Evidence for an indirect mechanism of aflatoxin B1 inhibition of rat liver nuclear RNA polymerase II activity in vivo.

Previous studies suggested multiple sites of action of aflatoxin B1 (AFB1) in vivo to inhibit rat liver nuclear RNA synthesis--it impairs nucleolar DNA template function and inhibits RNA polymerase II activity. We have previously shown that AFB1 activated in vitro inhibits nucleolar RNA synthesis. The question is whether AFB1 can inhibit RNA polymerase II under these in vitro conditions. Male Sprague-Dawley rats, 200 g, were injected i.p. with 0.6 mg AFB1 and liver nuclei were isolated 2 h later. When the total nuclear free RNA polymerases were extracted and assayed in the absence and presence of alpha-amanitin (3.2 micrograms/ml), we found that only alpha-amanitin-sensitive (i.e., RNA polymerase II) activity was inhibited (97%). DEAE-Sephadex chromatography confirmed this result. When total nuclear free RNA polymerases were incubated with AFB1 activated in vitro under conditions producing 70% inhibition of nucleolar RNA synthesis, no inhibition was observed for either alpha-amanitin-sensitive or -resistant activities. Similar results were obtained with low and high (28 and 167 micrograms/ml) concentrations of AFB1. This was further confirmed using highly purified RNA polymerase II. We conclude that AFB1 inhibition of RNA polymerase II activity in vivo is not a result of direct interaction of AFB1 to the enzyme.