Previous studies from others as well as from our own have established that aflatoxin B1 (AFB1) after activation either in vivo or in vitro strongly inhibits rat liver nuclear and nucleolar RNA synthesis.
The specific aims of this proposal are: 1, to determine whether the inhibition of RNA synthesis by AFB: is a result of RNA chain termination or of decreased rate of RNA synthesis; 2, to study the steric effect of AFB1 on the inhibition of RNA synthesis in vitro; 3, to study AFB1 induced infidelity of RNA synthesis in vitro. To accomplish Specific Aim 1, we propose to directly measure the chain length of the RNA transcripts from both the control and AFB1 treated rat liver nuclei, nucleoli, and P-3 fractions by autoradiography after gel electrophoresis. We will perform these experiments: 1) after AFB1 administration in vivo; and 2) after AFB1 activation in vitro. To accomplish Specific Aim 2, we propose: 1 to establish the concept of steric inhibition of AFB1 on RNA synthesis using polydG-polydC and polydI- polydC templates by confirming indeed AFB1 when bound to one strand of the DNA duplex is able to interfere with RNA synthesis of the complementary, non-binding strand; and 2) to study the mechanism of steric inhibition by AFB1 on RNA synthesis whether it is due to chain termination or reduced rate of RNA synthesis by autoradiography after gel electrophoresis. To accomplish Specific Aim 3, we propose 1) to establish the phenomenon of AFB: induced infidelity of RNA synthesis by studying the errors of incorporation of the non-complementary, radioactively labeled nucleo-tides into RNA using severAl single- and double-stranded DNA templates of known base sequences; and 2) to study the mechanism of AFB: induced infidelity of RNA synthesis by assessing the potentially contributing factors such as: a) transcriptional infi-delity from the formation of apurinic sites in DNA after AFB1 treatment; b) transcriptional infidelity from the formation of AFB1-FAPyr (2,3- dihydro-2-(N7-formy1-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3- hydroxy-AFB1) in DNA after AFB1 treatment; and c transcriptional infidelity from steric inhibition due to AFB1-adducts formation on the opposite side of the transcribing DNA strand after AFB1 treatment. We believe that these proposed experiments will provide insights into the mechanisms of AFB1 inhibition of rat liver nuclear and nucleolar RNA synthesis and of AFB1 induced carcinogenesis.
