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 aflatox-inB₁ (AFB₁) in vivo to inhibit rat liver nuclear RNA synthesis- it impairs nucleolar DNA template function and inhibits RNAPolymerase II activity. We have previously shown that AFB₁ activatedin vitro inhibits nucleolar RNA synthesis. The question is whetherAFB₁ can inhibit RNA polymerase II under these in vitro conditions.Male Sprague-Dawley rats, 200 g, were injected i.p. with 0.6mg AFB₁ and liver nuclei were isolated 2 h later. When the totalnuclear free RNA polymerases were extracted and assayed in theabsence and presence of a-amanitin (3.2 µg/ml), we foundthat only -amanitin-sensitive (i.e., RNA polymerase II) activitywas inhibited (97%). DEAE-Sephadex chromatography confirmedthis result. When total nuclear free RNA polymerases were incubatedwith AFB₁ activated in vitro under conditions producing 70%inhibition of nucleolar RNA synthesis, no inhibition was observedfor either -amanitin-sensitive or -resistant activities. Similarresults were obtained with low and high (28 and 167 µg/ml)concentrations of AFB₁. This was further confirmed using highlypurified RNA polymerase II. We conclude that AFB₁ inhibitionof RNA polymerase II activity in vivo is not a result of directinteraction of AFB₁ to the enzyme.