T.Colin Campbell

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.

Aflatoxin inhibition of rat liver mitochondria

Abstract When aflatoxin B1 (AFB1) is added to an actively respiring rat liver mitochondrial preparation, 25–44% inhibition of electron transport is produced with concentrations ranging from 2.5–4.8 middot; 10−4M, respectively. The degree of inhibition levels off at 4.8 middot; 10−4M, which was shown to be in agreement with the critical micelle concentration. Submitochondrial or Gregg particles exhibit a maximum of 63% inhibition. Weanling rats maintained on a 5% casein semipurified diet for 15 days showed an approximate 30–50% reduction in the degree of aflatoxin inhibition for both mitochondria and Gregg particles compared to control animals fed a 20% casein diet ad libitum. The mitochondria of the protein-deprived animals had similar respiratory control ratios to normal animals. Dietary protein deficiency appears to exert its effect primarily at the site of action of aflatoxin rather than to alterations in membrane transport. The major site of inhibition of electron transport appeared to be between cytochromes b and c (c1) as indicated by comparison of systems employing various substrates which donate their electrons to various portions of the electron transport system. At concentrations just below critical micelle formation, AFB1 also reduced the ADP:O ratio, which was partially relieved by protein deficiency. The relevance of these findings to liver cell necrosis promoted by aflatoxin is discussed.

On mechanisms affecting species susceptibility to aflatoxin

1. 1. Use of mouse- and rat-liver slice preparations permitted a determination of the rates of transport of aflatoxin B1 into the cell followed by subsequent rates of metabolism of aflatoxin B1 to M1 by microsomal preparations. 2. 2. The acute toxicity-response difference between these species, as measured by the concentration required to caused cell death, is 2.1-fold; that is 2.1 times as much B1 is required to kill the mouse-liver cell, giving the mouse the advantage in resistance, in part due to faster metabolic rates and in part due to slower transport rates. 3. 3. A mathematical model based upon rates of transport and metabolism was developed in order to project internal B1 and M1 concentrations as functions of initial external B1 concentrations and/or exposure times. The maximum internal concentrations of B1 achieved just prior to cell death are 158 μM for the rat and 174 μM for the mouse. This small difference, in spite of the rather divergent initial extracellular concentrations generating them, indicates equivalence of these two species in terms of intracellular target-site sensitivity. 4. 4. The carcinogenic-response difference, as measured in the model by the half-life of decay of a small internal concentration developed over a short period of time, is 4.9–5.8-fold; that is, it takes 5–6 times longer for a given concentration (equivalent for each species) to be reduced to one-half its value in the rat as compared to the mouse, again giving the mouse the advantage. 5. 5. These experiments support the hypothesis that the rate of cellular transport is a considerably more important aspect of the pathological effects of affatoxin than the subsequent rate of metabolism and support the hypothesis that aflatoxin B1 has a much greater role in the toxic lesions than aflatoxin M1.

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.

Preliminary study of in vitro aflatoxin B1 metabolism by human liver

[14C]Aflatoxin B1 (ring labeled) was incubated for 6.5 hr with 9000 g supernatant of human liver homogenate of tissue obtained from biopsy and at autopsy. The chloroform-extractable metabolites, after purification and identification by thin layer chromatography, were quantitated by liquid scintillation counting. The sample from biopsy yielded, as percent of the initial concentration: aflatoxin B1, 15.4%; aflatoxin M1, 0.6%; and aflatoxin P1 (free phenol), 1.3%. The sample from autopsy yielded: aflatoxin B1, 11.9%; aflatoxin M1, 1.1%; aflatoxin P1 (free phenol), undetected; and 2 aflatoxin metabolites less polar than aflatoxin B1, 0.9% and 1.7%.

Gossypol Detoxication by Fungi

An unidentified Diplodia isolate representative of several fungi tested and grown for 13 days on gossypol-containing cottonseed reduced the amount of free gossypol by 90 percent and detoxified the cottonseed for weanling rats and chickens. Residual free gossypol was also less toxic than equivalent amounts of normal free gossypol.