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Structure-Activity Relationships in DNA Binding and Nuclear Effects of Aflatoxin and Analogs¹

Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

The ability of aflatoxins B₁, B₂, and G₁ to inhibit RNA polymerase activity and decrease the RNA content in rat hepatocyte nuclei was found to be qualitatively similar to the carcinogenic and acute and subacute toxic actions of these compounds. Aflatoxin B₁ was approximately 3 times more effective than G₁ in reducing nRNA synthesis; aflatoxin B₂ had no effect on RNA polymerase activity at a dose of 200 mg/kg. Similar activity ratios also were observed regarding loss of rat liver nRNA following aflatoxin dosing; aflatoxin B₁ was more potent than G₁, while aflatoxin B₂ had no effect on nRNA content.

Aflatoxins B₁ and G₁ were shown by electron microscopy to cause a rapid macrosegregation of the fibrillar and granular portions of the hepatocyte nucleolus. Aflatoxin B₂ (200 mg/kg) induced only minimal segregation of these components (microsegregation). Coumarin, tetrahydrodeoxyaflatoxin B₁, and the inactive aflatoxin analogs, 5,7-dimethoxycyclopentene[c]coumarin, 5,7-dimethoxycyclopentenone[3,2-c]coumarin, 5,7-dimethoxycyclopentenone[2,3-c]coumarin caused no observable ultrastructural alterations.

The ability of aflatoxins B₁ and B₂, tetrahydrodeoxyaflatoxin B₁, and 5,7-dimethoxycyclopentenone[2,3-c]coumarin to bind to native calf thymus DNA was studied by equilibrium dialysis. While all 4 compounds have the same value for their theoretical saturation binding concentration (1 mole of ligand per 25 moles of nucleotide), their association constants, K, vary significantly. 5,7-Dimethoxycyclopentenone[2,3-c]coumarin has the highest affinity for DNA (K = 5.4 x 10⁴) followed, in decreasing order, by aflatoxin B₁ (K = 2.1 x 10⁴), aflatoxin B₂ (K = 1.2 x 10⁴), and tetrahydrodeoxyaflatoxin B₁ (K = 6.9 x 10³). The high affinity of 5,7-dimethoxycyclopentenone[2,3-c]coumarin for DNA is also reflected by a shift in its absorption peak near 360 nm to a longer wavelength and a marked hypochromism in the presence of DNA. The in vitro binding of these aflatoxin analogs does not accurately reflect their in vivo potency as toxins and carcinogens, as reported in the preceding paper (44).

A comparative study on the tissue distribution and excretion showed that twice as much radioactivity derived from aflatoxin B₁-¹⁴C was present in rat liver 3 hr after injection than the amount derived from aflatoxin B₂-¹⁴C under comparable conditions. The larger concentration of aflatoxin B₁-¹⁴C in liver was accounted for by a smaller urinary excretion of the label. All other organs and routes of excretion showed no differences in content of radioactivity derived from the 2 toxins. The 2-fold difference in liver content of ¹⁴C from aflatoxin B₁ versus aflatoxin B₂ was not sufficient to account for large differences in acute activities of these 2 compounds on the basis of concentration alone.

A marked parallelism between the acute and carcinogenic potency of a number of aflatoxin analogs has been reported in this and the preceding paper. Possible reasons for this parallelism and the potential value of the short-term effects as predictors of carcinogenic potency are discussed.

¹ This is Paper 2 of a series. Financial support was provided by National Cancer Institute Contract PH 43-62-468 and by USPHS Training Grant ES 00056-05.

² Present address: Department of Cell Biology, Rockefeller University, New York, N. Y. 10021.

³ Present address: National Cancer Institute, NIH, Bethesda, Md. 20014.

Received 4/20/71. Accepted 7/20/71.