Student Research: Marc Stifelman
MS, , 1998
Faculty Advisor: David L. Eaton
In vitro Characterization of Canine Hepatic Aflatoxin Metabolism
Aflatoxin B1 (AFB1) is a potent dietary carcinogen in rats and certain other species, although mice are highly resistant. The relative susceptibility of canines to the carcinogenic effects of AFB1 is not known, although canines are sensitive to acute aflatoxin toxicity, as evidenced by an LD50 1mg/kg. Most species differences in AFB1 carcinogenesis have been attributed to differences in biotransformation. An understanding of canine hepatic biotransformation in AFB1 is relevant to canine exposures and may benefit our understanding of aflatoxin carcinogenesis in humans. Susceptibility to AFB1-induced hepatocellular carcinoma is related to the proportion of AFB1 bio-activated to AFB1-8, 9-epoxide (AFBO) and the subsequent ability to conjugate AFBO with glutathione (GSH). Relative rates of production and detoxification of AFBO predict that canines are more sensitive to AFB1-induced liver cancer than humans.
Microsomes and cytosol prepared from liver collected from six beagles has been used to measure rates of cytochrome P-450 (P-450) mediated generation of AFBO and glutathione S-transferase (GST) mediated conjugation of AFBO. Observed AFB1 activities were correlated against marker substrates to identify the major P-450 family enzymes active toward AFB1. Canine liver microsomes produced AFBO at a mean rate of 286 pmol/mg/min, but cytosolic canine liver GSTs were ineffective at forming the AFBO-GSH conjugate. Canine AFM1 production correlated with Western blots probed with a polyclonal antibody raised against rabbit CYP1A! and 1A2 (R2=0.60;p=0.07). Production of AFM1 is attributable to CYP1A2 in humans. CYP3A4 produces AFBO and AFQ1 in humans. Western blots probed with an antibody raised against rat CYP3A correlated with AFQ1 activities (R2=0.88;p=0.05) with AFM1 activities. Phenacetin, also a CYP1A substrate, selectively inhibited AFM1 formation. Rates of AFBO production correlated with coumarin-7 hydroxlase (R2=0.62;p=0.06) and testosterone 16a hydroxolase activities (R2=0.64;p=0.05). These activities are associated with CYP2A and CYP2B/2C respectively. Surprisingly, rates of erythromycin N-demethylase activities, characteristic of CYP3A family enzymes, correlated negatively with AFBO production (R2= -0.87;p=0.02). Combinations of inhibitors for CYP2A, CYP2B, and CYP3A inhibited greater that 75% of AFBO production.