Johnnie R. Hayes

The role of aflatoxin metabolism in its toxic lesion

The public health significance and fundamental mechanism of action of aflatoxin has been extensively examined since its discovery over 15 yr ago. It is comprised of a group of bis-furano-coumarins, which can be metabolized by drug-metabolizing enzyme system in addition to other enzymes. Most current literature supports the concept that microsomal enzyme activation is required before cytotoxicity can be expressed. Many aflatoxin B1 metabolites have been identified and their toxicological properties examined. Therefore the role for metabolism of aflatoxin B1 is crucial to its eventual toxicity, and an understanding of this rather complex metabolism scheme would further elucidate the toxicological significance of this compound as well as possibly identify the critical molecular interaction initiating the lesion. The metabolism of aflatoxin is herein reviewed.

Effect of protein deficiency on the inducibility of the hepatic microsomal drug-metabolizing enzyme system—I: Effect on substrate interaction with cytochrome P-450

Abstract Male, weanling rats divided into three groups were maintained for 15 days on a semipurified diet containing either 5% casein fed ad lib. (group 1), 20% casein pair-fed to group 1 (group 2), or 20% casein fed ad lib. (group 3). After each group was further subdivided, animals were injected i.p. on days 11, 12, 13 and 14 with either 0.9% saline or phenobarbital (80 mg/kg) in 0.9% saline. Twenty-four hr after the last injection, animals were decapitated and liver microsomes were prepared. Contents of microsomal protein, phosphatidylcholine and cytochrome P-450 were measured and used as bases of expression for spectral dissociation constants ( K s ) and maximal spectral changes ( ΔA max ) associated with the binding of ethylmorphine and aniline to the cytochrome P-450 hemoprotein of microsomes. Phenobarbital administration increased microsomal protein, cytochrome P-450, and phosphatidylcholine in all three dietary groups; however, in all groups, the increase in P-450 was relatively greater than that for phosphatidylcholine. Protein deficiency (group 1 vs 2) decreased P-450 and microsomal protein, but had no effect on phosphatidylcholine contents. The effect of total food restriction (group 2 vs 3) on each of these parameters was not significant. These data suggest that a portion of the induced cytochrome P-450 binding sites may be dependent on an association with phosphatidylcholine. The fraction of such phosphatidylcholine-associated sites relative to the total sites was greater during protein deficiency and was in agreement with a greater ΔA max per nanomole P-450 for ethylmorphine. Phenobarbital induction decreases the proposed fraction of phosphatidylcholine-associated P-450 sites relative to the total P-450 sites and results in a decrease in the ΔA max per nanomole P-450 for ethylmorphine. Phenobarbital increased the ΔA max per milligram of microsomal protein for aniline, which paralleled the increase in total P-450, thus indicating that the type II site may be independent of any association of cytochrome P-450 with phosphatidylcholine. These results indicate that phosphatidylcholine may play an important role in distinguishing the effects of dietary deficiency on type I substrate binding and the corresponding capacity for induction of the rat liver microsomal enzyme system.

Effect of protein deficiency on the inducibility of the hepatic microsomal drug-metabolizing enzyme system—II: Effect on enzyme kinetics and electron transport system☆

Abstract Male, weanling rats divided into three groups were maintained for 15 days on a semipurified diet containing either 5% casein fed ad lib . (group 1), 20% casein pair-fed to group 1 (group 2), or 20% casein fed ad lib . (group 3). After each group was further subdivided, animals were injected i.p. on days 11–14 with either 0.9% saline or phenobarbital (80 mg/kg) in 0.9 % saline. Twenty-four hr after the last injection, animals were decapitated and liver microsomes were prepared. Apparent V max and apparent K m kinetic constants were determined for ethylmorphine and aniline. The V max per milligram of microsomal protein was 64–66 per cent lower in the protein-deficient group. Equivalent reductions of the content of cytochrome P-450 and activities of cytochrome P-450 and c reductases were also observed. Phenobarbital induction increased specific enzyme activities ( V max per milligram of microsomal protein) in all groups with slightly greater percentage increases seen in the protein-deficient animals. Increases were also noted for the cytochrome P-450 content and cytochromes P-450 and c reductase activities. It was suggested that phosphatidylcholine and cytochrome P-450 both play important roles in the kinetics of metabolism determined after protein deficiency or phenobarbital induction, or both.

Effect of protein deficiency on the inducibility of the hepatic microsomal drug-metabolizing enzyme system—III: Effect of 3-methylcholanthrene induction on activity and binding kinetics

Abstract Male, weanling rats divided into three groups were maintained for 15 days on a semipurified diet containing either 5% casein fed ad lib. (group 1), 20% casein pair-fed to group 1 (group 2), or 20% casein fed ad lib. (group 3). After each group was further subdivided, animals were injected i.p. on days 13 and 14 with either corn oil or 3-methylcholanthrene (3-MC) (20 mg/kg) in corn oil. Twenty-four hr after the last injection, animals were decapitated and liver microsomes were prepared. Contents of microsomal protein, phosphatidylcholine and hemoprotein (cytochrome P-450 or cytochrome P-448 or both) were measured and used as the basis of expression for spectral and enzyme kinetic constants; the activities of cytochrome c and P-450 reductases were also determined. Protein deficiency reduced microsomal protein and hemoprotein, but did not alter phosphatidylcholine (PC); 3-MC treatment produced increases in protein, hemoprotein and PC/mg of protein, but a decrease in PC/hemoprotein. In general, 3-MC produced greater percentage increases in the above parameters with protein deficiency, although the absolute amounts remained considerably below that achieved with groups 2 and 3. The 3-MC-induced groups showed the characteristic spectral shift for the P-450-CO complex to 448 nm. Ethylmorphine (EM) metabolism was decreased by both protein deficiency and 3-MC induction, whereas aniline (AN) metabolism was decreased by protein deficiency but increased by 3-MC treatment. Spectral binding ( Δ A max ) of EM was decreased by protein deficiency and unchanged by 3-MC when expressed on a protein basis, but was reduced when expressed on a hemoprotein basis, indicating the loss of the type I site in the 3-MC-induced hemoprotein. Aniline binding, when based on protein, was decreased by protein deficiency but remained unchanged when expressed on a hemoprotein basis, whereas 3-MC treatment increased the Δ A max when expressed on a protein basis, but did not change this parameter when expressed on a hemoprotein basis, indicating the retention of the type II site after 3-MC induction. Protein deficiency reduced both cytochrome c and P-450 reductase activities, whereas 3-MC treatment did not alter these parameters. Based on these and previous data, a role for the type I binding site as a “substrate effector site” was proposed.

Interaction of endrin and dieldrin with hepatic microsomal cytochrome P-450 from the rat, mouse, and endrin-susceptible and resistant pine voles☆

Abstract Studies were undertaken to determine whether binding of endrin and dieldrin to hepatic microsomal cytochrome P-450 was related to the pesticide resistance phenomenon demonstrated in two strains of pine voles. Both endrin and dieldrin produce type I spectral changes upon interaction with cytochrome P-450 in both strains of vole as well as in the rat and the mouse. The K s or Δ A max /nmol P-450 for the binding of both endrin and dieldrin to vole cytochrome P-450 did not differ for these two strains of vole which were shown to differ in their resistance to these pesticides. The differences in endrin toxicity in the vole do not appear to be related to its interaction with cytochrome P-450 at the type I binding site.