23 Dietary Casein Levels and Aflatoxin B Metabolism in 1
Rainbow Trout (Salmo gairdneri) W. T. STOTT and R. O. SINNHUBER
Downloaded by UNIV OF ARIZONA on March 14, 2017 | http://pubs.acs.org Publication Date: May 24, 1979 | doi: 10.1021/bk-1979-0099.ch023
Department of Food Science and Technology, Oregon State University, Corvallis, OR 97331
The influence of nutrition on chemical carcinogenesis has been reviewed by Clayson (1). Dietary protein reportedly affects the toxicity and carcinogenicity of a variety of chemical carcinogens in animals, presumably by altering the activities of enzymes involved in their activation and/or detoxification (2, 3, 4). Aflatoxin B (AFB) is a mold metabolite which has been observed to be acutely toxic and carcinogenic to a wide variety of animals (5, 6) and has been implicated in human primary hepatic carcinoma (7, 8). Diets deficient in protein have been reported to increase the susceptibility of mammals to acute AFB toxicity and the induction of cancer (2, 9, 10, 11, 12, 13). Increased dietary proteins have increased the carcinogenic activity of AFB fed to rats (14) and trout (15). Supportive of this latter finding has been the reported direct relationship between dietary protein content and AFB-DNA adduct formation in vivo in rats (16, 17). AFB has been shown to require metabolic activation to its ultimate carcinogenic species (18, 19, 20, 21, 22) which is believed to be a 2,3-epoxide form of AFB (OAFB) (19, 23-29). This epoxidation of AFB has been associated with aldrin epoxidase (AE) activity in trout (30). As with other epoxide carcinogens, OAFB may be a substrate Tor epoxide metabolizing enzyme systems such as epoxide hydrase (EH) (EC4.2.1.63) and glutathione-Sepoxide transferase (GTr) (EC4.4.1.7) found in mammals and fish (31, 32, 33, 34). AFB also undergoes a variety of other reactions, generally to less toxic metabolites depending on the species of animal involved (35, 36). The primary AFB metabolite in rainbow trout has been shown to be a reduced form of AFB, aflatoxicol (AFL) (24). The present study was undertaken to determine the influence of several levels of dietary casein upon the activities of trout hepatic enzyme systems which may be involved in the in vitro activation and detoxification of AFB. In addition, the effect of technical Paper No. 4883, Oregon Agricultural Experiment Station. 1
0-8412-0489-6/79/47-099-389$05.00/0 © 1979 American Chemical Society Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
390
PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS
d i e t a r y casein upon the conversion of AFB to an a c t i v e mutagen by t r o u t hepatic enzymes i s d e s c r i b e d . Experimental Procedures
Downloaded by UNIV OF ARIZONA on March 14, 2017 | http://pubs.acs.org Publication Date: May 24, 1979 | doi: 10.1021/bk-1979-0099.ch023
Diets. Semi p u r i f i e d d i e t s , as shown i n Table I, were prepared as described by Sinnhuber et a l . (37). Casein at l e v e l s of 32, 42, 52 and 62% (dry weight) provided the p r o t e i n source along with an 8% g e l a t i n b i n d e r . A l l d i e t s were i s o c a l o r i c and each t e s t group received the same amount of feed each day. Fish. One year o l d Mt. Shasta s t r a i n rainbow t r o u t (Salmo g a i r d n e r i ) weighing an average of 140g were used f o r the study and housed in c i r c u l a r tanks supplied with 15.2 1 o f well water/min. at 1 1 - 1 2 ° C . Fish were fed twice d a i l y f o r seven months p r i o r to s a c r i f i c e at which time they weighed an average of 800g. Hepatic Enzyme P r e p a r a t i o n s . Fish were k i l l e d by a c r a n i a l blow between 6 and 8 A.M. L i v e r s were immediately removed, weighed and oerfused with i c e c o l d s a l i n e (0.9%) and then homogenized with four volumes of 0.25M sucrose s o l u t i o n in a P o t t e r Elvejhem apparatus by four complete passes of the p e s t l e . Homogenates were c e n t r i f u g e d f o r 15 minutes at 12,000xg, the supernatant recovered (PMF) and r e c e n t r i f u g e d at 105,000xg f o r one hour. Fatty l a y e r s were discarded and the 105,000xg supernatant recovered. Microsomal p e l l e t s were resuspended in an o r i g i n a l weight of e i t h e r 0.25M sucrose or 0.073M potassium phosphate b u f f e r (pH 7 . 6 ) . A l l steps were c a r r i e d out at 1°C and i s o l a t e d f r a c t i o n s were frozen in dry i c e and stored at -45°C u n t i l used (
