. al., 1988; Phillips et al., 1995; Lindemann et al., 1997 . Examination of Smith’s data
supported his hypothesis, and initiated this study. Ž
. Winfree and Allred 1992 developed a method to detect and measure clay–aflatoxin
Ž .
or substance–aflatoxin interactions, and calculated a numerical absorption coefficient based on a substance’s capacity to remove aflatoxin from a mixture of feed and water.
Sodium bentonite exhibited a high affinity for AFB , and preliminary feeding trials
1
indicated that dietary bentonite could reduce the percentage of fish developing tumors Ž
. after consuming AFB
unpublished data . Bentonite is commonly employed as a feed
1
Ž .
Ž .
binder, and previous work by Smith 1980 and Reinitz 1984 have established the mineral to be safe for trout at dietary inclusion levels up to 10.
The current study was initiated to trace the absorption, metabolism, and elimination of dietary aflatoxin in fish consuming AFB contaminated diets, with or without 2
1
dietary bentonite added as an adsorbent. Our purpose was to determine whether Ž
. bentonite’s demonstrated ability to adsorb AFB in vitro Winfree and Allred, 1992
1
would block absorption of the toxin in vivo.
2. Materials and methods
2.1. Diets Two test diets were prepared. Each contained a casein-based diet, basal diet H440
Ž .
Ž .
NRC, 1973 . Aflatoxin AFB amounting to 20 mgrkg were added to each diet. The
1
Ž .
clay diet contained 20 mgrkg AFB , 98 H440, and 2 sodium bentonite Volclaye .
1
The non-clay diet contained 20 mgrkg AFB and 100 H440. The two diets prior to
1
adding the AFB were vacuum-dried overnight at 608C. One gram of either diet was
1
loaded into a hollow gelatin capsule supported upright in a drilled board. Exactly 0.100 Ž
. ml of a methanol solution containing 20 ng aflatoxin AFB
radio-labeled with 0.2 mCi
1 3
ŽŽ .
H Moravek Biochemicale was added to the 1.00-g diet in the gelatin capsule. The
capsules were dried overnight in vacuo at 608C. The AFB rmethanol solution and
1
Ž .
3
several finished pellets without bentonite were analyzed for AFB and H, respec-
1
tively, to ensure specified concentration. 2.2. Feed trials and collection of excreta
Ž .
Male rainbow trout, Oncorhyncus mykiss, 266 12.6 g BW were conditioned to the H440 diets for 2 weeks before the experiment was started. The feeding trials were
conducted in acrylic plastic metabolism chambers, one fish per chamber, using the Ž
. system designed by Smith et al. 1980 . A total of 20 fish were used in the study. A
group of five fish were fed the AFB diet without clay and five fish were fed the AFB
1 1
diet with clay for 7 days. This procedure was repeated with 10 more fish the following week. The metabolism chambers were designed for separate collection of gill, fecal, and
Ž .
urinary excretions. Each chamber was divided into anterior head water and posterior Ž
. tail water portions using a sleeve cut from a latex surgical glove. Sleeves were sized,
fitted, and sutured to the dorsal fin in each fish. Fish were maintained at 158C and a bottled-oxygen diffuser was installed in each head tank and oxygen levels were
monitored daily with a YSI oxygen meter. Head water samples were changed daily. Only 1-day head water sample was saved for
3
H analysis. The fish were fed one capsule daily at the same time the head water was exchanged. Feeding was done by gently
inserting a polished glass tube through the mouth and into the esophagus and ejecting a single feed capsule by means of a plunger.
Urine and fecal material were collected once daily. Urine was collected continuously with a flexible polyethylene catheter, inserted into the fish’s urinary bladder and
draining to a sample bottle outside the chamber. Head and tail waters were concentrated by rotary evaporation at 508C, frozen in glass cylinders by means of a rotary shell, and
freeze-dried to constant weight. Long-term storage of excreta, body fluids, and tissue samples, prior to analysis was at y608C in airtight polypropylene bottles or zipper-lock-
ing polyethylene sample bags.
2.3. Euthanasia and tissue collection At 7 days after the first feeding, the fish were removed and anaesthetized with
MS-222. Blood samples were taken by direct cardiac puncture or through the caudal Ž
artery, using a heparinized 18 gauge hypodermic needle and 60 ml syringe 10,000 units .
porcine sodium heparinrsyringe . The fish were then euthanized by severing the spinal cord and pithing neural material. The abdominal cavity was surgically opened and the
contents of the urinary and gall bladders were withdrawn by hypodermic syringes and tuberculin needles. The liver and the intestinal tract from the esophagus to vent were
removed. Kidneys were teased from the dorsal cavity by scalpel and a blunt probe. Blood was immediately centrifuged to separate plasma from the cellular material. The
carcass, minus body fluids and tissue samples, was frozen for analysis.
2.4. Analysis of tissue and body fluids
3
H from the labeled AFB was counted in a Beckman LS 6800 liquid scintillation
1
counter. The liver, kidney, lower GI tract, spleen and blood cells were extracted with chloroform–methanol. Minced tissue amounting to 0.5 g was combined with 1.8 ml of
methanol and 0.9 ml of chloroform. After homogenization, the materials were cen- trifuged at 12,000 = g for 15 min. The supernatant was transferred to a separatory
funnel and 2.5 ml of a 4:2:1, methanolrchloroformrwater mixture was added. Follow- ing homogenization and centrifugation two more times, the supernatants were added to
the separatory funnel and the tissue residue saved. A total of 10 ml of chloroform and 10 ml water were added to the separatory funnel and then left overnight. One milliliter of
each fraction was then combined with 10 ml of Beckman Ready-Solv Hprb. This was set aside for a minimum of 3 days and then counted.
Urine amounting to 1 ml and 0.1 ml bile were directly counted in 10 ml of the above liquid scintillation cocktail.
2.5. Combustion analysis of fecal and extracted tissue residues Ž
. The method used was a modification by Sheppard and Rodegker 1962 . The dry
sample was pressed into a pellet and placed in a Parre oxygen bomb, which was charged with 30 atm oxygen, submerged in a water bath and electrically combusted. The
bomb was then cooled to condense the vapors, followed by slowly discharging the exhaust through a cold water trap. The bomb was then opened, flushed with absolute
ethanol and the rinsate collected for
3
H analysis. The percentage recovery was approxi- mately 75 3.0.
2.6. Statistical analysis Ž
. The data expressed in counts per minute CPM recovered from the urine, bile, GI
tract, and liver from the clay-fed and non-clay-fed fish were subjected to the Mann– Ž
. Whitney U-test Siegel, 1956 . CPM data from the fecal matter obtained from these two
Ž .
groups were subjected to evaluation by the Student’s t-distribution McGhee, 1985 .
3. Results
