 5, or 10% MO (from MSU Vet Clinic), added to the diet on a w/w basis; 0,15, 30, or 45% feed restriction of ad libitum controls; or a combination of MO + FR. A 5 x 3 x 4 factorial design was followed. The treatments were continued for 21 d, at which time the rats were killed and frozen. Three rats were started in each group of the 3 x 4 factorial representing those fed control diets for the 14-d contamination period. There were 6 rats per group in each cell of the 3 x 4 factorial arrangement representing those fed the PBB diets of 0.1 to 100 ppm, as indicated by the outline within Tables 1 and 2.
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The diet was Purina Chow 5002, ordered ground so that the PBBs could be distributed uniformly in it. The PBBs were introduced into the diet via 4-kg premixes that were then blended in a small drum-type commercial feed mixer (Mix-Mill, Inc., Bluffton, III.) to 30 kg. The premixes were made according to standard dry mixing procedures by which small quantities of chemicals, such as vitamins or purified amino acids, are blended into already mixed diets. Individual feed containers were heavyduty white glass wide-mouth jars. Any spillage was collected twice each week by sifting the bedding. The spilled feed was weighed and the feed intake determined once each week. Water and feed were allowed ad libitum during the exposure period, but only water was allowed ad libitum during the withdrawal period. PBBs were determined by a modification of the procedure of Price et al. (1986). Frozen rats were ground in a Hobart grinder. The ground samples were refrozen until analyzed for PBBs. Aliquots (50 g) were taken and frozen in liquid nitrogen in a Waring blender, the excess liquid nitrogen poured off, and the frozen tissue blended to a fine powder. PBBs were analyzed in 20-g aliquots of the powdered tissue weighed into a beaker and mixed thoroughly with 100 g of granular sodium sulfate until dry. A 25 mm ID x 300 mm chromatography column was packed with approximately 2 g Na2SO4, and the dry Na2SO4 tissue homogenate mixture was packed lightly into the column. The sample container and the column were rinsed with 10 ml of extracting solvent, diethyl ether/petroleum ether (1 :1), and an additional 190 ml of the extracting solvent was added to the top of the column. The eluate was collected into a preweighed beaker at 5-6 ml/min. The solvent was evaporated under N2 with gentle heating, and the lipid content of the tissue was determined by differential weight. A 0.25-g aliquot of fat was dissolved in 1.0 ml hexane and applied to the top of a 9 x 200 mm glass column packed with 5 g silica gel 60. The sample was eluted onto the column with 15 ml hexane, and the effluent was discarded. The PBBs were eluted with 45 ml hexane and concentrated by evaporation in Snyder tubes. The PBBs were quantified by chromatography on a 2 mm ID x 180 cm column of 3% OV-1 on Gas Chrom Q at 240°C and detected by electron capture with a 63 Ni detector. Peak areas were integrated automatically by a Spectra Physics 4100 integrator. A total of nine peaks was calculated, each dependent on its own standard curve. Total PBBs were determined from the sum of the congeners. Standard curves were run for each day that samples were processed, and the procedure was monitored for each run by including a sample from a large known meat source contaminated with PBBs. Body burdens were derived from the concentrations of PBBs in the fat, the concentration of fat in the carcass, and the body weight of the rat. Values are reported on a wet weight body basis and were determined on individual rats unless specified otherwise. Body burdens, and not concentration of PBBs, were emphasized to adjust for changes in body weight, in
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most situations substantial, that occurred during the experiment. Final concentrations of PBBs were also reported. (When lipid and protein are removed from the body through restricted feeding and PBBs are also removed, then upon refeeding one would expect the PBB concentrations to be reduced as they are diluted with newly formed lipid and protein clear of PBBs.) Values were adjusted for percent recovery derived from analyzing spiked samples. Statistical analyses were conducted using a Macintosh Plus computer (Apple Computer Co.) with the program StatView512+ (Brain Power Inc., Calabasa, Calif.). Statistical significance was accepted at p < .05. In a subsequent experiment using the same procedures as above, rats were fed a diet with 100 ppm PBBs for 14 d followed by treatment with 0 or 10% MO, without or with the addition of vitamins, or with 3.5% safflower oil (SO). The vitamin mix (details in text) was prepared in our laboratory, and the SO was purchased at a local food store. Three different sources of MO were evaluated (details in text). At the end of the withdrawal period, remaining animals were weighed and killed with excess CO2 and examined for clinical signs. RESULTS Experiment 1: Controls
Rats fed no PBBs had detectable amounts of PBBs in their bodies averaging 4.1 and 17.5 /¿g/rat, on a wet weight basis, at d 14 and 35, respectively, of the experiment (Table 1). These values correspond to 16 and 58 /¿g/kg body weight (parts per billion, ppb). Day 35 was d 21 of withdrawal for those rats that were fed diets with PBBs. The control rats had accumulated trace amounts of PBBs from dust particles in the room, as dust on the room air filters had PBBs. Control diets were not mixed in the equipment used to prepare the PBB diets, and were stored, as were TABLE 1. Average Body Burdens of PBBs in Rats Fed for 14 d Diets Containing PBBs from 0 to 100 ppm, and in Rats 21 d after the Withdrawal of These Diets PBBs
Day of treatment regimen, PBBs (fig/rat)
in aiei
(ppm)
Day 14 on
Day 35 off
Day 35/day 14
None 0.1 1.0 10.0 100.0
4.1 (3)a 26.7 (6) 112.1 (6) (6) 1086.8 (6) 12426.0
17.56 (2) 44.4 (6) (6) 102.4 (6) 865.1 (6) 8321
427 166 91 80 67
a b
Number of rats. Omitted outlyer value of 128.7 ^ig.
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201
all diets, in closed containers, each with its own scoop for feeding. One rat from the control group had a body burden of 128.7 /¿g, an amount in the range for rats fed 1.0 ppm PBBs. That value was excluded from the statistical analysis as an outlyer. Analysis of PBBs in rats at d 35 (d 21 of withdrawal) revealed (Table 2) that those treated with MO and/or FR regimen had significantly (p < .01) decreased body burdens characterized by a statistically significant interaction (p < .01). The latter statistics indicated that the combination of the treatments was better than either one alone in reducing the body burdens of PBBs in the contaminated control rats. With increasing FR, or increasing MO, the body burdens of PBBs were reduced. Control rats treated with 10% MO + 45% FR had the lowest body burdens, 0.8 /¿g/rat, and the greatest percentage decline (Table 2). Experiment 1: Rats Contaminated with PBBs
Body Burdens Feeding PBBs at 0.1 to 100.0 ppm to rats for 14 d resulted in body burdens ranging from 26.7 to 12426 /ug/rat (Table 1). The dose-response relationship was linear on a log-log basis yielding the equation Y = 0.91X + 2.179 (? = 0.974), where X is the log of PBB concentration in diet (ppm), and V is the log of PBB body burden (/¿g). Generally, the body burdens in rats from the 1,10, and 100 ppm dietary levels of PBBs differed by a factor of 10. Diets with 0.1 ppm PBBs produced body burdens 25% less than the value from the 1.0 ppm diet. At d 21 of withdrawal, those rats not treated with MO and/or FR had body burdens that ranged from 17.5 to 8321 ^g (Table 1), which on a log-log linear relationship was represented by the equation Y = 0.787X + 2.218 (r2 = .95). Nontreated rats previously fed control diet and those fed 0.1 ppm PBB diet had burdens of PBBs at d 21 off that were greater than those when PBB diets were removed. Their low values were influenced by the dust in the room. Another factor involved in these rats with very low burdens was the wide variation in body burden values having a pooled standard deviation of ±10 (converted from a log basis). Rats fed diets with 1-100 ppm PBBs had body burdens that were less on d 21 of withdrawal than those on d 14 of feeding the contaminated diets (Table 2). Thus, these rats were able to remove some of their body burdens without external treatment. PBBs could not be detected in rats kept in a separate room and fed control diet. Withdrawal treatments of MO and FR produced some loss of PBB body burdens depending on the treatment and on the level of contamination. 1. Rats that had been fed 0.1 ppm and then placed on the withdrawal regimen had significant reductions in their PBB burdens by both MO and FR treatments (Table 2). The lowest average body burden resulted from treating with 10% MO + 30% FR, a reduction of 69% when the
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TABLE 2. Body Burdens of Rats on d 21 of Withdrawal Following the Feeding of Diets with 0, 0.1, 1.0,10.0, or 100.0 ppm PBBs Mineral oil
Feed restriction (%) 30
15
45
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Control diet (no prior PBBs) d!
0 5 10
(2)
17.5'
(3) (1)
9.0 4.2
0 5 10
(6) (6) (6)
44.4 26.1 23.0
(3) (3) (3)
15.0 2.1 2.1
(3) (3) (3)
0.5 1.1 1.3
(3) (3) (3)
2.3 1.3 0.8
29.0 29.0 13.6
(6) (6) (6)
24.2 16.2 17.1
83.8 83.8 71.4
(6)
98.6
(6) (6)
75.4 59.0
Withdrawal from 0.1 p p m PBBs (6) (6) (5)
25.7 20.9 20.9
(6) (6) (6)
Withdrawal from 1.0 p p m PBBs 0
(6)
93.0
(6)
(5)
102.4 65.9
(6)
5
(6)
(6)
10
(5)
96.1
(6)
117.4 71.7
(6)
Withdrawal from 10.0i p p m PBBs 0 5 10
(6) (6) (2)
865 777 782
0 5 10
(6) 8321 (1) 9425 (1) 6669
(6) (6) (4)
849 845 767
772 682 690
(6) (6) (5)
(6) (6) (6)
716 658 627
Withdrawal from 100.0 ppm PBBs (6) 10430 (3) 7998 — (0)
(6)
(6) 8199 (4) 8007 (5) 7104
10059 9846 7248
(4) (2)
Summary of body Iburdens vs. treatment Change in
Former treatment (ppm)
Best withdrawal treatment
(%)
From
To
.05) reduce body weights (317 vs. 322 g), whereas, 30 and 45% FR reduced body weights progressively to 290 and 251 g, respectively (Table 3). Rats not fed PBBs and then continued on diets with no MO or FR weighed at the end of the withdrawal period 297 g. Prior PBB feeding did not have an effect on body weights during the withdrawal period, as the rats weighed from 309 to 331 g. There was no significant interaction (p > .05) to indicate that as a result of the prior PBB treatment MO or FR produced a greater effect than no withdrawal treatment. Body Fat Fat in control rats averaged 7.04%. The average body fat in rats not subject to feed restriction was 6.69% (Table 4), whereas the fat in rats not given MO was 5.50% (Table 4, footnote a), the latter value reflecting the averaging in of the FR effect. Five and 10% MO caused body fat percentage to be significantly (p < .05) reduced to 4.86 and 4.36%, respectively, values that were 88 and 79% of 0% MO. Body fat was progressively and significantly reduced by 15, 30, and 45% FR, with the latter
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D. POLIN ET AL.
TABLE 3. Body Weights of Rats on d 21 of Withdrawal from Diets with 0, 0.1, 1.0, 10.0, and 100.0 ppm PBBs Feed restriction (%) KA \ nar^ \ /Vlli itrl dl
oil (%)a
0
15
30
45
Withdrawal from control diet (no prior PBBs)
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0 5 10
(3) 297 (3) 307 (1) 319
(3) 324 (3) 321 (3) 313
(3) 298 (3) 297 (3) 286
(3) 252 (3) 260 (3) 250
(6) 288 (6) 295 (6) 285
(6) 258 (6) 257 (6) 240
(6) 291
(6) 275
(6) (6)
288 280
(6) (6)
2S5
(6) (6) (5)
309 291 292
(6) (6) (6)
263 248 239
301 272 260
(6) (4)
Withdrawal from 0.1 ppm PBBs 0 5 10
(6) 331 (6) 328 (6) 322
(6) 324 (6) 321 (5) 322
Withdrawal from 1.0 ppm PBBs 0
(6) 309
5
(5)
10
336 (5) 346
0 5 10
(6) 329 (6) 320 (2) 291
(6) 318 (6) 331 (6) 280
239
Withdrawal from 10.0 ppm PBBs (6) 324 (6) 324 (4) 324
Withdrawal from 100.0 ppmi PBBs 0 5 10 Mean weight (g)b
(6) 326 (1) 347 (1) 264
(6) 322 (3) 291 (0) -
(63) 322a
(69) 317a
(6) (4) (2) (74)
290b
253 250 (5) 222 (78) 251c
Noie. Some were treated with mineral oil at 5 or 10% and/or restricted in feed intake to 15, 30, or 45% of control ad libitum fed rats. Values given are number of rats (in parentheses) and body weight (g). a Overall effect of mineral oil: 0% - (108) 300; 5% = (96) 296; 10% = (81) 280. Significant effect (p < .05) of mineral oil in rats withdrawn from 10 and 100 ppm PBBs. b Mean values with different letters are significantly different at p < .05.
treatment producing an average body fat of 2.60% (Table 4), a value only 39% of the value for 0% FR. PBBs Concentration Rats not fed PBBs or not treated with MO or FR during the withdrawal period had contamination concentrations of PBBs of 0.186 ppm (Table 5). Treating these rats with 15, 30, or 45% FR reduced the mean PBB concentrations markedly (Table 5). The same overall pattern was detected for the rats that had been fed 0.1 ppm PBBs and treated with MO and/or FR. Thus, the FR produced a significant decline
ELIMINATION OF PBBs IN RATS
205
at these very low PBB concentrations. In the groups withdrawn from 1.0 and 10.0 ppm PBBs, FR did not reduce PBB concentrations (Table 5). In the group withdrawn from 100 ppm PBBs, the PBB concentrations were significantly increased by an average of 25%, as a result of the FR, but concentrations were not changed by MO treatment (Table 5). Mortality During the withdrawal period from PBBs, mortality averaged 0, 13.6, and 35.8% for the rats fed 0, 5, and 10% MO, respectively (Table 6, footnote a). It averaged 25,15, 8.6, and 3.7% for rats undergoing
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TABLE 4. Percentage of Fat in Rats on d 21 of Withdrawal from Diets with 0, 0.1,1.0,10.0, and 100.0 ppm PBBs Feed restriction (%) Mineral oil (%)a
0
15
30
45
Withdrawal from control diet (no prior PBBs) 0 5 10
(3) (3) (1)
7.04 6.20 7.82
(3) (3) (3)
7.15 6.11 5.66
5.42 3.74 3.30
(3) (3) (3)
2.61 2.71 2.28
5.28 5.01 4.24
(6) (6) (6)
2.52 2.26 2.34
(6) 5.45 (6) 2.76 (6) 4.26
(6) (6) (6)
2.56 2.48 1.80
4.22 3.93 4.81
(6) (6) (6)
2.79 3.31 2.07
(6) (4) (2)
5.28 5.25 5.06
(6) (4) (5)
2.72 3.73 3.17
(74)
4.53c
(78)
(3) (3) (3)
Withdrawal from 0.1 ppm PBBs 0
5 10
(6) 6.71 (6) 5.60 (6) 6.02
(6) 6.24 (6) 6.25 (5) 6.11
(6) (6) (6)
Withdrawal from 1.0 ppm PBBs 0
5 10
(6) (5) (5)
7.13 6.47 6.42
(6) (6) (6)
6.84 7.10 6.03
Withdrawal from 10.0i ppm PBBs 0 5 10
(6) (6) (2)
7.26 6.46 7.76
(6) (1) (1)
7.40 7.34 7.27
(63)
6.69a
(6) 7.99 (6) 6.35 (4) 4.30
(6) (6) (5)
Withdrawal from 100.0 ppm PBBs 0 5 10 Mean fat (%)b
(6) (3) (0) (69)
7.59 6.92 6.55b
2.60d
Note. Some were treated with mineral oil at 5 or 10% and/or restricted in feed intake to 15, 30, or 45% of control ad libitum fed rats. Values given are numbers of rats (in parentheses) and carcass fat (%). a Overall effect of mineral oil: 0% = (108) 5.50afa; 5% - (94) 4.86b; 10% - (87) 4.36c. Significant effect (p S .05) of mineral oil in rats withdrawn from 10 and 100 ppm PBBs. È Mean values with different letters are significantly different at p < .05.
D. POLIN ET AL.
206
TABLE 5. PBB Concentrations in Rats on d 21 of Withdrawal from Diets with 0, 0.1, 1.0, 10.0, and 100.0 ppm PBBs Feed restrictions (%) Mineral oil (%)a
15
0
45
30
Withdrawal from control diet (no prior PBBs) 0 5 10
(3) (3) (1)
0.186 0.029 0.013
(6) (6) (6)
0.134 0.079 0.072
(3) (3) (3)
0.045 0.006 0.0065
(3) (3) (3)
0.0018 0.0037 0.0044
(3) (3) (3)
0.0090 0.0051 0.0033
0.061 0.099 0.048
(6) (6) (6)
0.082 0.063 0.071
0.288 0.291 0.256
(6) (6) (6)
0.381 0.293 0.247
2.50 2.36 2.37
(6) (6) (6)
2.73 2.66 2.63
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Withdrawal from 0.1 ppm PBBs 0 5 10
(6) (6) (5)
0.081 0.065 0.060
(6) (6) (6)
Withdrawal from 1.0 ppm PBBs 0 5 10
(6) (5) (5)
0.329 0.197 0.278
(6) (6) (6)
0.296 0.352 0.254
(6) (6) (6)
Withdrawal from 10.0 ppm PBBs 2.64 2.45 2.76
0 5 10
(6) (6) (2)
0 5 10
(6) 25.5 (1) 27.2 (1) 25.3
(6) (6) (4)
2.64 2.62 2.35
(6) (6) (5)
Withdrawal ( rom 100.0 ppm PBBs
Mean (/ig/g)b
(53)
3.94a
(6) 32.5 (3) 27.5 (0) (69)
4.71a
(6) 33.4 (4) 36.7 (2) 28.3 (74)
6.01b
(6) 32.6 (4) 31.9 (5) 32.1 (78)
6.91b
Note. Some were treated with mineral oil at 5 or 10% and/or restricted in feed intake to 15, 30, or 45% of control ad libitum fed rats. Values given are number of rats (in parentheses) and PBBs a Overall effect of mineral oil: 0% - 7.57a6; 5% - .(95) 4.77b; 10% - (81) 3.60c. Significant effect (p < .05) of mineral oil in rats withdrawn from 10 and 100 ppm PBBs. ö Mean values with different letters are significantly different at p < .05.
FR at 0, 15, 30, and 45%, respectively (Table 6). Thus, increasing MO increased mortality, while increasing FR reduced overall mortality. Mortality increased markedly for rats previously fed 10 and 100 ppm PBBs: 5.5 (2/36), 4.1 (3/72), 2.8 (2/72), 9.7 (7/72), and 38.9% (28/72), for prior PBB contamination of 0, 0.1, 1.0, 10, and 100 ppm, respectively (Table 6). Clinical signs in the affected rats were paralyzed rear legs, bloody urine, wet abdominal skin, bloody discharge from the nostrils, rhinorrhagia, exophthalmos, and subcutaneous edema and lethargy.
ELIMINATION OF PBBs IN RATS
207
Experiment 2
Average starting weight for all rats was 360 g. During the 14-d contamination period, the 105 rats fed PBBs gained as much as the 12 control rats, with body weights averaging 389 g. Treating the rats during the withdrawal period with MO, types A, B, and C (Table 7), reduced the weights of the rats to 350 g. Rats treated with MO + FR averaged 315 g TABLE 6. Number of Rats That Died during the 21 d of Withdrawal from Diets with 0, 0.1,1.0,10.0, and 100.0 ppm PBBs
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Feed restriction (%) Mineral oil (%)a
0
15
30
45
Withdrawal from control diet (no prior PBBs) 0 5 10
(3) (3) (3)
0 0 2
(3) 0 (3) 0 (3) 0
(3) 0 (3) 0 (3) 0
(6) 0 (6) 0 (6) 0
(6) 0 (6) 0 (6) 0
(6) 0 (6) 0 (6) 0
(6) 0 (6) 0 (6) 0
(6) 0 (6) 0 (6) 1
(6) 0 (6) 0 (6) 0
0 3 6
(6) 0 (6) 2 (6) 4
(6) 0 (6) 2 (6) 1
(81) 12 15
(81) 7 8.(
(81) 3 3.7
(3) (3) (3)
0 0 0
Withdrawal from 0.1 ppm PBBs 0 5 10
(6) (6) (6)
0 0 2
(6) (6) (6)
0 0 1
Withdrawal from 1.0 ppm PBBs 0 5 10
(6) (6) (6)
0 5 10
(6) (6) (6)
0 1 1
(6) (6) (6)
0 0 0
Withdrawal from 10.0 ppm PBBs 0 0 4
(6) (6) (6)
0 0 2
Withdrawal f rom 100.0 ppm PBBs 0 5 10 (Number)6 dead % Dead
(6) (6) (6)
0 5 5
(81) 20 2Í
(6) (6) (6)
Note. Some were treated with mineral oil at 5 or 10% and/or restricted in feed intake to 15, 30, or 45% of control ad libitum fed rats. Values are number of rats at the start (in parentheses) and mortality (number of rats). a Overall effect of mineral oil: 0% MO - (108) 0.0% dead; 5% MO = (95) 13.6% dead; 10% MO = (81) 35.8% dead. Significant effect (p < .05) of mineral oil in rats withdrawn from 10 and 100 ppm PBBs. 6 Rats at the start.
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TABLE 7. Summary of the Histopathologic Lesions Detected in Rats Treated with Mineral Oil (MO), 30% Feed Restriction (FR), Vitamins (VIT), and/or Safflower Oil (SO) during Withdrawal from Diets with 100 ppm Polybrominated Biphenyls (PBBs) Histopathologic lesions detected* 6
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Lesions No histologie lesions Focal alveolar hemorrhage Mild midzonal hepatocellular fatty change Prominent splenic histocytic population Mild hemosiderosis Eosinophilic proteinaceous fluid in renal tubules Diffuse centrilobular hepatocellular Necrosis Vacuolar change Thinning of centrilobular hepatic cords with necrosis Hemorrhage and edema in connective tissue of Epididymis Urinary bladder Esophagus Esophageal necrosis, exudation of fibrin, lymphocytic infiltration, and fibrosis Distention of renal tubules with eosinophilic fluid Hemorrhage and congestion of lamina propria of urinary bladder Hemorrhage within muscular layer of urinary bladder Interstitial testicular hemorrhage Interstitial epididymal hemorrhage Epididymal edema and neutrophilic infiltration a
Control (5)
MO C Hv FRd + VITe (6)
MO + FR + SO' (6)
MO (5)
5 0 0 0 0
1 0 2 0 0
2 1 3 0 0
0 0 0 3 1
0
0
0
1
0 0
0 0
0 0
1 1
0
0
0
1
0 0 0
0 0 0
0 0 0
1 1 1
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0 0
0 0 0 0
0 0 0 0
0 1 1 0
Many of the hemorrhages were within the same rats. ^Clinical signs include paralysis of the hindlegs, bloody urine, wet abdominal area, rhinorrhagia, exophthalmos, subcutaneous edema, and lethargy. c Type A - Fisher Scientific (Livonia, Mich.), a paraffin light oil catalog number 0121; Type B USDA Laboratory (Mt. Hope Road, E. Lansing, Mich.); Type C - Amoco Oil Co (from a distributor in Lansing, Mich.); each added at 10% (w/w). d Feed restriction at 30% of ad libitum controls. e Vitamin mix to supply per kg diet: A, 35,883 USP units; D3, 1800 ICU; E, 4.5 III; menadione sodium bisulfate complex (K activity), 12.6 mg; B12 supplement, 0.135 mg; riboflavin, 18 mg; dcalcium pentothenate, 6.3 mg; niacin, 126 mg; thiamine mononitrate, 2.6 mg; pyridoxine HCI, 12.5 me; folie acid, 3.6 mg; ascorbic acid, 15 mg; d-biotin, 0.63 mg. ^Safflower oil added at 3.5%.
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with no difference among the MO types A, B, and C. Statistical analysis revealed that weight loss was primarily attributed to restricting feed, and a borderline effect from feeding MO. Rats alive at the end of the withdrawal period had hemorrhages in various organs if they were fed MO without FR [81% (13/16)], or MO + FR [76% (16/21)]. In the groups fed only "Type A MO + FR" but also vitamins or SO, mortality was 0% and the incidence of hemorrhages was 11% (2/19). The hemorrhages in the rats fed MO alone or with FR were mostly in the kidney (28.6%), urinary bladder (21.4%), testis (14.3%), epididymis (14.3%), and brain (12.5%), with the remainder detected in heart, pancreas, lung, and thymus. Of the 56 rats examined from the PBB treatments, 6 (10.7%) had pale livers while none had pale kidneys. Table 7 contains a summary of the histopathologic lesions detected in the rats treated with MO, MO + FR, or MO + FR + (vitamins or SO). No histopathologic lesions were detected in the controls. Two of the six rats from MO + FR + vitamins and three of six from MO + FR + SO had mild midzonal hepatocellular fatty alterations. This change was detected only in one rat from MO + FR and in no rats from MO alone. On the other hand, treatment with MO or MO + FR caused many histopathologic changes that were not detected in rats given vitamins or SO. DISCUSSION The efficacy of the MO + FR treatment depended on the extent of the PBB contamination, as well as the particular strength of the treatment. Rats having low burdens eliminated PBBs to the extent observed with chickens (Polin et al., 1989). MO at 10% of the diet with 30 or 45% FR, the latter causing a marked reduction in body fat, were the effective combinations, possibly indicating that utilization of body fat is necessary for removal of the xenobiotic. That either MO or FR alone was rather ineffective in removing PBBs from highly contaminated rats, but was effective to some extent for rats with very low contamination of PBBs, suggested that PBB storage and metabolism may differ depending upon the level of contamination, or possibly related to the adverse clinical effects and mortality caused by much greater contamination. Other dietary approaches, such as limiting caloric consumption only, may serve the same purpose as FR to enhance fat mobilization with less severe nutritional consequences. Kimbrough et al. (1980) were unable to reduce adipose and blood concentrations of PBBs in rats given a single large dose of PBBs followed 3 weeks later by MO for 3 mo or fed diets with 4 and 20% fiber. Our rats contaminated with large amounts of PBBs and subsequently treated with FR also had increased PBB concentrations, though body burdens were reduced as much as 30% as a reflection of weight loss. Because body burdens were not presented by Kimbrough et al. (1980), the effect of the
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fiber and MO on excretion was not known. Based on data from other animals treated with MO alone, the ability to reduce halogenated compounds was minimally to partially effective (Richter et al., 1977; Richter and Schafer, 1981; Rozman et al., 1982a, 1982b, 1983) and seems to require utilization of stored lipid, as presented in this report and by Polin et al. (1985,1986,1989). Body fat showed a trend to decline in rats treated with MO, and this may account for researchers occasionally detecting some enhanced withdrawal of body burdens when MO was fed. The experimental conditions may account for the difference that highly contaminated chickens lost up to 80% PBBs (Polin and Leavitt, 1984; Polin et al., 1985, 1986) while rats similarly contaminated lost far less. Rats were dying, particularly when fed MO at 10% of the diet or 5 or 10% of the diet if previously treated with 10 or 100 ppm PBBs, or showing pathological signs, which did not occur with chickens. Those clinical signs suggested a vitamin deficiency, a conclusion strengthened by reversal of mortality and clinical signs by the addition of vitamins or SO to reverse the effect of MO + FR to rats previously fed 100 ppm PBBs. Why did not the chickens in previous experiments show vitamin deficiencies when they were fed up to 100 ppm PBBs? The reason may be that the chickens were fed diets with vitamin concentrations three- to fourfold above NRC requirements (National Research Council, 1984), whereas the commercial diet used for the rats was most likely formulated close to NRC requirements. Darjono et al. (1983) overcame in rats fed 100 ppm PBBs an induced vitamin A deficiency by adding excess vitamin A to a diet with adequate vitamin A. Thus, high intakes of PBBs appear to be interfering with vitamin activity, including vitamin A, as indicated, and presumably other vitamins, as suggested by the clinical signs of hindlimb paralysis, bloody urine, rhinorrhagia, and exophthalamos reported by us. Other than loss of body weight, these signs were not characteristic of PBB toxicity (Render et al., 1982). Also, these are not typical signs of vitamin A deficiency, which include epithelial hyperplasia, hyperkeratosis, and squamous metaplasia (Darjono et al., 1983), and were not observed in the present study. Interference with nutrients in high-fiber diets was speculated as the probable cause of enhanced liver pathology by PBBs (Kimbrough et al., 1980). Their observation added to those above would be additional evidence to consider PBBs interference with vitamin utilization. The question that arises is whether the MO + FR technique is a viable approach to remove xenobiotics if the animal is subjected to possible vitamin deficiency from use of MO. Evidently, the MO + FR treatment is viable when the diet contains adequate vitamins. The property of MO that enhances the removal of xenobiotics is supposedly through its interference with bile absorption, thus enhancing the nonbiliary intestinal secretion of xenobiotics (Yoshimura and Yamamoto, 1975; Richter et al., 1977; Rozman et al., 1982a, 1982b). Interference with bile absorption
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also reduces uptake of lipid- and fat-soluble vitamins and would explain the higher mortality and morbidity detected as the level of MO increased, and the lower mortality as the FR increased reducing MO intake. Having excess vitamins in the diet when MO was used did not interfere with xenobiotic removal in chickens (Polin et al., 1985, 1989) or goats (Polin et al., 1987), and, in contrast, may enhance its efficacy because the animals were healthy. Rats with low PBB burdens lost a considerable percentage of PBBs, but there were no data to indicate if the reason was the low level of contamination or their state of health. Rats practice coprophagy and this may also have some effect on MO + FR to reduce body burdens. Amounts of PBBs removed at the highest level of contamination far exceeded the body burdens of lower levels of contamination. Thus, the potential exists for removal of all of the xenobiotics from less contaminated animals. REFERENCES Coon, C. W., and Couch, J. R. 1973. The effects of lipotropic agents in depletion of chlorinated biphenyls in laying hens. Poult. Sci. 52:2014 (abstr.). Cregar, C. R., and Kubena, L. F. 1971. The effect of lipotropic agents on depletion of chlorinated hydrocarbons from tissues of poultry. Poult. Sei. 50:1567-1568. Darjono, Sleight, S. D., Stowe, H. D., and Aust, S. D. 1983. Vitamin A status, polybrominated biphenyl (PBB) toxicosis, and common bile duct hyperplasia in rats. Toxicol. Appl. Pharmacol. 71:184-193. Donaldson, W. F., Sheets, T. S., and Jackson, M. D. 1971. Influence of iodinated casein on DDT residues in chicks. Poult. Sci. 47:237-243. Egebertson, K. A., and Davison, K. L. 1971. Dieldrin accumulation and excretion by rats fed phenobarbital and charcoal. Fed. Proc. 30:562 (abstr.). Kimbrough, R. D., Korver, M. P., Burse, V. W., and Grace, D. F. 1980. The effect of different diets and mineral oil on liver pathology and polybrominated biphenyl concentration in tissues. Toxicol. Appl. Pharmacol. 52:442-453. National Research Council. 1978. Nutrient Requirements of Laboratory Animals, 3rd ed., National Academy of Sciences, Washington, D.C. Polin, D., and Leavitt, R. 1984. Colestipol and energy restriction as an approach to hasten removal of PBBs from chickens. J. Toxicol. Environ. Health 13:659-671. Polin, D., Lehning, E., Pullen, D., Bursian, S., and Leavitt, R. 1985. Procedures to enhance withdrawal of xenobiotics from chickens. J. Toxicol. Environ. Health 16:243-254. Polin, D., Olsen, B., Bursian, S., and Lehning, E. 1986. Enhanced withdrawal from chickens of hexachlorobenzene (HCB) and pentachlorophenol (PCP) by colestipol, mineral oil, and/or restricted feeding. J. Toxicol. Environ. Health 19:359-368. Polin, D., Underwood, M., Lehning, E., Bursian, S., and Wiggers, P. 1987. PCBs in goats. Hastening withdrawal using mineral oil. Toxicologist 7:272. Polin, D., Underwood, M., Lehning, E., Olsen, B., and Bursian, S. 1989. Enhanced withdrawal of polychlorinated biphenyls: A comparison of colestipol, mineral oil, propylene glycol, and petroleum jelly with or without restricted feeding. Poult. Sci. 68:885-890. Price, H. A., Welch, R. L., Scheel, R. H., and Warren, L. A. 1986. Modified multiresidue method for chlordane, toxaphene, and polychlorinated biphenyls in fish. Bull. Environ. Contam. Toxicol. 37:1-9. Render, J. A., Aust, S. D., and Sleight, S. D. 1982. Acute pathologic effects of 3,3',4,4',5,5'hexabromobiphenyl in rats: Comparison of its effects with Firemaster BP-6 and 2,2',4,4',5,5'hexabromobiphenyl. Toxicol. Appl. Pharmacol. 62:428-444.
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Richter, E., Lay, J. P., Klein, W., and Korte, F. 1977. Enhanced elimination of hexachlorobenzene in rats by light liquid paraffin. Chemosphere 6:357-369. Richter, E., and Schafer, S. G. 1981. Intestinal excretion of hexachlorobenzene in rats. Arch. Toxicol. 47:233-239. Rozman, K. K., Rozman, T. A., Williams, J., and Greim, H. A. 1982a. Effect of mineral oil and/or cholestryamine in the diet on biliary and intestinal elimination of 2,4,5,2',4',5'hexabromobiphenyl in the rhesus monkey. J. Toxicol. Environ. Health 9:611-618. Rozman, K., Rozman, T., Greim, H., Neiman, I. J., and Smith, G. S. 1982b. Use of aliphatic hydrocarbons in feed to decrease body burdens of lipophilic toxicants in livestock. J. Agric. Food Chem. 30:98-100. Rozman, K., Rozman, T., and Greim, H. 1983. Stimulation of non-biliary intestinal secretion of hexachlorobenzene in rhesus monkeys by mineral oil. Toxicol. Appl. Pharmacol. 70:255-261. Sell, J. L., Davidson, K. L., and Bristol, M. 1977. Depletion of dieldrin from turkeys. Poult. Sci. 56:2045-2051. Wilson, K. A., Cook, R. M., and Emory, R. S. 1968. Effect of charcoal feeding on dieldrin excretion in ruminants. Fed. Proc. 27:558 (abstr.). Wyss, P. A., Muhlebach, S., and Bickel, M. H. 1982. Pharmacokinetics of 2,2',4,4',5,5'hexachlorobiphenyl (6-CB) in rats with decreasing adipose tissue mass. 1. Effects of restricted food intake two weeks after administration of 6-CB. Drug Metab. Dispos. 10:657-661. Yoshimura, H., and Yamamoto, H. A. 1975. A novel route of excretion of 2,4,2',4'tetrachlorobiphenyl in rats. Bull. Environ. Contam. Toxicol. 13:681-688.
Received May 2, 1990 Accepted December 14, 1990
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Elimination of PBBs in rats. effect of mineral oil and/or feed restriction D. Polin , S. J. Bursian , M. S. Underwood , P. A. Wiggers , N. Biondo , I. Su , W. E. Braselton & J. A. Render To cite this article: D. Polin , S. J. Bursian , M. S. Underwood , P. A. Wiggers , N. Biondo , I. Su , W. E. Braselton & J. A. Render (1991) Elimination of PBBs in rats. effect of mineral oil and/or feed restriction, Journal of Toxicology and Environmental Health, 33:2, 197-212, DOI: 10.1080/15287399109531518 To link to this article: http://dx.doi.org/10.1080/15287399109531518
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Date: 10 November 2015, At: 18:43
ELIMINATION OF PBBs IN RATS. EFFECT OF MINERAL OIL AND/OR FEED RESTRICTION D. Polin, S. J. Bursian, M. S. Underwood, P. A. Wiggers, N. Biondo Department of Animal Science, Michigan State University, East Lansing, Michigan
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I. Su, W. E. Braselton Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan J. A. Render Department of Pathology, Michigan State University, East Lansing, Michigan Rats were fed polybrominated biphenyls (PBBs) at 0.1 to 100.0 ppm for 14 d and then treated to hasten the removal of PBBs with 0, 5, or 10% mineral oil (MO) and/or 0, 15, 30, or 45% feed restriction (FR) for 21 d. PBB body burdens were determined at d 14 and expressed on a log-log basis by Y = 0.91X + 2.179 (r2 = 0.974), where X = log of PBB concentration in diet (ppm) and Y = log of PBB body burden (μg). After 21 d withdrawal, body burdens were expressed by the equation Y = 0.787X + 2.218 (r2 = 0.95). The most effective withdrawal treatment was 10% MO + 45% FR producing a reduction of body burdens inversely related to prior body burdens (69% at 0.1 ppm to 23% at 100 ppm). Body weights and fat content were significantly (p .05) reduced by feed restriction, with fat content only 39% of controls at 21 d off. Mortality averaged 0, 13.6, and 35.8% for rats fed 0, 5, or 10% MO, and 25, 15, 8.6, and 3.7% for rats feed restricted at 0, 15, 30, and 45%, respectively. Histopathology of the dead and moribund rats indicated that the clinical signs were not characteristic of PBB toxicity. In a second experiment, safflower oil at 3.5% or excess vitamins prevented the mortality and clinical signs associated with MO during withdrawal from 100 ppm PBBs. Based on these data and those in the literature, PBBs interfere with vitamin utilization.
INTRODUCTION The long-term retention of certain xenobiotics results in continuous exposure of target organs even after cessation of external exposure. This possibility poses a long-term risk to humans and/or food animals harboring the xenobiotics. As a result, there has been considerable attention directed toward reducing the body burdens of xenobiotics. Such studies used treatments involving charcoal, phénobarbital, lipotropic agents and Support came from the Michigan Department of Public Health and the Michigan Agricultural Experiment Station. Requests for reprints should be sent to Donald Polin, Department of Animal Science, Room 132 Anthony Hall, Michigan State University, East Lansing, Ml 48824. 197 Journal of Toxicology and Environmental Health, 33:197-212, 1991 Copyright © 1991 by Hemisphere Publishing Corporation
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bile acid binding resins, but all with uncertain and conflicting results (Wilson et al., 1968; Donaldson et al., 1971; Cregar and Kubena, 1971; Egebertson and Davison, 1971; Coon and Couch, 1973; Sell et al., 1977; Wyss et al., 1982; Polin and Leavitt, 1984). Polin and Leavitt (1984) revealed the need to determine the role of varying the dosages of these compounds and the length of treatment to determine if these treatments could be effective. In chickens almost 70% of the body burdens of polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs), and hexachlorobenzene (HCB), and 100% of the pentachlorophenol (PCP) body burdens were eliminated in 21 d (Polin et al., 1985; Polin et al., 1986). The combination of 45% feed restriction (FR) and 10% mineral oil (MO) in the diet appeared to be the best combination of several investigated (Polin et al., 1989). The purpose of this study was to determine if in the rat the MO + FR treatment would be a viable approach when used over a wide range of dietary PBBs. The effects of other dietary concentrations of MO and lesser amounts of FR were also studied. PROCEDURES AND METHODS Male Sprague-Dawley rats, 6 wk of age, were assigned to an environmentally controlled room (21°C). Rats were weighed weekly to the nearest gram. Thirty-nine rats were fed control diet, and 78 rats were fed either 0.1,1.0,10.0, or 100.0 mg PBBs/kg diet (as FireMaster; see Polin and Leavitt, 1984, for details on product). The rats were housed in groups of three per cage. The cages were plastic bases with stainless steel metal wire tops. There were no wire floors to keep the rats off the bedding (coprophagy could be practiced by the rats). The diets containing the PBBs were fed for 14 d. Diets were withdrawn from 3 control rats, and from 6 rats fed each of the levels of PBBs on the evening before d 14 so that the digestive contents would be at a minimum and not contribute PBBs to the body burden measurements. The rats were euthanized on d 14 with excess CO2. Their carcasses were frozen, and stored in a freezer at -20°C until processed for fat and PBBs determinations. On d 14 of exposure PBB feeding was discontinued. Each of the 5 groups exposed to the different dietary levels of PBBs was then treated with 0, 5, or 10% MO (from MSU Vet Clinic), added to the diet on a w/w basis; 0,15, 30, or 45% feed restriction of ad libitum controls; or a combination of MO + FR. A 5 x 3 x 4 factorial design was followed. The treatments were continued for 21 d, at which time the rats were killed and frozen. Three rats were started in each group of the 3 x 4 factorial representing those fed control diets for the 14-d contamination period. There were 6 rats per group in each cell of the 3 x 4 factorial arrangement representing those fed the PBB diets of 0.1 to 100 ppm, as indicated by the outline within Tables 1 and 2.
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The diet was Purina Chow 5002, ordered ground so that the PBBs could be distributed uniformly in it. The PBBs were introduced into the diet via 4-kg premixes that were then blended in a small drum-type commercial feed mixer (Mix-Mill, Inc., Bluffton, III.) to 30 kg. The premixes were made according to standard dry mixing procedures by which small quantities of chemicals, such as vitamins or purified amino acids, are blended into already mixed diets. Individual feed containers were heavyduty white glass wide-mouth jars. Any spillage was collected twice each week by sifting the bedding. The spilled feed was weighed and the feed intake determined once each week. Water and feed were allowed ad libitum during the exposure period, but only water was allowed ad libitum during the withdrawal period. PBBs were determined by a modification of the procedure of Price et al. (1986). Frozen rats were ground in a Hobart grinder. The ground samples were refrozen until analyzed for PBBs. Aliquots (50 g) were taken and frozen in liquid nitrogen in a Waring blender, the excess liquid nitrogen poured off, and the frozen tissue blended to a fine powder. PBBs were analyzed in 20-g aliquots of the powdered tissue weighed into a beaker and mixed thoroughly with 100 g of granular sodium sulfate until dry. A 25 mm ID x 300 mm chromatography column was packed with approximately 2 g Na2SO4, and the dry Na2SO4 tissue homogenate mixture was packed lightly into the column. The sample container and the column were rinsed with 10 ml of extracting solvent, diethyl ether/petroleum ether (1 :1), and an additional 190 ml of the extracting solvent was added to the top of the column. The eluate was collected into a preweighed beaker at 5-6 ml/min. The solvent was evaporated under N2 with gentle heating, and the lipid content of the tissue was determined by differential weight. A 0.25-g aliquot of fat was dissolved in 1.0 ml hexane and applied to the top of a 9 x 200 mm glass column packed with 5 g silica gel 60. The sample was eluted onto the column with 15 ml hexane, and the effluent was discarded. The PBBs were eluted with 45 ml hexane and concentrated by evaporation in Snyder tubes. The PBBs were quantified by chromatography on a 2 mm ID x 180 cm column of 3% OV-1 on Gas Chrom Q at 240°C and detected by electron capture with a 63 Ni detector. Peak areas were integrated automatically by a Spectra Physics 4100 integrator. A total of nine peaks was calculated, each dependent on its own standard curve. Total PBBs were determined from the sum of the congeners. Standard curves were run for each day that samples were processed, and the procedure was monitored for each run by including a sample from a large known meat source contaminated with PBBs. Body burdens were derived from the concentrations of PBBs in the fat, the concentration of fat in the carcass, and the body weight of the rat. Values are reported on a wet weight body basis and were determined on individual rats unless specified otherwise. Body burdens, and not concentration of PBBs, were emphasized to adjust for changes in body weight, in
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most situations substantial, that occurred during the experiment. Final concentrations of PBBs were also reported. (When lipid and protein are removed from the body through restricted feeding and PBBs are also removed, then upon refeeding one would expect the PBB concentrations to be reduced as they are diluted with newly formed lipid and protein clear of PBBs.) Values were adjusted for percent recovery derived from analyzing spiked samples. Statistical analyses were conducted using a Macintosh Plus computer (Apple Computer Co.) with the program StatView512+ (Brain Power Inc., Calabasa, Calif.). Statistical significance was accepted at p < .05. In a subsequent experiment using the same procedures as above, rats were fed a diet with 100 ppm PBBs for 14 d followed by treatment with 0 or 10% MO, without or with the addition of vitamins, or with 3.5% safflower oil (SO). The vitamin mix (details in text) was prepared in our laboratory, and the SO was purchased at a local food store. Three different sources of MO were evaluated (details in text). At the end of the withdrawal period, remaining animals were weighed and killed with excess CO2 and examined for clinical signs. RESULTS Experiment 1: Controls
Rats fed no PBBs had detectable amounts of PBBs in their bodies averaging 4.1 and 17.5 /¿g/rat, on a wet weight basis, at d 14 and 35, respectively, of the experiment (Table 1). These values correspond to 16 and 58 /¿g/kg body weight (parts per billion, ppb). Day 35 was d 21 of withdrawal for those rats that were fed diets with PBBs. The control rats had accumulated trace amounts of PBBs from dust particles in the room, as dust on the room air filters had PBBs. Control diets were not mixed in the equipment used to prepare the PBB diets, and were stored, as were TABLE 1. Average Body Burdens of PBBs in Rats Fed for 14 d Diets Containing PBBs from 0 to 100 ppm, and in Rats 21 d after the Withdrawal of These Diets PBBs
Day of treatment regimen, PBBs (fig/rat)
in aiei
(ppm)
Day 14 on
Day 35 off
Day 35/day 14
None 0.1 1.0 10.0 100.0
4.1 (3)a 26.7 (6) 112.1 (6) (6) 1086.8 (6) 12426.0
17.56 (2) 44.4 (6) (6) 102.4 (6) 865.1 (6) 8321
427 166 91 80 67
a b
Number of rats. Omitted outlyer value of 128.7 ^ig.
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all diets, in closed containers, each with its own scoop for feeding. One rat from the control group had a body burden of 128.7 /¿g, an amount in the range for rats fed 1.0 ppm PBBs. That value was excluded from the statistical analysis as an outlyer. Analysis of PBBs in rats at d 35 (d 21 of withdrawal) revealed (Table 2) that those treated with MO and/or FR regimen had significantly (p < .01) decreased body burdens characterized by a statistically significant interaction (p < .01). The latter statistics indicated that the combination of the treatments was better than either one alone in reducing the body burdens of PBBs in the contaminated control rats. With increasing FR, or increasing MO, the body burdens of PBBs were reduced. Control rats treated with 10% MO + 45% FR had the lowest body burdens, 0.8 /¿g/rat, and the greatest percentage decline (Table 2). Experiment 1: Rats Contaminated with PBBs
Body Burdens Feeding PBBs at 0.1 to 100.0 ppm to rats for 14 d resulted in body burdens ranging from 26.7 to 12426 /ug/rat (Table 1). The dose-response relationship was linear on a log-log basis yielding the equation Y = 0.91X + 2.179 (? = 0.974), where X is the log of PBB concentration in diet (ppm), and V is the log of PBB body burden (/¿g). Generally, the body burdens in rats from the 1,10, and 100 ppm dietary levels of PBBs differed by a factor of 10. Diets with 0.1 ppm PBBs produced body burdens 25% less than the value from the 1.0 ppm diet. At d 21 of withdrawal, those rats not treated with MO and/or FR had body burdens that ranged from 17.5 to 8321 ^g (Table 1), which on a log-log linear relationship was represented by the equation Y = 0.787X + 2.218 (r2 = .95). Nontreated rats previously fed control diet and those fed 0.1 ppm PBB diet had burdens of PBBs at d 21 off that were greater than those when PBB diets were removed. Their low values were influenced by the dust in the room. Another factor involved in these rats with very low burdens was the wide variation in body burden values having a pooled standard deviation of ±10 (converted from a log basis). Rats fed diets with 1-100 ppm PBBs had body burdens that were less on d 21 of withdrawal than those on d 14 of feeding the contaminated diets (Table 2). Thus, these rats were able to remove some of their body burdens without external treatment. PBBs could not be detected in rats kept in a separate room and fed control diet. Withdrawal treatments of MO and FR produced some loss of PBB body burdens depending on the treatment and on the level of contamination. 1. Rats that had been fed 0.1 ppm and then placed on the withdrawal regimen had significant reductions in their PBB burdens by both MO and FR treatments (Table 2). The lowest average body burden resulted from treating with 10% MO + 30% FR, a reduction of 69% when the
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TABLE 2. Body Burdens of Rats on d 21 of Withdrawal Following the Feeding of Diets with 0, 0.1, 1.0,10.0, or 100.0 ppm PBBs Mineral oil
Feed restriction (%) 30
15
45
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Control diet (no prior PBBs) d!
0 5 10
(2)
17.5'
(3) (1)
9.0 4.2
0 5 10
(6) (6) (6)
44.4 26.1 23.0
(3) (3) (3)
15.0 2.1 2.1
(3) (3) (3)
0.5 1.1 1.3
(3) (3) (3)
2.3 1.3 0.8
29.0 29.0 13.6
(6) (6) (6)
24.2 16.2 17.1
83.8 83.8 71.4
(6)
98.6
(6) (6)
75.4 59.0
Withdrawal from 0.1 p p m PBBs (6) (6) (5)
25.7 20.9 20.9
(6) (6) (6)
Withdrawal from 1.0 p p m PBBs 0
(6)
93.0
(6)
(5)
102.4 65.9
(6)
5
(6)
(6)
10
(5)
96.1
(6)
117.4 71.7
(6)
Withdrawal from 10.0i p p m PBBs 0 5 10
(6) (6) (2)
865 777 782
0 5 10
(6) 8321 (1) 9425 (1) 6669
(6) (6) (4)
849 845 767
772 682 690
(6) (6) (5)
(6) (6) (6)
716 658 627
Withdrawal from 100.0 ppm PBBs (6) 10430 (3) 7998 — (0)
(6)
(6) 8199 (4) 8007 (5) 7104
10059 9846 7248
(4) (2)
Summary of body Iburdens vs. treatment Change in
Former treatment (ppm)
Best withdrawal treatment
(%)
From
To
.05) reduce body weights (317 vs. 322 g), whereas, 30 and 45% FR reduced body weights progressively to 290 and 251 g, respectively (Table 3). Rats not fed PBBs and then continued on diets with no MO or FR weighed at the end of the withdrawal period 297 g. Prior PBB feeding did not have an effect on body weights during the withdrawal period, as the rats weighed from 309 to 331 g. There was no significant interaction (p > .05) to indicate that as a result of the prior PBB treatment MO or FR produced a greater effect than no withdrawal treatment. Body Fat Fat in control rats averaged 7.04%. The average body fat in rats not subject to feed restriction was 6.69% (Table 4), whereas the fat in rats not given MO was 5.50% (Table 4, footnote a), the latter value reflecting the averaging in of the FR effect. Five and 10% MO caused body fat percentage to be significantly (p < .05) reduced to 4.86 and 4.36%, respectively, values that were 88 and 79% of 0% MO. Body fat was progressively and significantly reduced by 15, 30, and 45% FR, with the latter
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D. POLIN ET AL.
TABLE 3. Body Weights of Rats on d 21 of Withdrawal from Diets with 0, 0.1, 1.0, 10.0, and 100.0 ppm PBBs Feed restriction (%) KA \ nar^ \ /Vlli itrl dl
oil (%)a
0
15
30
45
Withdrawal from control diet (no prior PBBs)
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0 5 10
(3) 297 (3) 307 (1) 319
(3) 324 (3) 321 (3) 313
(3) 298 (3) 297 (3) 286
(3) 252 (3) 260 (3) 250
(6) 288 (6) 295 (6) 285
(6) 258 (6) 257 (6) 240
(6) 291
(6) 275
(6) (6)
288 280
(6) (6)
2S5
(6) (6) (5)
309 291 292
(6) (6) (6)
263 248 239
301 272 260
(6) (4)
Withdrawal from 0.1 ppm PBBs 0 5 10
(6) 331 (6) 328 (6) 322
(6) 324 (6) 321 (5) 322
Withdrawal from 1.0 ppm PBBs 0
(6) 309
5
(5)
10
336 (5) 346
0 5 10
(6) 329 (6) 320 (2) 291
(6) 318 (6) 331 (6) 280
239
Withdrawal from 10.0 ppm PBBs (6) 324 (6) 324 (4) 324
Withdrawal from 100.0 ppmi PBBs 0 5 10 Mean weight (g)b
(6) 326 (1) 347 (1) 264
(6) 322 (3) 291 (0) -
(63) 322a
(69) 317a
(6) (4) (2) (74)
290b
253 250 (5) 222 (78) 251c
Noie. Some were treated with mineral oil at 5 or 10% and/or restricted in feed intake to 15, 30, or 45% of control ad libitum fed rats. Values given are number of rats (in parentheses) and body weight (g). a Overall effect of mineral oil: 0% - (108) 300; 5% = (96) 296; 10% = (81) 280. Significant effect (p < .05) of mineral oil in rats withdrawn from 10 and 100 ppm PBBs. b Mean values with different letters are significantly different at p < .05.
treatment producing an average body fat of 2.60% (Table 4), a value only 39% of the value for 0% FR. PBBs Concentration Rats not fed PBBs or not treated with MO or FR during the withdrawal period had contamination concentrations of PBBs of 0.186 ppm (Table 5). Treating these rats with 15, 30, or 45% FR reduced the mean PBB concentrations markedly (Table 5). The same overall pattern was detected for the rats that had been fed 0.1 ppm PBBs and treated with MO and/or FR. Thus, the FR produced a significant decline
ELIMINATION OF PBBs IN RATS
205
at these very low PBB concentrations. In the groups withdrawn from 1.0 and 10.0 ppm PBBs, FR did not reduce PBB concentrations (Table 5). In the group withdrawn from 100 ppm PBBs, the PBB concentrations were significantly increased by an average of 25%, as a result of the FR, but concentrations were not changed by MO treatment (Table 5). Mortality During the withdrawal period from PBBs, mortality averaged 0, 13.6, and 35.8% for the rats fed 0, 5, and 10% MO, respectively (Table 6, footnote a). It averaged 25,15, 8.6, and 3.7% for rats undergoing
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TABLE 4. Percentage of Fat in Rats on d 21 of Withdrawal from Diets with 0, 0.1,1.0,10.0, and 100.0 ppm PBBs Feed restriction (%) Mineral oil (%)a
0
15
30
45
Withdrawal from control diet (no prior PBBs) 0 5 10
(3) (3) (1)
7.04 6.20 7.82
(3) (3) (3)
7.15 6.11 5.66
5.42 3.74 3.30
(3) (3) (3)
2.61 2.71 2.28
5.28 5.01 4.24
(6) (6) (6)
2.52 2.26 2.34
(6) 5.45 (6) 2.76 (6) 4.26
(6) (6) (6)
2.56 2.48 1.80
4.22 3.93 4.81
(6) (6) (6)
2.79 3.31 2.07
(6) (4) (2)
5.28 5.25 5.06
(6) (4) (5)
2.72 3.73 3.17
(74)
4.53c
(78)
(3) (3) (3)
Withdrawal from 0.1 ppm PBBs 0
5 10
(6) 6.71 (6) 5.60 (6) 6.02
(6) 6.24 (6) 6.25 (5) 6.11
(6) (6) (6)
Withdrawal from 1.0 ppm PBBs 0
5 10
(6) (5) (5)
7.13 6.47 6.42
(6) (6) (6)
6.84 7.10 6.03
Withdrawal from 10.0i ppm PBBs 0 5 10
(6) (6) (2)
7.26 6.46 7.76
(6) (1) (1)
7.40 7.34 7.27
(63)
6.69a
(6) 7.99 (6) 6.35 (4) 4.30
(6) (6) (5)
Withdrawal from 100.0 ppm PBBs 0 5 10 Mean fat (%)b
(6) (3) (0) (69)
7.59 6.92 6.55b
2.60d
Note. Some were treated with mineral oil at 5 or 10% and/or restricted in feed intake to 15, 30, or 45% of control ad libitum fed rats. Values given are numbers of rats (in parentheses) and carcass fat (%). a Overall effect of mineral oil: 0% = (108) 5.50afa; 5% - (94) 4.86b; 10% - (87) 4.36c. Significant effect (p S .05) of mineral oil in rats withdrawn from 10 and 100 ppm PBBs. È Mean values with different letters are significantly different at p < .05.
D. POLIN ET AL.
206
TABLE 5. PBB Concentrations in Rats on d 21 of Withdrawal from Diets with 0, 0.1, 1.0, 10.0, and 100.0 ppm PBBs Feed restrictions (%) Mineral oil (%)a
15
0
45
30
Withdrawal from control diet (no prior PBBs) 0 5 10
(3) (3) (1)
0.186 0.029 0.013
(6) (6) (6)
0.134 0.079 0.072
(3) (3) (3)
0.045 0.006 0.0065
(3) (3) (3)
0.0018 0.0037 0.0044
(3) (3) (3)
0.0090 0.0051 0.0033
0.061 0.099 0.048
(6) (6) (6)
0.082 0.063 0.071
0.288 0.291 0.256
(6) (6) (6)
0.381 0.293 0.247
2.50 2.36 2.37
(6) (6) (6)
2.73 2.66 2.63
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Withdrawal from 0.1 ppm PBBs 0 5 10
(6) (6) (5)
0.081 0.065 0.060
(6) (6) (6)
Withdrawal from 1.0 ppm PBBs 0 5 10
(6) (5) (5)
0.329 0.197 0.278
(6) (6) (6)
0.296 0.352 0.254
(6) (6) (6)
Withdrawal from 10.0 ppm PBBs 2.64 2.45 2.76
0 5 10
(6) (6) (2)
0 5 10
(6) 25.5 (1) 27.2 (1) 25.3
(6) (6) (4)
2.64 2.62 2.35
(6) (6) (5)
Withdrawal ( rom 100.0 ppm PBBs
Mean (/ig/g)b
(53)
3.94a
(6) 32.5 (3) 27.5 (0) (69)
4.71a
(6) 33.4 (4) 36.7 (2) 28.3 (74)
6.01b
(6) 32.6 (4) 31.9 (5) 32.1 (78)
6.91b
Note. Some were treated with mineral oil at 5 or 10% and/or restricted in feed intake to 15, 30, or 45% of control ad libitum fed rats. Values given are number of rats (in parentheses) and PBBs a Overall effect of mineral oil: 0% - 7.57a6; 5% - .(95) 4.77b; 10% - (81) 3.60c. Significant effect (p < .05) of mineral oil in rats withdrawn from 10 and 100 ppm PBBs. ö Mean values with different letters are significantly different at p < .05.
FR at 0, 15, 30, and 45%, respectively (Table 6). Thus, increasing MO increased mortality, while increasing FR reduced overall mortality. Mortality increased markedly for rats previously fed 10 and 100 ppm PBBs: 5.5 (2/36), 4.1 (3/72), 2.8 (2/72), 9.7 (7/72), and 38.9% (28/72), for prior PBB contamination of 0, 0.1, 1.0, 10, and 100 ppm, respectively (Table 6). Clinical signs in the affected rats were paralyzed rear legs, bloody urine, wet abdominal skin, bloody discharge from the nostrils, rhinorrhagia, exophthalmos, and subcutaneous edema and lethargy.
ELIMINATION OF PBBs IN RATS
207
Experiment 2
Average starting weight for all rats was 360 g. During the 14-d contamination period, the 105 rats fed PBBs gained as much as the 12 control rats, with body weights averaging 389 g. Treating the rats during the withdrawal period with MO, types A, B, and C (Table 7), reduced the weights of the rats to 350 g. Rats treated with MO + FR averaged 315 g TABLE 6. Number of Rats That Died during the 21 d of Withdrawal from Diets with 0, 0.1,1.0,10.0, and 100.0 ppm PBBs
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Feed restriction (%) Mineral oil (%)a
0
15
30
45
Withdrawal from control diet (no prior PBBs) 0 5 10
(3) (3) (3)
0 0 2
(3) 0 (3) 0 (3) 0
(3) 0 (3) 0 (3) 0
(6) 0 (6) 0 (6) 0
(6) 0 (6) 0 (6) 0
(6) 0 (6) 0 (6) 0
(6) 0 (6) 0 (6) 0
(6) 0 (6) 0 (6) 1
(6) 0 (6) 0 (6) 0
0 3 6
(6) 0 (6) 2 (6) 4
(6) 0 (6) 2 (6) 1
(81) 12 15
(81) 7 8.(
(81) 3 3.7
(3) (3) (3)
0 0 0
Withdrawal from 0.1 ppm PBBs 0 5 10
(6) (6) (6)
0 0 2
(6) (6) (6)
0 0 1
Withdrawal from 1.0 ppm PBBs 0 5 10
(6) (6) (6)
0 5 10
(6) (6) (6)
0 1 1
(6) (6) (6)
0 0 0
Withdrawal from 10.0 ppm PBBs 0 0 4
(6) (6) (6)
0 0 2
Withdrawal f rom 100.0 ppm PBBs 0 5 10 (Number)6 dead % Dead
(6) (6) (6)
0 5 5
(81) 20 2Í
(6) (6) (6)
Note. Some were treated with mineral oil at 5 or 10% and/or restricted in feed intake to 15, 30, or 45% of control ad libitum fed rats. Values are number of rats at the start (in parentheses) and mortality (number of rats). a Overall effect of mineral oil: 0% MO - (108) 0.0% dead; 5% MO = (95) 13.6% dead; 10% MO = (81) 35.8% dead. Significant effect (p < .05) of mineral oil in rats withdrawn from 10 and 100 ppm PBBs. 6 Rats at the start.
D. POLIN ET AL.
208
TABLE 7. Summary of the Histopathologic Lesions Detected in Rats Treated with Mineral Oil (MO), 30% Feed Restriction (FR), Vitamins (VIT), and/or Safflower Oil (SO) during Withdrawal from Diets with 100 ppm Polybrominated Biphenyls (PBBs) Histopathologic lesions detected* 6
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Lesions No histologie lesions Focal alveolar hemorrhage Mild midzonal hepatocellular fatty change Prominent splenic histocytic population Mild hemosiderosis Eosinophilic proteinaceous fluid in renal tubules Diffuse centrilobular hepatocellular Necrosis Vacuolar change Thinning of centrilobular hepatic cords with necrosis Hemorrhage and edema in connective tissue of Epididymis Urinary bladder Esophagus Esophageal necrosis, exudation of fibrin, lymphocytic infiltration, and fibrosis Distention of renal tubules with eosinophilic fluid Hemorrhage and congestion of lamina propria of urinary bladder Hemorrhage within muscular layer of urinary bladder Interstitial testicular hemorrhage Interstitial epididymal hemorrhage Epididymal edema and neutrophilic infiltration a
Control (5)
MO C Hv FRd + VITe (6)
MO + FR + SO' (6)
MO (5)
5 0 0 0 0
1 0 2 0 0
2 1 3 0 0
0 0 0 3 1
0
0
0
1
0 0
0 0
0 0
1 1
0
0
0
1
0 0 0
0 0 0
0 0 0
1 1 1
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0 0
0 0 0 0
0 0 0 0
0 1 1 0
Many of the hemorrhages were within the same rats. ^Clinical signs include paralysis of the hindlegs, bloody urine, wet abdominal area, rhinorrhagia, exophthalmos, subcutaneous edema, and lethargy. c Type A - Fisher Scientific (Livonia, Mich.), a paraffin light oil catalog number 0121; Type B USDA Laboratory (Mt. Hope Road, E. Lansing, Mich.); Type C - Amoco Oil Co (from a distributor in Lansing, Mich.); each added at 10% (w/w). d Feed restriction at 30% of ad libitum controls. e Vitamin mix to supply per kg diet: A, 35,883 USP units; D3, 1800 ICU; E, 4.5 III; menadione sodium bisulfate complex (K activity), 12.6 mg; B12 supplement, 0.135 mg; riboflavin, 18 mg; dcalcium pentothenate, 6.3 mg; niacin, 126 mg; thiamine mononitrate, 2.6 mg; pyridoxine HCI, 12.5 me; folie acid, 3.6 mg; ascorbic acid, 15 mg; d-biotin, 0.63 mg. ^Safflower oil added at 3.5%.
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ELIMINATION OF PBBs IN RATS
209
with no difference among the MO types A, B, and C. Statistical analysis revealed that weight loss was primarily attributed to restricting feed, and a borderline effect from feeding MO. Rats alive at the end of the withdrawal period had hemorrhages in various organs if they were fed MO without FR [81% (13/16)], or MO + FR [76% (16/21)]. In the groups fed only "Type A MO + FR" but also vitamins or SO, mortality was 0% and the incidence of hemorrhages was 11% (2/19). The hemorrhages in the rats fed MO alone or with FR were mostly in the kidney (28.6%), urinary bladder (21.4%), testis (14.3%), epididymis (14.3%), and brain (12.5%), with the remainder detected in heart, pancreas, lung, and thymus. Of the 56 rats examined from the PBB treatments, 6 (10.7%) had pale livers while none had pale kidneys. Table 7 contains a summary of the histopathologic lesions detected in the rats treated with MO, MO + FR, or MO + FR + (vitamins or SO). No histopathologic lesions were detected in the controls. Two of the six rats from MO + FR + vitamins and three of six from MO + FR + SO had mild midzonal hepatocellular fatty alterations. This change was detected only in one rat from MO + FR and in no rats from MO alone. On the other hand, treatment with MO or MO + FR caused many histopathologic changes that were not detected in rats given vitamins or SO. DISCUSSION The efficacy of the MO + FR treatment depended on the extent of the PBB contamination, as well as the particular strength of the treatment. Rats having low burdens eliminated PBBs to the extent observed with chickens (Polin et al., 1989). MO at 10% of the diet with 30 or 45% FR, the latter causing a marked reduction in body fat, were the effective combinations, possibly indicating that utilization of body fat is necessary for removal of the xenobiotic. That either MO or FR alone was rather ineffective in removing PBBs from highly contaminated rats, but was effective to some extent for rats with very low contamination of PBBs, suggested that PBB storage and metabolism may differ depending upon the level of contamination, or possibly related to the adverse clinical effects and mortality caused by much greater contamination. Other dietary approaches, such as limiting caloric consumption only, may serve the same purpose as FR to enhance fat mobilization with less severe nutritional consequences. Kimbrough et al. (1980) were unable to reduce adipose and blood concentrations of PBBs in rats given a single large dose of PBBs followed 3 weeks later by MO for 3 mo or fed diets with 4 and 20% fiber. Our rats contaminated with large amounts of PBBs and subsequently treated with FR also had increased PBB concentrations, though body burdens were reduced as much as 30% as a reflection of weight loss. Because body burdens were not presented by Kimbrough et al. (1980), the effect of the
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D.POLIN ET AL.
fiber and MO on excretion was not known. Based on data from other animals treated with MO alone, the ability to reduce halogenated compounds was minimally to partially effective (Richter et al., 1977; Richter and Schafer, 1981; Rozman et al., 1982a, 1982b, 1983) and seems to require utilization of stored lipid, as presented in this report and by Polin et al. (1985,1986,1989). Body fat showed a trend to decline in rats treated with MO, and this may account for researchers occasionally detecting some enhanced withdrawal of body burdens when MO was fed. The experimental conditions may account for the difference that highly contaminated chickens lost up to 80% PBBs (Polin and Leavitt, 1984; Polin et al., 1985, 1986) while rats similarly contaminated lost far less. Rats were dying, particularly when fed MO at 10% of the diet or 5 or 10% of the diet if previously treated with 10 or 100 ppm PBBs, or showing pathological signs, which did not occur with chickens. Those clinical signs suggested a vitamin deficiency, a conclusion strengthened by reversal of mortality and clinical signs by the addition of vitamins or SO to reverse the effect of MO + FR to rats previously fed 100 ppm PBBs. Why did not the chickens in previous experiments show vitamin deficiencies when they were fed up to 100 ppm PBBs? The reason may be that the chickens were fed diets with vitamin concentrations three- to fourfold above NRC requirements (National Research Council, 1984), whereas the commercial diet used for the rats was most likely formulated close to NRC requirements. Darjono et al. (1983) overcame in rats fed 100 ppm PBBs an induced vitamin A deficiency by adding excess vitamin A to a diet with adequate vitamin A. Thus, high intakes of PBBs appear to be interfering with vitamin activity, including vitamin A, as indicated, and presumably other vitamins, as suggested by the clinical signs of hindlimb paralysis, bloody urine, rhinorrhagia, and exophthalamos reported by us. Other than loss of body weight, these signs were not characteristic of PBB toxicity (Render et al., 1982). Also, these are not typical signs of vitamin A deficiency, which include epithelial hyperplasia, hyperkeratosis, and squamous metaplasia (Darjono et al., 1983), and were not observed in the present study. Interference with nutrients in high-fiber diets was speculated as the probable cause of enhanced liver pathology by PBBs (Kimbrough et al., 1980). Their observation added to those above would be additional evidence to consider PBBs interference with vitamin utilization. The question that arises is whether the MO + FR technique is a viable approach to remove xenobiotics if the animal is subjected to possible vitamin deficiency from use of MO. Evidently, the MO + FR treatment is viable when the diet contains adequate vitamins. The property of MO that enhances the removal of xenobiotics is supposedly through its interference with bile absorption, thus enhancing the nonbiliary intestinal secretion of xenobiotics (Yoshimura and Yamamoto, 1975; Richter et al., 1977; Rozman et al., 1982a, 1982b). Interference with bile absorption
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ELIMINATION OF PBBs IN RATS
211
also reduces uptake of lipid- and fat-soluble vitamins and would explain the higher mortality and morbidity detected as the level of MO increased, and the lower mortality as the FR increased reducing MO intake. Having excess vitamins in the diet when MO was used did not interfere with xenobiotic removal in chickens (Polin et al., 1985, 1989) or goats (Polin et al., 1987), and, in contrast, may enhance its efficacy because the animals were healthy. Rats with low PBB burdens lost a considerable percentage of PBBs, but there were no data to indicate if the reason was the low level of contamination or their state of health. Rats practice coprophagy and this may also have some effect on MO + FR to reduce body burdens. Amounts of PBBs removed at the highest level of contamination far exceeded the body burdens of lower levels of contamination. Thus, the potential exists for removal of all of the xenobiotics from less contaminated animals. REFERENCES Coon, C. W., and Couch, J. R. 1973. The effects of lipotropic agents in depletion of chlorinated biphenyls in laying hens. Poult. Sci. 52:2014 (abstr.). Cregar, C. R., and Kubena, L. F. 1971. The effect of lipotropic agents on depletion of chlorinated hydrocarbons from tissues of poultry. Poult. Sei. 50:1567-1568. Darjono, Sleight, S. D., Stowe, H. D., and Aust, S. D. 1983. Vitamin A status, polybrominated biphenyl (PBB) toxicosis, and common bile duct hyperplasia in rats. Toxicol. Appl. Pharmacol. 71:184-193. Donaldson, W. F., Sheets, T. S., and Jackson, M. D. 1971. Influence of iodinated casein on DDT residues in chicks. Poult. Sci. 47:237-243. Egebertson, K. A., and Davison, K. L. 1971. Dieldrin accumulation and excretion by rats fed phenobarbital and charcoal. Fed. Proc. 30:562 (abstr.). Kimbrough, R. D., Korver, M. P., Burse, V. W., and Grace, D. F. 1980. The effect of different diets and mineral oil on liver pathology and polybrominated biphenyl concentration in tissues. Toxicol. Appl. Pharmacol. 52:442-453. National Research Council. 1978. Nutrient Requirements of Laboratory Animals, 3rd ed., National Academy of Sciences, Washington, D.C. Polin, D., and Leavitt, R. 1984. Colestipol and energy restriction as an approach to hasten removal of PBBs from chickens. J. Toxicol. Environ. Health 13:659-671. Polin, D., Lehning, E., Pullen, D., Bursian, S., and Leavitt, R. 1985. Procedures to enhance withdrawal of xenobiotics from chickens. J. Toxicol. Environ. Health 16:243-254. Polin, D., Olsen, B., Bursian, S., and Lehning, E. 1986. Enhanced withdrawal from chickens of hexachlorobenzene (HCB) and pentachlorophenol (PCP) by colestipol, mineral oil, and/or restricted feeding. J. Toxicol. Environ. Health 19:359-368. Polin, D., Underwood, M., Lehning, E., Bursian, S., and Wiggers, P. 1987. PCBs in goats. Hastening withdrawal using mineral oil. Toxicologist 7:272. Polin, D., Underwood, M., Lehning, E., Olsen, B., and Bursian, S. 1989. Enhanced withdrawal of polychlorinated biphenyls: A comparison of colestipol, mineral oil, propylene glycol, and petroleum jelly with or without restricted feeding. Poult. Sci. 68:885-890. Price, H. A., Welch, R. L., Scheel, R. H., and Warren, L. A. 1986. Modified multiresidue method for chlordane, toxaphene, and polychlorinated biphenyls in fish. Bull. Environ. Contam. Toxicol. 37:1-9. Render, J. A., Aust, S. D., and Sleight, S. D. 1982. Acute pathologic effects of 3,3',4,4',5,5'hexabromobiphenyl in rats: Comparison of its effects with Firemaster BP-6 and 2,2',4,4',5,5'hexabromobiphenyl. Toxicol. Appl. Pharmacol. 62:428-444.
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Richter, E., Lay, J. P., Klein, W., and Korte, F. 1977. Enhanced elimination of hexachlorobenzene in rats by light liquid paraffin. Chemosphere 6:357-369. Richter, E., and Schafer, S. G. 1981. Intestinal excretion of hexachlorobenzene in rats. Arch. Toxicol. 47:233-239. Rozman, K. K., Rozman, T. A., Williams, J., and Greim, H. A. 1982a. Effect of mineral oil and/or cholestryamine in the diet on biliary and intestinal elimination of 2,4,5,2',4',5'hexabromobiphenyl in the rhesus monkey. J. Toxicol. Environ. Health 9:611-618. Rozman, K., Rozman, T., Greim, H., Neiman, I. J., and Smith, G. S. 1982b. Use of aliphatic hydrocarbons in feed to decrease body burdens of lipophilic toxicants in livestock. J. Agric. Food Chem. 30:98-100. Rozman, K., Rozman, T., and Greim, H. 1983. Stimulation of non-biliary intestinal secretion of hexachlorobenzene in rhesus monkeys by mineral oil. Toxicol. Appl. Pharmacol. 70:255-261. Sell, J. L., Davidson, K. L., and Bristol, M. 1977. Depletion of dieldrin from turkeys. Poult. Sci. 56:2045-2051. Wilson, K. A., Cook, R. M., and Emory, R. S. 1968. Effect of charcoal feeding on dieldrin excretion in ruminants. Fed. Proc. 27:558 (abstr.). Wyss, P. A., Muhlebach, S., and Bickel, M. H. 1982. Pharmacokinetics of 2,2',4,4',5,5'hexachlorobiphenyl (6-CB) in rats with decreasing adipose tissue mass. 1. Effects of restricted food intake two weeks after administration of 6-CB. Drug Metab. Dispos. 10:657-661. Yoshimura, H., and Yamamoto, H. A. 1975. A novel route of excretion of 2,4,2',4'tetrachlorobiphenyl in rats. Bull. Environ. Contam. Toxicol. 13:681-688.
Received May 2, 1990 Accepted December 14, 1990
