We have found, by cytospectrophotometric methods, that a small number of rat liver nodules induced by an initiation-promotion regimen have an aneuploid modal DNA content. Over the past two years we have not only confirmed the initial finding, but also determined that aneuploidy is present when either choline deficiency or phenobarbital are given separately after the same initiation treatment with diethylnitrosamine. We now have preliminary evidence, which will have to be validated in future experiments, that the administration of diethylnitrosamine alone or the feeding of a choline deficient diet by itself produces aneuploidy in some enzyme altered foci. The quantitative alterations in the genome that result in aneuploidy are in fact a manifestation of the PROGRESSION stage during transformation into malignancy. Since aneuploidy results in a heterogeneous cell population, the subsequent ability of some tumors to metastasize may have been decided at the beginning of the carcinogenic process and not later, as a consequence of changes that follow overt malignancy. Cytospectrophotometry is a grudging, slow methodology. The advent of Computerized Digital Image Analysis, together with the complete automation of the system we propose in this application, should overcome most of these problems. The second phase of our investigations will be at first devoted mostly to the originally planned experiments to study the relationship between ploidy distribution, nodule persistence and hepatoma development. Treatment of these animals has already been performed in the first funding cycle. With the use of Computerized Image Analysis we will also determine whether aneuploidy is seen with either promoting treatment or with diethylnitrosamine when each is applied separately. In the last part of our studies we will examine, using the same methodology, whether aneuploidy is caused by clastogenic changes due to the administration of methyl-N- nitros-urea/phenobarbital or aflatoxin B1/phenobarbital or by agents that produce abnormal or arrested mitotic spindles, such as diethylstilbestrol. The experiments proposed will help to understand the mechanisms of PROGRESSION in the rat liver in vivo (as opposed to cells in culture) and may result in less restrictive but more veritable regulations for in vivo tests of hepatocarcinogenesis.
