Mechanistic and quantitative aspects of liver tumour promotion in mice

A variety of xenobiotic compounds is known to induce characteristic changes in the livers of laboratory animals. These changes include enlargement of the liver, usually as a result of cell enlargement (hypertrophy) or Increased cell replication (hyperplasia), induction of drugmetabolizing enzymes and proliferation of the smooth endoplasmic reticulum (SER). Such changes are usually not accompanied by evidence of liver damage and thus are reversible upon withdrawal and elimination of the compound. Consequently, most authors regard this phenomenon as an adaptive response of the organ to increased functional demands.<p>However, chronic exposure of various strains of mice to dieldrin, phenobarbitone, DDT and the α-, β- and γ-stereoisomers of hexachlorocyclohexane (HCH, also known as benzenehexachloride, MC) may lead to the development of liver tumours.<p>The tumorigenic effects of microsomal enzyme Inducers In mice may result from (A) a weak carcinogenic action of the xenobiotics themselves or (h) an enhancing (promoting) action of xenobiotics on a pre-existing oncogenic factor in mouse liver. The first objective of this study was to discriminate between these two possible types.<p>Druckrey and his associates have established both theoretically and experimentally the dose-response characteristics of chemical carcinogens:<p>D.T <sup>n</SUP>= constant (1)<p>where D = daily dose, T = the median tumour Induction period and n = an exponent, always>1.<p>Since the mechanisms by which enhancers or promotors of carcinogenesis operate is quite different from the one used by carcinogens, it is be conceivable that promotors also exhibit different dose-response characteristics.<p>The dose-response characteristics of dieldrin-mediated enhancement of liver tumour formation in CF-1 mice were analysed, using existing tumour data from chronic feeding studies at six exposure levels of dieldrin (a model compound for microsomal enzyme induction). It was found that the dose- response relationship can be expressed as:<p>(d <sub>o</sub> + δx).t = constant (2)<p>where d <sub>o</sub> stands for the background dose equivalent required for the induction of spontaneous liver tumours, δx represents the actual dieldrin dose (ppm in the diet) and t the median tumour induction period in the respective treatment groups. It was also established that the dose<br/>response characteristics of limited dieldrin exposures and those of de layed exposure were consistent with equation (2), which is a Druckrey relation where n = 1.<p>From these findings it is concluded that dieldrin interacts reversibly with its receptors, resulting in an acceleration of tumour formation (which is essentially Irreversible); dieldrin may thus be regarded as a tumour promotor. The validity of equation (2) for both chronic and limited dieldrin exposure Indicates that ( <u>a</u> ) the velocity of liver tumour development is proportional to the daily dose level (δx), ( <u>b</u> ) the total tumorigenic dose is constant across all doses, ( <u>c</u> ) the effects of dieldrin on the neoplastic process In mouse liver are essentially irreversible and cumulative, and ( <u>d</u> ) there is no evidence for a threshold level.<p>Tumour formation Is a dose- and time-dependent process. The induction of liver enlargement, microsomal enzyme systems and proliferation of the smooth endoplasmic reticulum by dieldrin are only dose-dependent. In contrast, polyploldization Is dose- and time-dependent. To establish a<br/>possible link between microsomal enzyme induction, nuclear polyploidization and liver tumour formation, nuclear polyploidization in livers of CF-1 mice was studied at five different dieldrin dose levels from 1.85 months up to tumour development. Nuclear polyploidization, expressed in the proportion of octaploid (8c) nuclei, was found to be characterized by a linear increase with age in untreated control CF-1 mice. Dieldrin treatment induced a dose-dependent increase in the proportion of 8c-nuclei in the initial phases of treatment. In "steady-state" situations nuclear polyploidization (as expressed by the percentage of 8c-nuclei) was maintained on a dose-dependent, higher level, and the percentage was was observed to increase with age, the velocity of which was the same as in untreated controls. Tumour formation was found to be associated with a constant degree of nuclear polyploidization In all treatment groups Including controls. The observed quantitative link between nuclear polyploidization and tumour formation leads to the question whether or not a causal relationship between the two exists. Assuming that polyploidization reflects the ageing process, the data suggest that liver tumour formation Is Imminent at a constant biological age and that dieldrin could operate by advancing the biological age of CF-1 mouse liver.<p>Further support for this hypothesis was obtained from the determination of cytoplasmic alanine amino transferase (AAT) isoenzymes. The expression of the isoenzyme decreases with age In untreated control CF-1 mice. Dieldrin treatment was found to enhance (accelerate) this process in a dose-dependent manner.<p>Although the nature of the development of "spontaneous" liver tumours in CF-1 mice remains unknown, the decrease In the tetraplold(4c)-diplold (2c) ratio of liver nuclei, observed in the study of polyploidization, may be related to tumour formation. The decrease was observed in all<br/>treatment groups, including controls, and its onset was dose-dependently advanced by dieldrin treatment, occurring approximately 4 months before the median liver tumour induction period in all cases. Two mechanisms are proposed that may explain the tumorigenic features of a decrease<br/>in the 4c-2c ratio.<br/>1. Tetraploid cells could be more sensitive to accumulative toxic stress. Thus, their turnover may be Increased. To replace one tetraploid cell a diploid cell has to divide twice; the loss of tetraploid cells would therefore result in a proliferative response of the diploid<br/>population (resulting tumour formation).<br/>2. A reduction in the 4c-2c ratio could be induced by the occurrence of amitotic nuclear divisions in the tetraploid cells. Evidence for this possibility was obtained from experiments with 3H-thymidine-labelled nuclei. Amitotic nuclear divisions could give rise to chromosomal<br/>re-arrangements, resulting in the expression of the intrinsic neoplastic potential of CF-1 mouse liver.<br/>Both hypotheses imply that the diploid population is the source of liver<br/>tumours. The determination of nuclear polyploidization in liver tumours confirmed that these tumours originate from the diploid liver cell population.<p><TT></TT&gt