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Effects of dietary paraffin, squalane and sucrose polyester on residue disposition and elimination of hexachlorobenzene in rats
Chem.-Biol. Interactions, 40 (1982) 335--344
335
Elsevier/North-Holland Scientific Publishers Ltd.
EFFECTS OF DIETARY P A R A F F I N , SQUALANE AND SUCROSE POLYESTER ON RESIDUE DISPOSITION AND ELIMINATION OF HEXACHLOROBENZENE IN RATS
E. RICHTER, B. FICHTL and S.G. SCHAFER Institut ffir Pharmakologie und Toxikologie der Ludwig-Maximilians-Universit~it Milnchen, Nussbaumstrasse 26, D-8000 Miinchen 2 (F.R.G.)
(Received December 24th, 1982) (Accepted January 16th, 1982) SUMMARY Previous studies have shown addition of light liquid paraffin to enhance the elimination of organochlorine xenobiotics. In the present study the effect of paraffin on the elimination of [14C]hexachlorobenzene (HCB) was compared with the effect of possible alternative compounds, squalane and sucrose polyester (SPE). Four groups of 7 rats were fed a diet containing 1.5 ppm [14C]HCB for 4 days followed by 10 days on HCB-free diet. Thereafter one group (control) remained on this diet whereas the other 3 groups received a diet supplemented with 8% (w/w) paraffin, squalane or SPE, respectively. Radioactivity in urine and faeces was measured daily and at the end of the experiment in samples of abdominal fat, muscle, liver, kidney and blood. Dietary treatment with either paraffin, squalane or SPE markedly enhanced faecal excretion of [14C]HCB, whereas urinary excretion was not affected. Both the time course as well as the extent of faecal [ 14C]HCB elimination were similar in the treated groups. After 3 weeks of treatment the a m o u n t of [ ~4C]HCB excreted with faeces was about three times higher in treated animals than in controls. The half-life (tin) of [~4C]HCB elimination from the body was markedly decreased in treated animals {mean 34--38 days) compared to controls (110 days). [14C]HCB concentrations in some major tissues were significantly reduced to the same extent by all three dietary regimens. Thus squalane and SPE are as effective as paraffin in removing HCB from contaminated animals.
INTRODUCTION The persistence of organochlorine xenobiotic residues in human organs Abbreviations: GLC, gas liquid chromatography; HCB, hexachlorobenzene; LSD, least significant difference; SPE, sucrose polyester; TLC, thin-layer chromatography. 0009--2797/82/0000---0000/$02.75 © 1982 Elsevier/North-Holland Scientific Publishers Ltd.
336 and in animal tissues intended for human food consumption raises the problem of effecting their removal from the body. The elimination of those residues may be hastened by liver enzyme induction [1,2]. This al~proach, however, is not necessarily of benefit. The metabolic degradation of some of those c o m p o u n d s most probably involves arene oxide intermediates, which have been implicated with toxicity and/or carcinogenicity [3]. Indeed, Koss et al. [4] have shown that metabolism of HCB is a prerequisite for its porphyrinogenic action. Therefore it appears to be much more desirable to accelerate the elimination of the unchanged c o m p o u n d instead of stimulating the formation of possible hazardous metabolites. Feeding light liquid paraffin or mineral oil has proved to be a promising approach to this goal. It has been reported that this procedure markedly enhances the elimination of 2,4,6,2',4'-pentachlorobiphenyl [5] and ~-hexachlorocyclohexane [6] in rats and the elimination of HCB in rats [7,8], beagle dogs [9] and rhesus monkeys [10]. Paraffin or mineral oil, however, is partially absorbed from the intestinal tract and may cause a variety of untoward effects [11]. Furthermore it has been claimed that paraffins may contain variable amounts of carcinogenic impurities [12]. It seemed necessary, therefore, to look for alternative compounds. In the present study the influence of light liquid paraffin, squalane and SPE on the elimination of HCB in rats was compared. MATERIALS AND METHODS
Animals The experiments were performed using male Sprague--Dawley rats of a b o u t 400 g b o d y wt. (breeder: Versuchstierzucht WIGA, Sulzfeld, F.R.G.). The animals were housed individually in stainless steel metabolism cages in an animal room {temperature 21°C; humidity 55%; 12 h light per day). The rats had free access to food and drinking water. A finely ground diet (Altromin 1321, obtained from Altrogge, Lage, F.R.G.) was used. Chemicals [~4C]HCB (spec. act. 23.73 mCi/mmol) was purchased from the New England Nuclear Co. The radiochemical purity was > 9 9 % as assessed by thin-layer chromatography (TLC). [ 14C]HCB was diluted with the unlabelled c o m p o u n d (purity > 9 9 % by GLC) to a specific activity of 13.4 mCi/mmol. It was added to the diet at 1.5 ppm. Light liquid paraffin (DAB 7) was obtained from Merck (Darmstadt, F.R.G.). Squalane (Prosynth ® ) was purchased from Riedel~le Ha6n (Seelze, F.R.G.). SPE was donated by Procter and Gamble (Cincinnati, OH). All other chemicals were of analytical grade. Study protocol The rats were subdivided into 4 groups of 7 rats and adapted to the metabolism cages for 10 days. The animals were then fed the HCB-forti-
337 fied diet for 4 days. This period and the first day after withdrawal from the HCB-containing diet is referred to as the accumulation phase. This was followed by another 9 days on HCB-free diet (pretreatment phase). During the following 3 weeks (treatment phase) the control group remained on this diet whereas the other 3 groups received the diets supplemented with 8% (w/w) paraffin, squalane and SPE, respectively. F o o d consumption was measured daily and b o d y weights were recorded weekly. Urine and faeces were collected daily. After 3 weeks on the experimental diets the animals were killed under ether anaesthesia by bleeding from the aorta abdominalis. The weight of liver and kidneys was recorded and samples of these organs and of abdominal fat, muscle and blood were taken for determination of [ 14C]HCB content.
A naly tical procedures Radioactivity in urine was determined by the addition of 0.2--0.5 ml of the urine sample to a dioxane scintillator (Bray's solution) and counting in a Packard Tri-Carb liquid scintillation counter. Faeces, tissue and f o o d samples were combusted in a Packard Tri-Carb B 306 sample oxidizer. Samples of urine, faeces and tissues were analyzed in duplicate and an average taken for further calculations. The radioactivity in the HCB-fortified diet was calculated from 8 determinations. Erythrocytes and leukocytes were determined by a Coulter counter. Data evaluation Tissue concentrations of [ 14C]HCB are expressed as HCB-equivalents. The b o d y load of HCB at the beginning of the pretreatment period was calculated from the difference between the amount of [14C]HCB ingested during the accumulation phase and the amount excreted during this period [6]. Body load during the study period was calculated by subtracting the cumulative amount excreted. For the calculation of elimination half-life times mono- or biexponential equations were fitted to the data b y means of non-linear regression. For this purpose a published programme was adapted [ 13]. The choice of the proper equation (mono-vs. biexponential) was based on the plots of the residuals and the F-ratio test according to Boxenbaum et al. [14]. The goodness of fit was assessed by the coefficient of determination (r 2) which was > 0 . 9 9 in all b u t one animal. Mean values are given with S.E.M. Differences between group means were assessed by one way analysis of variance and the 'least significant difference' (LSD)-test [15]. RESULTS Mean initial b o d y wt. was 407 + 6 g. There was a steady increase in b o d y wt. throughout the study period in all b u t one animal (see below). The experimental diets did n o t influence b o d y wt. gain (mean 45.4 + 2.9 g) and food consumption. Likewise there were no significant differences between the study groups with respect to white and red blood cell c o u n t and the weight of liver and kidneys.
338 During the accumulation phase the rats accumulated 120.8 + 3.6/~g HCBequivalents on average. Figure 1 shows the time course o f ['4C]HCB excretion in faeces and urine. During the pretreatment phase the groups did not differ from each other. Faecal excretion accounted mainly for [14C]HCB excretion, the amount of radioactivity found in urine being only some 13% of the total amount excreted (Table I). Dietary treatment with either paraffin, squalane or SPE markedly enhanced faecal excretion. During the treatment phase, both the time course and extent of faecal [ 14C]HCB elimination were similar in the treated groups. As compared to controls the elimination rate of [~4C]HCB was significantly ( P < 0.001) increased from the first day and reached its maximum at day 3. It thereafter decreased slightly, stabilising within 1 week at a level 3 times the control value (Fig. 2). This led to a significant reduction of HCB body burden with respect to controls as early as 4 days after the beginning of treatment. The total amount of ['4C]HCB
40
o
Squalane
a
Control
~ ~.
-~
~
30 Feces
.= 20
illjllilillll=r '
10 Urine
0 ~.
5 pre-
treatment
10 I
15 Day
20 treatment
25
30 I
Fig. 1. C u m u l a t i v e e x c r e t i o n o f [14C]HCB in u r i n e a n d faeces o f rats as p e r c e n t a g e o f t h e t o t a l b o d y load a f t e r t h e a c c u m u l a t i o n phase. S h a d e d areas r e p r e s e n t 95% c o n f i d e n c e limits. F o r u r i n e d a t a t h e w i d t h o f c o n f i d e n c e interval was b e l o w 2% t h r o u g h o u t . V i r t u a l l y identical curves as for t h e s q u a l a n e - t r e a t e d animals were o b t a i n e d in t h e paraffin- a n d S P E - t r e a t e d groups.
EXCRETION
OF ["C]HCB
1 . 5 6 -+ 0 . 3 3 1 1 . 4 9 -+ 1 . 0 3 13.05 ± 1.35
Total
8.18 + 0.53
Total
Urine Faeces
1.13 + 0.19 7 . 0 5 -+ 0 . 4 4
Urine Faeces
34.59 ± 1.65 c
1.19 + 0.12 33.40 ± 1.66 c
7.76 + 0.51
0.97 + 0.13 6.79 ± 0.45
Paraffin
34.16 ± 2.73 c
1.03 + 0.11 33.13 + 2.62 c
8.44 ± 1.12
1.10 ± 0.15 7.35 ± 0.98
Squalane
a All groups fed control diet. b Three weeks on control diet or diets supplemented with 8% paraffin, squalane or SPE, respectively. c S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l v a l u e (P ~ 0 . 0 0 1 ) .
Treatment phase b
Pretreatment phase a
Control
Treatment group
C u m u l a t i v e e x c r e t i o n is e x p r e s s e d a s p e r c e n t a g e o f t h e r a d i o a c t i v i t y s t o r e d in t h e b o d y d u r i n g t h e a c c u m u l a t i o n
CUMULATIVE
TABLE I
32.73 ± 1.90 c
0 . 9 6 -+ 0 . 0 9 31.76 ± 1.82 c
7.78 ± 0.68
0 . 9 7 -+ 0 . 1 0 6.81 ± 0.58
SPE
phase.
¢JD
340 3-
~.
o
SPE
o
Control
2tl
c
.o rE
1-
I l l l I l ~ l
0
5
.... 10
I .... 15
I
20
'
'
'
'
I
25
'
'
'
'
I
30
Day __
pretreatment
treatment
i
Fig. 2. Time course of [ ~4C]HCB elimination rate in rats. Elimination rate (total amount of [~4C]HCB excreted per day) is given as percentage of the amount of HCB present in body. Shaded areas represent 95% confidence limits. Elimination rate in paraffin- and squalane-treated rats did not differ significantly from SPE-treated animals.
excreted with faeces during the treatment period was a b o u t three times higher in treated animals than in controls (Table I). On the other hand urinary excretion of [14C]HCB was n o t affected by the different regimens (Fig. 1; Table I). One animal of the control group lost 67 g of b o d y wt. during the first half of the experiment. It was completely anorectic during days 3--6 of the pretreatment phase. Thereafter it recovered slowly and regained 26 g. At necroscopy the animal had only small amounts of adipose tissue b u t no other visible alterations. The white blood cell count was elevated 3-fold as compared to the six other control rats (30 000 vs. 9600 + 1400 cells/mm3). Interestingly this animal excreted more radioactivity than the other controls (total cumulative excretion 28.2 vs. 20.1 + 1.3%). During the period of weight loss in this rat both faecal and urinary excretion rate of [14C]HCB was markedly increased. Table II gives the tln for the decrease of the amount of [14C]HCB in the body. Semilogarithmic plots of total b o d y load of HCB vs. time were linear (r2> 0.99) during the pretreatment period, yielding a t~n in the order of 90 days. As reported previously [7] the b o d y load of HCB in the rat decreases biexponentially. Thus these figures u n d o u b t e d l y are biased towards
341 T A B L E II H A L F - L I F E T I M E S ( D A Y S ) O F [ ~4C]HCB E L I M I N A T I O N F R O M R A T S Treatment group
Pretreatment phase a Treatment phase b
Control
Paraffin
Squalane
SPE
84.5 ± 6.1 108.3 .-+ 9.6
98.3 -+ 7.2 34.2 + 1.7 c
92.5 2 11.4 37.5 ± 3.4 c
103.1 ± 10.5 35.3 ± 2.4 c
a B o d y l o a d ( A ) was f i t t e d b y a single e x p o n e n t i a l A = A , • e-Tt; t i n = In 2/7. T h e r e w e r e n o significant d i f f e r e n c e s d u r i n g this period. b During this p h a s e a b i e x p o n e n t i a l e q u a t i o n of t h e t y p e A = A I . e - a t + A~" e -~t p r o v i d e d a significant (P < 0.001 ) b e t t e r fit t h a n a single e x p o n e n t i a l in all b u t 3 a n i m a l s (1 squalane-, 2 S P E - t r e a t e d ) . H o w e v e r t h e s h o r t lived a - p h a s e ( m e a n t~n = 3 d a y s ) cont r i b u t e d o n l y slightly t o t h e t o t a l e l i m i n a t i o n , t h e r a t i o A~/A: b e i n g 0.07 o n t h e average. T h e r e f o r e t ~ 2 o f t h e p-phase o n l y is given. c Significantly d i f f e r e n t f r o m c o n t r o l g r o u p (P < 0.001).
lower values due to the short observation period of 9 days. Accordingly, in the control group tin increased to a b o u t 110 days during the 3-week treatment period, which is comparable to values reported previously [7]. On the other hand in the treated groups t,n was considerably shortened to about one third of the control group (Table II). The distribution of radioactivity in some major tissues at the end of the study is shown in Table III. This data provides evidence of the markedly reduced b o d y load of HCB following treatment. Obviously paraffin, squalane and SPE are equally effective in reducing the HCB content of tissues. DISCUSSION
In the present experiment HCB was again used as a model substance for several reasons. It is a pollutant of global occurrence [16], produces besides
T A B L E III D I S T R I B U T I O N O F [ ' 4 ] H C B IN R A T T I S S U E S Values are given as HCB e q u i v a l e n t s in tissues ( 1 0 -9 g • g - ' , w e t w t . ) at t h e e n d o f t h e t r e a t m e n t period.
Tissue
AbdominMfat Liver Kidney Muscle Blood
Treatment group Control
Paraffin
Squalane
SPE
222.1±9.2 59.1±8.1 38.5~5.1 8.3±1.3 4.9±1.3
114.6±5.2 33.3±1.4 25.221.4 2.720.3 2.9±0.2
95:1±6.1 32.1±2.7 21.0±1.6 2.6±0.3 2.2±0.2
103.3±7.2 43.2±4.4 24.4±1.8 2.7±0.4 2.2±0.1
342 liver cell tumours in various species [17--19] a variety of untoward effects [20] and has been implicated in the mass poisonings of humans [21]. As mentioned in the outset, paraffin has proved effectively to remove HCB from contaminated animals. These favourable results have been confirmed by the present study and in addition it was clearly demonstrated that squalane and SPE are equally effective as paraffin. This applies to both the time course and the extent of HCB-removal from the body. Thus both substances are possible alternatives to paraffin. This may have some bearing with regard to possible applications in man. Squalane is a commercially available liquid hydrocarbon which is widely used for incorporation into cosmetics and no untoward effects have been reported after application to skin [22]. Furthermore it is not absorbed orally by rats [23]. Likewise orally administered SPE is almost completely recovered in the stools [24]. At present the mechanism by which these substances increase HCBelimination is not well understood. Two basic mechanisms are conceivable. According to the c o m m o n equation
tin = 0 . 6 9 3 . Vd/C1, which relates elimination tin, volume of distribution (Vd) and clearance (C1), the observed decrease in t,n may be due to redistribution, i.e. a decrease in Vd, or due to increased elimination capacity, or both. Actually, there are several studies where an enhanced elimination of organochlorine residues was obviously due to a decrease in Vd. F o o d deprivation and hence loss of b o d y wt. leads to enhanced faecal HCB excretion in rats b u t also to increased concentrations of HCB in livers and brain and potentiated liver toxicity [25]. In rats treated with /~-['4C]hexachlorocyclohexane a negative correlation between b o d y wt. and urinary excretion was observed [6]. However, a change in Vd seems unlikely to be involved in our present experiments. Firstly it is difficult to imagine h o w a substance like squalane which is essentially n o t absorbed could influence Vd of HCB. Furthermore a decrease in Vd would lead to an increase in both renal and intestinal excretion. This was clearly demonstrated in one of our control animals, where b o d y fat and hence Vd of HCB was markedly reduced. The minimal degree of absorption of the c o m p o u n d s used in the present study and their rapid onset of action render enzyme induction as a major underlying mechanism improbable. Furthermore Villeneuve et al. [26] found a poor correlation only between induction of liver enzyme activity by phenobarbital and increased rate of disappearance of HCB residues in livers of rats. In HCB cont~-ninated steers feeding of phenobarbital at 2 g/100 kg b o d y wt. for 6 months did n o t have a n y e f f e c t u p o n HCB residues in adipose tissue [27]. Since biliary excretion of HCB is negligible [8--10], an interference with enterohepatic circulation of HCB does not seem probable. On the other hand, paraffin and squalane enhance the elimination of unchanged HCB in isolated peffused intestinal loops [28]. Thus the most likely mechanism
343 by which paraffin, squalane and SPE increase HCB elimination appears t o be increased intestinal elimination. Due to its rather limited solubility in aqueous solution, minute amounts of HCB suffice to saturate the aqueous phase of the gut content, thus abolishing any concentration gradient between blood and gut lumen. Most probably paraffin, squalane and SPE act as a 'sink' for the highly lipophilic HCB, maintaining a concentration gradient between blood and the aqueous phase of gut lumen. Nevertheless this explanation is speculative at present. Clearly the proposed hypothesis has to be challenged further by experiment. ACKNOWLEDGEMENT
The expert technical assistance of Mr. E. Wnendt is gratefully acknowledged. REFERENCES 1 J. Alary, P. Guay and J. Brodeur, Effect of phenobarbital pretreatment on the metabolism of DDT in the rat and the bovine, Toxicol. Appl. Pharmacol., 18 (1971) 457. 2 P.W. Ferguson, C.R. Clark, S.J. Gee and R.I. Krieger, Phenobarbital treatments lower DDT b o d y burden in rhesus monkeys, Arch. Environ. Contam. Toxicol., 10 (1981) 263. 3 H.B. Matthews and S. Kato, The metabolism and disposition of halogenated aromatics, Ann. N.Y. Acad. Sci., 320 (1979) 131. 4 G. Koss, S. Seubert, A. Seubert, W. Koransky, P. Kraus and H. Ippen, Conversion products of hexachlorobenzene and their role in the disturbance of the porphyrin pathway in rats, Int. J. Biochem., 12 (1980) 1003. 5 E. Richter, J.P. Lay, W. Klein and F. Korte, Paraffin-stimulated excretion of 2,4,6,2',4'-pentachlorobi(~4C)phenyl by rats, Toxicol. Appl. Pharmacol., 50 (1979) 17. 6 E. Richter, W. Luger, W. Klein, F. Korte and N. Weger, Excretion of ~-hexachlorocyclohexane by the rat as influenced by oral paraffin, squalane, and Lutrol E 400, Ecotoxicol. Environ. Saf., 5 (1981) 270. 7 E. Richter, J.P. Lay, W. Klein and F. Korte, Enhanced elimination of hexachlorobenzene in rats by light liquid paraffin, Chemosphere, 6 (1977) 357. 8 K. Rozman, T. Rozman and H. Greim, The mechanism of intestinal elimination Of hexachlorobenzene and its enhancement by hexadecane in the rat, Toxicol. Lett., Spec. Iss. 1 (1980) 145. 9 E. Richter, K. Rembold, N. Weger and F. Korte, Pharmacokinetics of hexachlorobenzene (HCB) in paraffin-treated beagle dogs with re-entrant bile duct catheter, Naunyn-Schmiedeberg's Arch. Pharmacol., 313, Suppl. (1§80) R57. 10 T. Rozman, K. Rozman and H. Greim, Quantitative determination of intestinal and biliary elimination of hexachlorobenzene in untreated and mineral oil treated rhesus monkeys with complete biliary bypasses, Toxicol. Lett., Spec. Iss. 1 (1980) 145. 11 E. Fingl, Laxatives and cathartics, in: A. Goodman Gilman, L.S. Goodman and A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, Macmillan Publishing Co., New York, 1980, pp. 1002--1012. 12 S. Monarca, F. Faglioli and G. Morozzi, Evaluation of the potential carcinogenicity of paraffins for medicinal and cosmetic uses -- Determination of polycyclic aromatic hydrocarbons, Sci. Total Environ., 17 (1981) 83.
344 13 M. Pfeffer, COMPT, a time-sharing program for nonlinear regression analysis of compartmental models of drug distribution, J. Pharmacokin. Biopharmaceut., 1 (1973) 137. 14 H.G. Boxenbaum, S. Riegelman and R.M. Elashoff, Statistical estimations in pharmacokinetics, J. Pharmacokin. Biopharmaceut., 2 (1974) 123. 15 L. Sachs, Angewandte Statistik, Springer, Berlin, Heidelberg, New York, 1978, p. 394. 16 M. Zell and K. Ballschmiter, Baseline studies of the global pollution, II. Global occurrence of hexachlorobenzene (HCB) and polychlorocamphenes (Toxaphene R) (PCC) in biological samples, Fresenius Z. Anal. Chem., 300 (1980) 387. 17 A.G. Smith and J.R. Cabral, Liver-cell tumours in rats fed hexachlorobenzene, Cancer Lett., 11 (1980) 169. 18 J.R.P. Cabral, P. Shubik, T. Mollner and F. Raitano, Carcinogenic activity of hexachlorobenzene in hamsters, Nature, 269 (1977) 510. 19 J.R.P. Cabral, T. Mollner, F. Raitano and R. Shubik, Carcinogenesis of hexachlorobenzene in mice, Int. J. Cancer, 23 (1979) 47. 20 K.D. Courtney, Hexachlorobenzene (HCB): A review, Environ. Res., 20 (1979) 225. 21 D.J. Cripps, A. Gocmen and H.A. Peters, Porphyria t u r c i c a - 20 years hexachlorobenzene intoxication, Arch. Dermatol., 116 (1980) 46. 22 T.R. Davis, Polysynlane: a novel synthetic substitute for squalane, Cosmet. Toilet., 91 (1976) 33. 23 P.W. Albro and L. Fishbein, Absorption of aliphatic hydrocarbons by rats, Biochim. Biophys. Acta, 219 (1970) 437. 24 R.W. Fallat, C.J. Glueck, R. Lutmer and F.H. Mattson, Short term study of sucrose polyester a nonabsorbable fat-like material as a dietary agent for lowering plasma cholesterol, Am. J. Clin. Nutr., 29 (1976) 1204. 25 D.C. Villeneuve, The effect of food restriction on the redistribution of hexachlorobenzene in the rat, Toxicol. Appl. Pharmacol., 31 (1975) 313. 26 D.C. Villeneuve, W.E. Phillips, L.G. Panopio, C.E. Mendoza, G.V. Hatina and D.L. Grant, The effects of phenobarbital and carbon tetrachloride on the rate of decline of body burdens of HCB in the rat, Arch. Environ. Contain. Toxicol., 2 (1974) 243. 27 F.G. Hembry, L.I. Smart and J.M. Dixon, Removal of HCB contamination from beef cattle, Louisiana Agric., 19 (1976) 6. 28 E. Richter and S.G. Sch~/fer, Intestinal excretion of hexachlorobenzene, Arch. Toxicol., 47 (1981) 233.
335
Elsevier/North-Holland Scientific Publishers Ltd.
EFFECTS OF DIETARY P A R A F F I N , SQUALANE AND SUCROSE POLYESTER ON RESIDUE DISPOSITION AND ELIMINATION OF HEXACHLOROBENZENE IN RATS
E. RICHTER, B. FICHTL and S.G. SCHAFER Institut ffir Pharmakologie und Toxikologie der Ludwig-Maximilians-Universit~it Milnchen, Nussbaumstrasse 26, D-8000 Miinchen 2 (F.R.G.)
(Received December 24th, 1982) (Accepted January 16th, 1982) SUMMARY Previous studies have shown addition of light liquid paraffin to enhance the elimination of organochlorine xenobiotics. In the present study the effect of paraffin on the elimination of [14C]hexachlorobenzene (HCB) was compared with the effect of possible alternative compounds, squalane and sucrose polyester (SPE). Four groups of 7 rats were fed a diet containing 1.5 ppm [14C]HCB for 4 days followed by 10 days on HCB-free diet. Thereafter one group (control) remained on this diet whereas the other 3 groups received a diet supplemented with 8% (w/w) paraffin, squalane or SPE, respectively. Radioactivity in urine and faeces was measured daily and at the end of the experiment in samples of abdominal fat, muscle, liver, kidney and blood. Dietary treatment with either paraffin, squalane or SPE markedly enhanced faecal excretion of [14C]HCB, whereas urinary excretion was not affected. Both the time course as well as the extent of faecal [ 14C]HCB elimination were similar in the treated groups. After 3 weeks of treatment the a m o u n t of [ ~4C]HCB excreted with faeces was about three times higher in treated animals than in controls. The half-life (tin) of [~4C]HCB elimination from the body was markedly decreased in treated animals {mean 34--38 days) compared to controls (110 days). [14C]HCB concentrations in some major tissues were significantly reduced to the same extent by all three dietary regimens. Thus squalane and SPE are as effective as paraffin in removing HCB from contaminated animals.
INTRODUCTION The persistence of organochlorine xenobiotic residues in human organs Abbreviations: GLC, gas liquid chromatography; HCB, hexachlorobenzene; LSD, least significant difference; SPE, sucrose polyester; TLC, thin-layer chromatography. 0009--2797/82/0000---0000/$02.75 © 1982 Elsevier/North-Holland Scientific Publishers Ltd.
336 and in animal tissues intended for human food consumption raises the problem of effecting their removal from the body. The elimination of those residues may be hastened by liver enzyme induction [1,2]. This al~proach, however, is not necessarily of benefit. The metabolic degradation of some of those c o m p o u n d s most probably involves arene oxide intermediates, which have been implicated with toxicity and/or carcinogenicity [3]. Indeed, Koss et al. [4] have shown that metabolism of HCB is a prerequisite for its porphyrinogenic action. Therefore it appears to be much more desirable to accelerate the elimination of the unchanged c o m p o u n d instead of stimulating the formation of possible hazardous metabolites. Feeding light liquid paraffin or mineral oil has proved to be a promising approach to this goal. It has been reported that this procedure markedly enhances the elimination of 2,4,6,2',4'-pentachlorobiphenyl [5] and ~-hexachlorocyclohexane [6] in rats and the elimination of HCB in rats [7,8], beagle dogs [9] and rhesus monkeys [10]. Paraffin or mineral oil, however, is partially absorbed from the intestinal tract and may cause a variety of untoward effects [11]. Furthermore it has been claimed that paraffins may contain variable amounts of carcinogenic impurities [12]. It seemed necessary, therefore, to look for alternative compounds. In the present study the influence of light liquid paraffin, squalane and SPE on the elimination of HCB in rats was compared. MATERIALS AND METHODS
Animals The experiments were performed using male Sprague--Dawley rats of a b o u t 400 g b o d y wt. (breeder: Versuchstierzucht WIGA, Sulzfeld, F.R.G.). The animals were housed individually in stainless steel metabolism cages in an animal room {temperature 21°C; humidity 55%; 12 h light per day). The rats had free access to food and drinking water. A finely ground diet (Altromin 1321, obtained from Altrogge, Lage, F.R.G.) was used. Chemicals [~4C]HCB (spec. act. 23.73 mCi/mmol) was purchased from the New England Nuclear Co. The radiochemical purity was > 9 9 % as assessed by thin-layer chromatography (TLC). [ 14C]HCB was diluted with the unlabelled c o m p o u n d (purity > 9 9 % by GLC) to a specific activity of 13.4 mCi/mmol. It was added to the diet at 1.5 ppm. Light liquid paraffin (DAB 7) was obtained from Merck (Darmstadt, F.R.G.). Squalane (Prosynth ® ) was purchased from Riedel~le Ha6n (Seelze, F.R.G.). SPE was donated by Procter and Gamble (Cincinnati, OH). All other chemicals were of analytical grade. Study protocol The rats were subdivided into 4 groups of 7 rats and adapted to the metabolism cages for 10 days. The animals were then fed the HCB-forti-
337 fied diet for 4 days. This period and the first day after withdrawal from the HCB-containing diet is referred to as the accumulation phase. This was followed by another 9 days on HCB-free diet (pretreatment phase). During the following 3 weeks (treatment phase) the control group remained on this diet whereas the other 3 groups received the diets supplemented with 8% (w/w) paraffin, squalane and SPE, respectively. F o o d consumption was measured daily and b o d y weights were recorded weekly. Urine and faeces were collected daily. After 3 weeks on the experimental diets the animals were killed under ether anaesthesia by bleeding from the aorta abdominalis. The weight of liver and kidneys was recorded and samples of these organs and of abdominal fat, muscle and blood were taken for determination of [ 14C]HCB content.
A naly tical procedures Radioactivity in urine was determined by the addition of 0.2--0.5 ml of the urine sample to a dioxane scintillator (Bray's solution) and counting in a Packard Tri-Carb liquid scintillation counter. Faeces, tissue and f o o d samples were combusted in a Packard Tri-Carb B 306 sample oxidizer. Samples of urine, faeces and tissues were analyzed in duplicate and an average taken for further calculations. The radioactivity in the HCB-fortified diet was calculated from 8 determinations. Erythrocytes and leukocytes were determined by a Coulter counter. Data evaluation Tissue concentrations of [ 14C]HCB are expressed as HCB-equivalents. The b o d y load of HCB at the beginning of the pretreatment period was calculated from the difference between the amount of [14C]HCB ingested during the accumulation phase and the amount excreted during this period [6]. Body load during the study period was calculated by subtracting the cumulative amount excreted. For the calculation of elimination half-life times mono- or biexponential equations were fitted to the data b y means of non-linear regression. For this purpose a published programme was adapted [ 13]. The choice of the proper equation (mono-vs. biexponential) was based on the plots of the residuals and the F-ratio test according to Boxenbaum et al. [14]. The goodness of fit was assessed by the coefficient of determination (r 2) which was > 0 . 9 9 in all b u t one animal. Mean values are given with S.E.M. Differences between group means were assessed by one way analysis of variance and the 'least significant difference' (LSD)-test [15]. RESULTS Mean initial b o d y wt. was 407 + 6 g. There was a steady increase in b o d y wt. throughout the study period in all b u t one animal (see below). The experimental diets did n o t influence b o d y wt. gain (mean 45.4 + 2.9 g) and food consumption. Likewise there were no significant differences between the study groups with respect to white and red blood cell c o u n t and the weight of liver and kidneys.
338 During the accumulation phase the rats accumulated 120.8 + 3.6/~g HCBequivalents on average. Figure 1 shows the time course o f ['4C]HCB excretion in faeces and urine. During the pretreatment phase the groups did not differ from each other. Faecal excretion accounted mainly for [14C]HCB excretion, the amount of radioactivity found in urine being only some 13% of the total amount excreted (Table I). Dietary treatment with either paraffin, squalane or SPE markedly enhanced faecal excretion. During the treatment phase, both the time course and extent of faecal [ 14C]HCB elimination were similar in the treated groups. As compared to controls the elimination rate of [~4C]HCB was significantly ( P < 0.001) increased from the first day and reached its maximum at day 3. It thereafter decreased slightly, stabilising within 1 week at a level 3 times the control value (Fig. 2). This led to a significant reduction of HCB body burden with respect to controls as early as 4 days after the beginning of treatment. The total amount of ['4C]HCB
40
o
Squalane
a
Control
~ ~.
-~
~
30 Feces
.= 20
illjllilillll=r '
10 Urine
0 ~.
5 pre-
treatment
10 I
15 Day
20 treatment
25
30 I
Fig. 1. C u m u l a t i v e e x c r e t i o n o f [14C]HCB in u r i n e a n d faeces o f rats as p e r c e n t a g e o f t h e t o t a l b o d y load a f t e r t h e a c c u m u l a t i o n phase. S h a d e d areas r e p r e s e n t 95% c o n f i d e n c e limits. F o r u r i n e d a t a t h e w i d t h o f c o n f i d e n c e interval was b e l o w 2% t h r o u g h o u t . V i r t u a l l y identical curves as for t h e s q u a l a n e - t r e a t e d animals were o b t a i n e d in t h e paraffin- a n d S P E - t r e a t e d groups.
EXCRETION
OF ["C]HCB
1 . 5 6 -+ 0 . 3 3 1 1 . 4 9 -+ 1 . 0 3 13.05 ± 1.35
Total
8.18 + 0.53
Total
Urine Faeces
1.13 + 0.19 7 . 0 5 -+ 0 . 4 4
Urine Faeces
34.59 ± 1.65 c
1.19 + 0.12 33.40 ± 1.66 c
7.76 + 0.51
0.97 + 0.13 6.79 ± 0.45
Paraffin
34.16 ± 2.73 c
1.03 + 0.11 33.13 + 2.62 c
8.44 ± 1.12
1.10 ± 0.15 7.35 ± 0.98
Squalane
a All groups fed control diet. b Three weeks on control diet or diets supplemented with 8% paraffin, squalane or SPE, respectively. c S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l v a l u e (P ~ 0 . 0 0 1 ) .
Treatment phase b
Pretreatment phase a
Control
Treatment group
C u m u l a t i v e e x c r e t i o n is e x p r e s s e d a s p e r c e n t a g e o f t h e r a d i o a c t i v i t y s t o r e d in t h e b o d y d u r i n g t h e a c c u m u l a t i o n
CUMULATIVE
TABLE I
32.73 ± 1.90 c
0 . 9 6 -+ 0 . 0 9 31.76 ± 1.82 c
7.78 ± 0.68
0 . 9 7 -+ 0 . 1 0 6.81 ± 0.58
SPE
phase.
¢JD
340 3-
~.
o
SPE
o
Control
2tl
c
.o rE
1-
I l l l I l ~ l
0
5
.... 10
I .... 15
I
20
'
'
'
'
I
25
'
'
'
'
I
30
Day __
pretreatment
treatment
i
Fig. 2. Time course of [ ~4C]HCB elimination rate in rats. Elimination rate (total amount of [~4C]HCB excreted per day) is given as percentage of the amount of HCB present in body. Shaded areas represent 95% confidence limits. Elimination rate in paraffin- and squalane-treated rats did not differ significantly from SPE-treated animals.
excreted with faeces during the treatment period was a b o u t three times higher in treated animals than in controls (Table I). On the other hand urinary excretion of [14C]HCB was n o t affected by the different regimens (Fig. 1; Table I). One animal of the control group lost 67 g of b o d y wt. during the first half of the experiment. It was completely anorectic during days 3--6 of the pretreatment phase. Thereafter it recovered slowly and regained 26 g. At necroscopy the animal had only small amounts of adipose tissue b u t no other visible alterations. The white blood cell count was elevated 3-fold as compared to the six other control rats (30 000 vs. 9600 + 1400 cells/mm3). Interestingly this animal excreted more radioactivity than the other controls (total cumulative excretion 28.2 vs. 20.1 + 1.3%). During the period of weight loss in this rat both faecal and urinary excretion rate of [14C]HCB was markedly increased. Table II gives the tln for the decrease of the amount of [14C]HCB in the body. Semilogarithmic plots of total b o d y load of HCB vs. time were linear (r2> 0.99) during the pretreatment period, yielding a t~n in the order of 90 days. As reported previously [7] the b o d y load of HCB in the rat decreases biexponentially. Thus these figures u n d o u b t e d l y are biased towards
341 T A B L E II H A L F - L I F E T I M E S ( D A Y S ) O F [ ~4C]HCB E L I M I N A T I O N F R O M R A T S Treatment group
Pretreatment phase a Treatment phase b
Control
Paraffin
Squalane
SPE
84.5 ± 6.1 108.3 .-+ 9.6
98.3 -+ 7.2 34.2 + 1.7 c
92.5 2 11.4 37.5 ± 3.4 c
103.1 ± 10.5 35.3 ± 2.4 c
a B o d y l o a d ( A ) was f i t t e d b y a single e x p o n e n t i a l A = A , • e-Tt; t i n = In 2/7. T h e r e w e r e n o significant d i f f e r e n c e s d u r i n g this period. b During this p h a s e a b i e x p o n e n t i a l e q u a t i o n of t h e t y p e A = A I . e - a t + A~" e -~t p r o v i d e d a significant (P < 0.001 ) b e t t e r fit t h a n a single e x p o n e n t i a l in all b u t 3 a n i m a l s (1 squalane-, 2 S P E - t r e a t e d ) . H o w e v e r t h e s h o r t lived a - p h a s e ( m e a n t~n = 3 d a y s ) cont r i b u t e d o n l y slightly t o t h e t o t a l e l i m i n a t i o n , t h e r a t i o A~/A: b e i n g 0.07 o n t h e average. T h e r e f o r e t ~ 2 o f t h e p-phase o n l y is given. c Significantly d i f f e r e n t f r o m c o n t r o l g r o u p (P < 0.001).
lower values due to the short observation period of 9 days. Accordingly, in the control group tin increased to a b o u t 110 days during the 3-week treatment period, which is comparable to values reported previously [7]. On the other hand in the treated groups t,n was considerably shortened to about one third of the control group (Table II). The distribution of radioactivity in some major tissues at the end of the study is shown in Table III. This data provides evidence of the markedly reduced b o d y load of HCB following treatment. Obviously paraffin, squalane and SPE are equally effective in reducing the HCB content of tissues. DISCUSSION
In the present experiment HCB was again used as a model substance for several reasons. It is a pollutant of global occurrence [16], produces besides
T A B L E III D I S T R I B U T I O N O F [ ' 4 ] H C B IN R A T T I S S U E S Values are given as HCB e q u i v a l e n t s in tissues ( 1 0 -9 g • g - ' , w e t w t . ) at t h e e n d o f t h e t r e a t m e n t period.
Tissue
AbdominMfat Liver Kidney Muscle Blood
Treatment group Control
Paraffin
Squalane
SPE
222.1±9.2 59.1±8.1 38.5~5.1 8.3±1.3 4.9±1.3
114.6±5.2 33.3±1.4 25.221.4 2.720.3 2.9±0.2
95:1±6.1 32.1±2.7 21.0±1.6 2.6±0.3 2.2±0.2
103.3±7.2 43.2±4.4 24.4±1.8 2.7±0.4 2.2±0.1
342 liver cell tumours in various species [17--19] a variety of untoward effects [20] and has been implicated in the mass poisonings of humans [21]. As mentioned in the outset, paraffin has proved effectively to remove HCB from contaminated animals. These favourable results have been confirmed by the present study and in addition it was clearly demonstrated that squalane and SPE are equally effective as paraffin. This applies to both the time course and the extent of HCB-removal from the body. Thus both substances are possible alternatives to paraffin. This may have some bearing with regard to possible applications in man. Squalane is a commercially available liquid hydrocarbon which is widely used for incorporation into cosmetics and no untoward effects have been reported after application to skin [22]. Furthermore it is not absorbed orally by rats [23]. Likewise orally administered SPE is almost completely recovered in the stools [24]. At present the mechanism by which these substances increase HCBelimination is not well understood. Two basic mechanisms are conceivable. According to the c o m m o n equation
tin = 0 . 6 9 3 . Vd/C1, which relates elimination tin, volume of distribution (Vd) and clearance (C1), the observed decrease in t,n may be due to redistribution, i.e. a decrease in Vd, or due to increased elimination capacity, or both. Actually, there are several studies where an enhanced elimination of organochlorine residues was obviously due to a decrease in Vd. F o o d deprivation and hence loss of b o d y wt. leads to enhanced faecal HCB excretion in rats b u t also to increased concentrations of HCB in livers and brain and potentiated liver toxicity [25]. In rats treated with /~-['4C]hexachlorocyclohexane a negative correlation between b o d y wt. and urinary excretion was observed [6]. However, a change in Vd seems unlikely to be involved in our present experiments. Firstly it is difficult to imagine h o w a substance like squalane which is essentially n o t absorbed could influence Vd of HCB. Furthermore a decrease in Vd would lead to an increase in both renal and intestinal excretion. This was clearly demonstrated in one of our control animals, where b o d y fat and hence Vd of HCB was markedly reduced. The minimal degree of absorption of the c o m p o u n d s used in the present study and their rapid onset of action render enzyme induction as a major underlying mechanism improbable. Furthermore Villeneuve et al. [26] found a poor correlation only between induction of liver enzyme activity by phenobarbital and increased rate of disappearance of HCB residues in livers of rats. In HCB cont~-ninated steers feeding of phenobarbital at 2 g/100 kg b o d y wt. for 6 months did n o t have a n y e f f e c t u p o n HCB residues in adipose tissue [27]. Since biliary excretion of HCB is negligible [8--10], an interference with enterohepatic circulation of HCB does not seem probable. On the other hand, paraffin and squalane enhance the elimination of unchanged HCB in isolated peffused intestinal loops [28]. Thus the most likely mechanism
343 by which paraffin, squalane and SPE increase HCB elimination appears t o be increased intestinal elimination. Due to its rather limited solubility in aqueous solution, minute amounts of HCB suffice to saturate the aqueous phase of the gut content, thus abolishing any concentration gradient between blood and gut lumen. Most probably paraffin, squalane and SPE act as a 'sink' for the highly lipophilic HCB, maintaining a concentration gradient between blood and the aqueous phase of gut lumen. Nevertheless this explanation is speculative at present. Clearly the proposed hypothesis has to be challenged further by experiment. ACKNOWLEDGEMENT
The expert technical assistance of Mr. E. Wnendt is gratefully acknowledged. REFERENCES 1 J. Alary, P. Guay and J. Brodeur, Effect of phenobarbital pretreatment on the metabolism of DDT in the rat and the bovine, Toxicol. Appl. Pharmacol., 18 (1971) 457. 2 P.W. Ferguson, C.R. Clark, S.J. Gee and R.I. Krieger, Phenobarbital treatments lower DDT b o d y burden in rhesus monkeys, Arch. Environ. Contam. Toxicol., 10 (1981) 263. 3 H.B. Matthews and S. Kato, The metabolism and disposition of halogenated aromatics, Ann. N.Y. Acad. Sci., 320 (1979) 131. 4 G. Koss, S. Seubert, A. Seubert, W. Koransky, P. Kraus and H. Ippen, Conversion products of hexachlorobenzene and their role in the disturbance of the porphyrin pathway in rats, Int. J. Biochem., 12 (1980) 1003. 5 E. Richter, J.P. Lay, W. Klein and F. Korte, Paraffin-stimulated excretion of 2,4,6,2',4'-pentachlorobi(~4C)phenyl by rats, Toxicol. Appl. Pharmacol., 50 (1979) 17. 6 E. Richter, W. Luger, W. Klein, F. Korte and N. Weger, Excretion of ~-hexachlorocyclohexane by the rat as influenced by oral paraffin, squalane, and Lutrol E 400, Ecotoxicol. Environ. Saf., 5 (1981) 270. 7 E. Richter, J.P. Lay, W. Klein and F. Korte, Enhanced elimination of hexachlorobenzene in rats by light liquid paraffin, Chemosphere, 6 (1977) 357. 8 K. Rozman, T. Rozman and H. Greim, The mechanism of intestinal elimination Of hexachlorobenzene and its enhancement by hexadecane in the rat, Toxicol. Lett., Spec. Iss. 1 (1980) 145. 9 E. Richter, K. Rembold, N. Weger and F. Korte, Pharmacokinetics of hexachlorobenzene (HCB) in paraffin-treated beagle dogs with re-entrant bile duct catheter, Naunyn-Schmiedeberg's Arch. Pharmacol., 313, Suppl. (1§80) R57. 10 T. Rozman, K. Rozman and H. Greim, Quantitative determination of intestinal and biliary elimination of hexachlorobenzene in untreated and mineral oil treated rhesus monkeys with complete biliary bypasses, Toxicol. Lett., Spec. Iss. 1 (1980) 145. 11 E. Fingl, Laxatives and cathartics, in: A. Goodman Gilman, L.S. Goodman and A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, Macmillan Publishing Co., New York, 1980, pp. 1002--1012. 12 S. Monarca, F. Faglioli and G. Morozzi, Evaluation of the potential carcinogenicity of paraffins for medicinal and cosmetic uses -- Determination of polycyclic aromatic hydrocarbons, Sci. Total Environ., 17 (1981) 83.
344 13 M. Pfeffer, COMPT, a time-sharing program for nonlinear regression analysis of compartmental models of drug distribution, J. Pharmacokin. Biopharmaceut., 1 (1973) 137. 14 H.G. Boxenbaum, S. Riegelman and R.M. Elashoff, Statistical estimations in pharmacokinetics, J. Pharmacokin. Biopharmaceut., 2 (1974) 123. 15 L. Sachs, Angewandte Statistik, Springer, Berlin, Heidelberg, New York, 1978, p. 394. 16 M. Zell and K. Ballschmiter, Baseline studies of the global pollution, II. Global occurrence of hexachlorobenzene (HCB) and polychlorocamphenes (Toxaphene R) (PCC) in biological samples, Fresenius Z. Anal. Chem., 300 (1980) 387. 17 A.G. Smith and J.R. Cabral, Liver-cell tumours in rats fed hexachlorobenzene, Cancer Lett., 11 (1980) 169. 18 J.R.P. Cabral, P. Shubik, T. Mollner and F. Raitano, Carcinogenic activity of hexachlorobenzene in hamsters, Nature, 269 (1977) 510. 19 J.R.P. Cabral, T. Mollner, F. Raitano and R. Shubik, Carcinogenesis of hexachlorobenzene in mice, Int. J. Cancer, 23 (1979) 47. 20 K.D. Courtney, Hexachlorobenzene (HCB): A review, Environ. Res., 20 (1979) 225. 21 D.J. Cripps, A. Gocmen and H.A. Peters, Porphyria t u r c i c a - 20 years hexachlorobenzene intoxication, Arch. Dermatol., 116 (1980) 46. 22 T.R. Davis, Polysynlane: a novel synthetic substitute for squalane, Cosmet. Toilet., 91 (1976) 33. 23 P.W. Albro and L. Fishbein, Absorption of aliphatic hydrocarbons by rats, Biochim. Biophys. Acta, 219 (1970) 437. 24 R.W. Fallat, C.J. Glueck, R. Lutmer and F.H. Mattson, Short term study of sucrose polyester a nonabsorbable fat-like material as a dietary agent for lowering plasma cholesterol, Am. J. Clin. Nutr., 29 (1976) 1204. 25 D.C. Villeneuve, The effect of food restriction on the redistribution of hexachlorobenzene in the rat, Toxicol. Appl. Pharmacol., 31 (1975) 313. 26 D.C. Villeneuve, W.E. Phillips, L.G. Panopio, C.E. Mendoza, G.V. Hatina and D.L. Grant, The effects of phenobarbital and carbon tetrachloride on the rate of decline of body burdens of HCB in the rat, Arch. Environ. Contain. Toxicol., 2 (1974) 243. 27 F.G. Hembry, L.I. Smart and J.M. Dixon, Removal of HCB contamination from beef cattle, Louisiana Agric., 19 (1976) 6. 28 E. Richter and S.G. Sch~/fer, Intestinal excretion of hexachlorobenzene, Arch. Toxicol., 47 (1981) 233.