Toxicology, 5 (1976) 351--358 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands DISTRIBUTION OF AFLATOXIN B1 IN TISSUES OF MINK (MUSTELA VISON)

CHENG-CHUN CHOU and ELMER H. MARTH Department of Food Science and The Food Research Institute, University of Wisconsin-Madison, Madison, Wis. 53706 (U.S.A.) (Received August 5th, 1975) (Revision received October 27th, 1975 ) (Accepted November 1st, 1975)

SUMMARY Seven female mink (Mustela vison) were injected intraperitoneally with a single dose of 100 pg aflatoxin B, ('4C-labeled and unlabeled). They were sacrificed 1, 2, 4, and 24 h after dosing. Liver, intestines, stomach, lung, kidney, brain, pancreas, spleen, urinary bladder, uterus, and bile were removed and examined for the retained radioactivity. 1 h after dosing, intestines and their contents retained the largest a m o u n t of '4C-radioactivity (18.9% of the a m o u n t that was administered) which was followed by liver (13.2%) and bile (10.8%). At this time all other tissues retained less than 1% of the administered radioactivity. Generally, the a m o u n t of radioactivity retained in all tissues declined with time. Only 1.2 and 0.6% of the administered radioactivity was found in intestines and bile, respectively, 24 h after dosing; however, the liver still contained 6.6% of the initial radioactivity. Examination of subcellular fractions of liver revealed that at all time intervals most of the radioactivity was associated with the microsomal supernatant fluid.

INTRODUCTION Aflatoxins are metabolites of some strains of Aspergillus flavus and Aspergillus parasiticus, and are toxic to many species of animals. Toxicity of aflatoxins varies with the species of animal. The duckling is most susceptible to effects of aflatoxin and the LDso value for the toxin when administered orally (single dose) to this animal is 0.34--=0.56 mg of aflatoxin B~/kg of b o d y weight. LDs0 values for aflatoxin when administered to the rat and hamster are 7.2 and 10.2 mg of B~/kg of body weight, respectively [1]. We found that mink are rather susceptible to the effects of aflatoxin and esti-

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mated the LDs0 of aflatoxins for this animal to be about 0.5--0.6 mg/kg of body weight [ 2 ]. Studies have been done to determine the fate of aflatoxin in some animal species. Radioactive toxin frequently has been used in such work and thus some quantitative information about the fate of aflatoxin in certain species of animals has become available [3--6]. Lijinsky et al. [7] used 3H-labeled toxins to study the interaction between aflatoxins B, and G, and rat tissues. ['4C] Aflatoxin was used by Wogan et al. [6], Dalezios and Wogan [3], and Mabee and Chipley [4,5] to study the fate of aflatoxin in the rat, m o n k e y , and chicken, respectively. Since the adult mink is rather sensitive to aflatoxin [2], we believed that studies on the fate of aflatoxin in this animal were needed to provide further insights on how this toxin affects animals. Data were lacking on how rapidly aflatoxin gets into and is cleared from tissues of mink and how it is distributed among the tissues of the animal after a single intraperitoneal injection of ['4C] aflatoxin B,. Experiments were done to obtain this information and results are reported in this paper. MATERIALS AND METHODS

Preparation and assay of aflatoxin B, and ['4C] aflatoxin B, Aspergillus parasiticus N R R L 2999 was grown in YES broth (20% sucrose and 2% yeast extract) [8], and aflatoxin was extracted from the broth with chloroform as described earlier [2]. Aflatoxin B, was then isolated and purified by applying the concentrated chloroform extract to a column (2.2 × 50 cm) containing Adsorbosil-5 (60 g) [9] (Applied Science Laboratories, Inc., State College, Pa., U.S.A.) mixed with 10 g celite (Sargent-Welch Scientific Company, Skokie, Ill., U.S.A.). The mixture was used to increase the porosity of the material when packed in the column. The column was eluted with about 500 ml benzene, followed by 500 ml chloroform:benzene (1:1 v/v). Fraction of eluate (10 ml/tube) were collected by means of a fraction collector (LKB-Produkter AB, Stockholm-Bromma 1, Sweden). ['4C]Aflatoxin B, was prepared with A. parasiticus N R R L 2999 essentially as outlined by Hsieh and Mateles [10], and using the medium of Adye and Mateles [11]. After the initial incubation in this procedure was completed, pellets of mycelium were collected by filtering the medium through sterile cheese cloth. 1 g of mycelium pellets was inoculated into each of several flasks containing 0.5 g glucose, 10 ml of replacement medium as described by Hsieh and Mateles [10], and 1 mCi of [1-'4C] sodium acetate (specific activity 57-58 pCi/pmole, Amersham/Searle, Arlington Heights, Ill., U.S.A.) plus unlabeled sodium acetate to give a final concentration Of 5.0 mM sodium acetate in the flask. Flasks were then incubated on the shaker at 200 strokes/ min for 15 h. After incubation, cultures were filtered and broth was extracted four times with equal amounts of chloroform (about 10 ml) in a separatory funnel. ['4C] Aflatoxin B, was then isolated and purified by two thinlayer chromatography (TLC) systems as described by Hsieh and Mateles

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[10]. The specific activity of ['4C]aflatoxin B, (purity 90--93%)from various preparations was in the range of 74--95 pCi/pmole. Aflatoxin B, was assayed quantitatively according to the method of Shih and Marth [12].

Experimental design Seven female mink (Mustela vison) that weighed 470--650 g were used in this experiment. They were injected intraperitoneally with [~4C] aflatoxin B~ and unlabeled aflatoxin BI, both dissolved in dimethylsulfoxide. Each mink received a total dose of 100 pg of aflatoxin B~/kg of body weight and 2.3--3.1 • 1 0 6 dpm of ~4C. Two mink were sacrificed 1, 4, and 24 h after dosing. One animal was sacrificed 2 h after injection. Organs and tissues were removed and examined for radioactivity.

Measurement of radioactivity Organs and tissues were first cut into small pieces with a dissecting scissors and mixed thoroughly. 100 to 200 mg of wet tissue were weighed into a scintillation counting vial. Then 2 ml of tissue solubilizer-Protosol (New England Nuclear, Boston, Mass.) were added and the mixture was incubated in a water bath at 55°C for 20 h. After cooling, the vial received 15 ml of scintillation solution [naphthalene 259.2 g, 2,5-diphenyloxazole 18.4 g, 2(1-naphthyl)-5-phenyloxazole 0.1839 g, xylene 1400 ml, dioxane 1400 ml, and ethanol 840 ml] and was counted in a Nuclear-Chicago Mark II Liquid Scintillation System (Nuclear-Chicago., Des Plaines, Ill., U.S.A.). To prepare subcellular fractions, liver was first homogenized in a 0.25 M sucrose solution. Then fractions were collected by successive centrifugation at 6 • 103 grain (nuclei and cell debris), 3.3 • 104 g-min (mitochondria), and 3 • 106 g-min (microsomes) [13]. The pellet of each fraction was resuspended in 0.25 M sucrose solution, recovered by centrifugation, resuspended again in the sucrose solution, and then recovered again by centrifugation. Measurement of radioactivity was done as just described. RESULTS Table I gives data on distribution of ~4C-radioactivity from aflatoxin in tissues of mink at intervals up to 24 h following administration of a single intraperitoneal dose of [~ac] aflatoxin BI. Data are given as the percentage of administered radioactivity that appeared in the entire organ. Since the size of organs varied considerably, data on distribution of ~4C also are expressed as dpm/100 mg wet tissue so that the concentration of radioactivity per unit of tissue also is apparent. Afiatoxin BI was rapidly translocated to some tisst~es. 1 h after administration of toxin all tissues that were examined contained some ~4C-radioactivity. Intestines and their contents contained the largest a m o u n t of any organs; the specific activity for 100 pg of liver was the same as that for an equivalent a m o u n t of intenstine and contents (1122 vs. 1121). A large

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TABLE I RADIOACTIVITY FROM [14 C ] A F L A T O X I N B 1 IN TISSUES OF MINK a Tissue

Percent of dose in entire organ

Dpm/100 mg wet tissue

lh Liver 13.2 Intestines and 18.9 contents Stomach and 0.6 contents Bile 10.8 Lung 0.6 Heart 0.4 Kidney 0.7 Brain 0.1 Pancreas 0.1 Spleen 0.1 Urinary bladder 0.1 Uterus G0.1 Total

45.6

2h

4h

24h

lh

2h

4h

24h

7.7 9.9

8.8 3.3

6.6 1.2

1122 1121

1028 985

1285 249

648 136

1.3

0.5

0.1

99

577

156

41

3.5 0.3 0.3 1.0 0.1 0.1 0.1 0.1 G0.1

1.4 0.3 0.5 0.3 0.1 0.1 0.1 ~0.1 G0.1

0.6 0.2 0.1 0.2 ~0.1 G0.1 ~0.1 G0.1 G0.1

188 182 327 51 231 96 223 152

128 102 613 32 128 82 389 102

136 220 178 27 71 88 61 57

103 48 112 9 42 34 33 25

24.4

15.4

8.9

.

.

.

.

a Values for 1,4, and 24 h are averages of data obtained from tests on two animals; values for 2 h were obtained from a single animal.

a m o u n t o f r a d i o a c t i v i t y , a p p r o x . 1 0 % o f t h e i n i t i a l d o s e , a p p e a r e d in b i l e t h a t w a s r e m o v e d f r o m t h e gall b l a d d e r . A t t h i s t i m e all o t h e r t i s s u e s c o n t a i n e d less t h a n 1% o f t h e r a d i o a c t i v i t y p r e s e n t in t h e i n i t i a l d o s e w i t h t h e u t e r u s retaining the least amount of radioactivity. 2 h l a t e r , t h e a m o u n t o f r a d i o a c t i v i t y d e c r e a s e d in all t i s s u e s e x c e p t t h e kidney and stomach where the concentration increased. Only 3.5% of the r a d i o a c t i v i t y in t h e i n i t i a l d o s e w a s n o w in b i l e . D e s p i t e a c o n s i d e r a b l e d e c r e a s e in r a d i o a c t i v i t y , i n t e s t i n e s a n d t h e i r c o n t e n t s still r e t a i n e d m o r e r a d i o activity (9.9%) than did any other organs. However, the radioactivity of liver t i s s u e e x c e e d e d v a l u e s f o r t h e i n t e s t i n e a n d o t h e r o r g a n s . T h i s is a l s o t r u e 4 and 24 h after toxin was administered. 4 h after mink were dosed, the liver contained more radioactivity than did a n y o f t h e o t h e r o r g a n s . T h e c o n c e n t r a t i o n o f r a d i o a c t i v i t y in t h e l i v e r w a s s i m i l a r t o t h a t o b s e r v e d a f t e r 2 h. R a d i o a c t i v i t y in i n t e s t i n e s a n d t h e i r c o n t e n t s a g a i n d e c r e a s e d , a n d o n l y a b o u t 3% o f t h e a m o u n t in t h e i n i t i a l d o s e r e m a i n e d . 2 4 h a f t e r t r e a t m e n t , o n l y 1 . 2 % o f t h e r a d i o a c t i v i t y in t h e i n i t i a l d o s e w a s f o u n d in i n t e s t i n e s a n d t h e i r c o n t e n t s . L i t t l e c h a n g e in r a d i o a c t i v i t y w a s n o t e d in l i v e r w h i c h still c o n t a i n e d 6 . 6 % o f t h e a m o u n t t h a t w a s a d m i n i s t e r e d i n i t i a l l y . A t t h e s a m e t i m e , r a d i o a c t i v i t y in all o t h e r t i s s u e s d e c r e a s e d even further. I t is a l s o e v i d e n t f r o m d a t a in T a b l e I t h a t a b o u t 4 5 % o f t h e a f l a t o x i n t h a t w a s a d m i n i s t e r e d a p p e a r e d in t i s s u e s t h a t w e r e e x a m i n e d 1 h l a t e r . T h i s w a s

354

reduced by nearly one-half after 2 h. After 24 h only about 9% remained in tissues. Inter-animal variation was minimal when samples from the uterus, urinary bladder, spleen, pancreas, brain, kidney, heart, and lung were tested. Furth er mo r e, the variation that did occur when these organs were examined was greatest 1 h after t r e a t m e n t of the animal and then diminished. For example, after 1 h the kidney from one animal contained 0.63% of the total dose of radioactivity, whereas the kidney from anot her mink contained 0.75%. After 4 h the values were 0.28 and 0.30%, and after 24 h t h e y were 0.18 and 0.23%, respectively. Variation among animals was greatest when tests were done on the liver, intestines and their contents, and stomach and its contents. Again, the variation ten d ed to decrease with time. Of these three organs, variation among animals was greatest when the stomach and its contents were tested. The values for the two animals at 1 h were 0.3 and 0.9% of the initial dose. After 4 h the values were 0.35 and 0.61% and after 24 h t h e y were 0.1 and 0.1%. The magnitude of variation among animals was less than that just described when tests were done on either the liver or the intestines and their contents. Fig. 1 provides data on distribution o f radioactivity among the particulate fractions o f the liver. Radioactivity was not distributed uni form l y among all fractions. At 1 h after toxin was injected, most radioactivity in the liver (45%) was associated with the microsomal supernatant fluid. The cell debris nuclei, mitochondria, and microsomes contained about 25, 14, and 16% of liver radioactivity, respectively. At intervals of 2 and 4 h after dosing, the

60 ~1

f~

H AFTER DOSING

N

2 H AFTER DOSING

o: 5O LLI

_>,

Al

O

[m~ 4 H AFTER DOSING ?-.4 H AFTER DOSING

Z40

(u

O

A

B

C

D

Fig. 1. Distribution of radioactivity in subceIlular fractions of mink liver. A, cell debris and nuclei; B, mitochondria; C, microsomes; D, microsomal supernatant fluid.

355

c o n t e n t of radioactivity in each fraction varied only slightly. After 24 h, a marked reduction in radioactivity occurred in the microsomal supernatant fluid, and an increase appeared in the microsomes and mitochondria. However, the microsomal supernatant fluid still retained more radioactivity (37%) than did any of the other fractions of the liver. DISCUSSION The a m o u n t and distribution pattern of radioactivity in mink tissue (Table I) are similar to those of the rat and m o n k e y [3,6]. Wogan et al. [6] observed that about 7.7% of administered radioactivity was retained in rat liver 24 h after intraperitoneal injection of radioactive toxin. Other rat tissues, except intestines, contained less than 0.1% of the administered radioactivity. Dalezios and Wogan [3] found 19, 0.9, and 0.3% of administered radioactivity in m o n k e y liver, kidney, and heart, respectively, 45 min after intraperitoneal injection of radioactive aflatoxin. 24 h later, the a m o u n t of radioactivity in all tissues decreased and liver, kidney, and heart retained 8.3, 0.1, and 0.1% of administered radioactivity, respectively. Distribution of 14C derived from ['4C] aflatoxin B, in tissues of layer and broiler chickens was studied by Mabee and Chipley [4,5]. They reported that 5 h after final dosing, the average a m o u n t of radioactivity detected in liver and heart was 9.8 and 4.3%, respectively, of the total a m o u n t retained by the broilers. On the other hand, 16.1 and 3.9% of total retained radioactivity was found in liver and heart, respectively, of layers. However, these data are not directly comparable to our results since Mabee and Chipley dosed chickens continuously for 14 days. The early appearance of large amounts of radioactivity in bile, liver, and intestines suggests that biliary secretion plays an important role in aflatoxin B, excretion by mink. It is believed that after intraperitoneal injection a large portion of aflatoxin B, and/or its derivatives were absorbed and transported to the liver through the portal system. They were then secreted into intestines from the gall bladder. This hypothesis also has been suggested by Wogan et al. [6] and Dalezios and Wogan [3]. The former investigators observed that the biliary excretion pattern correlated well with the pattern of distribution of radioactivity in gut contents after rats received an intraperitoneal dose of ['4C]aflatoxin B,. Thus it was believed that the biliary route is the primary pathway for excretion of aflatoxin B, by rats. The later investigators found t h a t radioactivity appeared in the small intestine, particularly in the duodenum, of the m o n k e y as early as 45 min after injection of ['4C]aflatoxin B, . 24 h later, radioactivity was found only in the ileum, suggesting ultimate excretion in feces. The considerable decrease of radioactivity in intestines of mink at 2, 4, and 24 h after dosing, as shown in Table I, probably resulted because radioactivity was excreted with feces. The relatively large a m o u n t of radioactivity observed in the liver demonstrated that this organ retained aflatoxin B, and/or its metabolites to a much

356

greater e x t e n t than did o t h e r tissues. At all times after dosing, the specific activity o f liver exceeded t hat of all ot her tissues. 24 h after t r e a t m e n t the liver had 5--72 times the specific activity found in any ot her tissue, and still contained ab out 6.6% of the administered radioactivity. The in vitro interaction of aflatoxin B, with liver nucleic acids and proteins has been report ed b y several investigators [ 1 4 , 1 5 ] . Lijinsky et al. [7] also dem onst rat ed t hat in vivo [3HI-aflatoxin B, interacted with liver cellular constituents. Hence, the greater and longer r e t e nt i on of radioactivity in mink liver may be the result of the interaction of liver cellular macromolecules and aflatoxin B, or B,-derivatives. This may also account for the p r o m i n e n t damage to mink liver caused by aflatoxins that was observed [2]. The study on aflatoxin in fractions of the liver yielded results t hat were somewhat different from those of Wogan et al. [6] who worked with rats. Th ey reported that most (60%) o f the radioactivity present in liver 0.5 h after injection was in the microsomal supernatant fluid. Within the first 2 h, increasing a m ount s of radioactivity were found in the microsomes. After 24 h, the microsomes contained 50% of liver radioactivity and 28% was present in the supernatant fluid. We did n o t observe much change in the a m o u n t o f radioactivity in the microsomal or in the cell debris plus nuclei fractions of mink liver during the 24 h after treatment. At 24 h after dosing, we f o u n d an increase in the a m o u n t of radioactivity in the microsomal supernatant fluid. Most (37%) o f the radioactivity in the liver was still associated with the microsomal supernatant fluid. It must be m ent i oned t hat our m et hods for preparing the liver fractions differed slightly from those used by Wogan et al. [ 6 ] . This might account for some of the differences between their and our observations. A not her cause of differences might be the biological variation between mink and rats. ACKNOWLEDGMENTS We thank Dr. R.M. Shackelford, D e p a r t m e n t of Meat and Animal Science, University o f Wisconsin, for providing the test animals. This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, by a grant from the Wisconsin D e p a r t m e n t o f Agriculture, and by Public Health Service Grant FD 00143 from the F o od and Drug Administration. REFERENCES 1 G.N. Wogan, Bacteriol. Rev., 30 (1966) 460. 2 C.C. Chou, Aflatoxin production and its effects on mink, Ph.D. Thesis, University of Wisconsin, Madison, 1974. 3 J.I. Dalezios and G.N. Wogan, Cancer Res., 32 (1972) 229"7. 4 M.S. Mabee and J.R. Chipley, Appl. Microbiol., 25 (1973) 763. 5 M.S. Mabee and J.R. Chipley, J. Food Sci., 38 {1973) 565. 6 G.N. Wogan, G.S. Edwards and R.C. Shank, Cancer Res., 27 (1967) 1729. 7 W. Lijinsky, K.Y. Lee and C.H. Gallagher, Cancer Res., 30 (1970) 2280.

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8 9 10 11 12 13 14 15

N.D. Davis, U.L. Diener and D.W. Eldridge, Appl. Microbiol., 14 (1966) 378. F.S. Chu, J. Assoc. Off. Anal. Chem., 54 (1971) 1304. D.P.H. Hsieh and R.I. Mateles, Appl. Microbiol., 22 (1971) 79. J. Adye and R.I. Mateles, Biochim. Biophys. Acta, 86 (1964) 418. C.N. Shih and E.H. Marth, J. Milk Food Technol., 32 (1969) 213. G. Feuer, L. Goldberg and J.R. Lepelley, Food Cosmet. Toxicol., 3 (1965) 235. H.S. Black and B. Jirgensons, Plant Physiol., 42 (1967) 732. M.B. Sporn, C.W. Dingnan, H.L. Phelps and G.N. Wogan, Science, 151 {1966) 1539.

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