Harold A. Campbell

Critical Parameters in the Quantitation of the Stages of Initiation, Promotion, and Progression in One Model of Hepatocarcinogenesis in the Rat

Critical parameters in the quantitation of altered hepatic foci (AHF) developing during multistage hepatocarcinogenesis in the rat include: 1) the enumeration of AHF induced by test agents as well as those AHF occurring spontaneously in livers of untreated animals; 2) the volume percentage or fraction of the liver occupied by all AHF as a reflection of the total number of altered cells within the liver and the degree of tumor promotion which has occurred; and 3) the phenotype of individual AHF as determined by multiple markers with serial sections. These parameters, especially the number of AHF, should be corrected by the presence of spontaneous AHF which increase with the age of the animal, more so in males than females. While accurate estimation of the background level of spontaneous AHF can be important in demonstrating that a carcinogenic agent does not possess the ability to increase the numbers of AHF above the background level, a better method to distinguish the effectiveness and relative potencies of agents as initiators or promoters is reviewed. The relative effectiveness of four different markers–γ-glutamyltranspeptidase (GGT), a placental form of glutathione S-transferase (GST), canalicular ATPase, and glucose 6-phosphatase (G6Pase)–was described for the chemicals C.I. Solvent Yellow 14 and chlorendic acid as promoting agents in males and females. C.I. Solvent Yellow 14 is a more effective promoting agent in females than males, and AHF exhibit extremely low numbers scored by GGT. On the other hand, the numbers of AHF present in livers of male rats promoted by this agent are more than twice those seen in livers of female animals, possibly owing to the effectiveness of this agent as an initiator in the male but not the female. Very few AHF, especially in the male, are scored by GGT during chlorendic acid promotion. The distribution of phenotypes with these markers also differs in the spontaneous AHF appearing in the livers of animals fed 0.05% phenobarbital on either a crude NIH-07 or AIN-76 purified diet. Such studies emphasize the extreme dependence of the promoting stage of hepatocarcinogenesis on environmental factors of sex, diet, and the molecular nature of the promoting agent itself. The hallmark of the final stage of progression in the development of hepatocellular carcinomas is aneuploidy, which may be reflected by phenotypic heterogeneity within individual AHF, termed foci-in-foci. The implications of such quantitative analyses during hepatocarcinogenesis induced by specific agents in relation to the specific action of the agent at one or more of the stages of hepatocarcinogenesis are discussed.

Diurnal variations in activity of four pyridoxal enzymes in rat liver during metabolic transition from high carbohydrate to high protein diet.

Abstract The diurnal variations in enzyme activities including tyrosine aminotransferase (TAT), ornithine decarboxylase (ODC), ornithine aminotransferase (OAT) and serine dehydratase (SDH) have been studied in rats trained to a 2 hour meal feeding schedule (″2+22″) during metabolic transition from 12.5 to 60% protein diets over a period of 21 days. Although the maximal TAT activity on the first day was slightly lower compared with other days, both TAT and ODC activities adapted rapidly to the increased dietary protein from the first day. The responses of TAT and ODC to the food were so rapid that the maximal value was observed only 4 hrs after the onset of feeding. After each feeding ODC activity decreased rapidly after 4 hours, while TAT activity declined only after 6 hours had elapsed. No clear diurnal rhythm was observed in either OAT or SDH, though OAT activity tended to decrease from the beginning of the dark period and to resume a slow adaptation after about four hours. In contrast to ODC and TAT both OAT and SDH required about 7 days to fully adapt to the high protein diet. The activities of the four enzymes were also compared after 4 groups of rats had been adapted to the ″2+22″ feeding of 12.5, 30 and 60% protein diets and to 60% diet, ad libitum , respectively. The enzyme activities were not directly proportional to the protein content of the diets although higher activity was observed on the high protein diets. The diurnal variations in both TAT and ODC were observed in all ″2+22″ groups although the timing of the peak values were slightly different from each other. The maximal activities of TAT were found at earlier times in 12.5 and 30% protein groups than in the 60% protein group. The peak time for ODC activity was found at a later time in the 12.5% protein group than in rats fed 30% and 60% protein. Ad libitum rats fed 60% protein maintained relatively high levels of TAT activity compared to the rats on the schedule. However, the maximal activity of ODC on the 60% ″2+22″ protein diet ad libitum was so low that a diurnal rhythm was not clearly evident.

The quantitation of altered hepatic foci during multistage hepatocarcinogenesis in the rat: Transforming growth factor α expression as a marker for the stage of progression

The experimental three-stage hepatocarcinogenesis protocol of initiation, promotion, and progression, coupled with the analytical technique of stereology, permits quantitative analysis of the carcinogenic process, including the derivation of biologically based risk assessment models. The aberrant expression of the placental isozyme of glutathione S-transferase (PGST) is an efficient marker for initiated, preneoplastic, and neoplastic hepatocytes. Putatively initiated cells and their clonal progeny can be identified, enumerated, and their growth characteristics determined on the basis of their aberrant expression of this protein. A lack of suitable markers has made the identification and quantitation of hepatocytes in the early stage of progression more difficult. One characteristic of cells in the stage of progression is the evolution of relatively autonomous growth. The alteration of growth factor signalling pathways may provide one mechanism for this observation. The expression of transforming growth factor alpha (TGF alpha) is seen in many malignancies. The initiation-promotion-progression protocol has been used to induce progression in the rat liver. The focal expression of TGF alpha was found to correlate with areas of progression in rats subjected to this protocol. The ability to identify and quantitate cells in the stage of progression should facilitate application of the Moolgavkar-Venzon-Knudson model for assessing human risk from carcinogens active at each of these three stages. Validation of this model will require determination of the number and growth characteristics of hepatocytes in the stage of progression.

AN INITIATION-PROMOTION ASSAY IN RAT LIVER AS A POTENTIAL COMPLEMENT TO THE 2-YEAR CARCINOGENESIS BIOASSAY

Several pharmaceutical agents, manufacturing chemicals, and environmental contaminants were found to act primarily as promoting agents in an initiation-promotion paradigm. The phenotypic distribution of four enzyme markers--placental glutathione-S-transferase (PGST), gamma-glutamyl transpeptidase (GGT), canalicular ATPase (ATPase), and glucose-6-phosphatase (G6Pase)--was analyzed in altered hepatic foci (AHF) by quantitative stereology. The number and volume distribution of AHF were determined for each promoter tested. For phenobarbital and 2,3,7,8-tetrachloro-p-dioxin, PGST and GGT together scored 100% of the AHF; for 1-(phenylazo)-2-naphthol (CI solvent yellow 14) and chlorendic acid, PGST alone marked 90% of the AHF; after chronic administration of WY-14,643, ATP and G6Pase were the predominant markers. In rats fed tamoxifen, G6P scored more than half of the AHF. Differences in the number of AHF promoted by each of these agents and in their phenotypic distributions may reflect the differentially responsive nature of individual initiated hepatocytes to the action of specific promoters. Since the chronic bioassay of suspected carcinogens does not allow one to differentiate between weak complete carcinogens and those carcinogenic agents that act in a reversible manner to promote the growth of previously initiated cells, the partial hepatectomy, altered-hepatic-focus model of cancer development is proposed as a supplement to the chronic bioassay for the identification of those carcinogenic agents that are primarily, if not exclusively, promoting agents in rat liver.

STEREO: A program on a PC-Windows 95 platform for recording and evaluating quantitative stereologic investigations of multistage hepatocarcinogenesis in rodents

The most common organ site of neoplasms induced by carcinogenic chemicals in the rodent bioassay is the liver. The development of cancer in rodent liver is a multistage process involving sequentially the stages of initiation, promotion, and progression. During the stages of promotion and progression, numerous lesions termed altered hepatic foci (AHF) develop. STEREO was developed for the purpose of efficient and accurate quantitation of AHF and related lesions in experimental and test rodents. The system utilized is equipped with a microcomputer (IBM-compatible PC running Windows 95) and a Summagraphics MICROGRID or SummaSketch tablet digitizer. The program records information from digitization of single or serial sections obtained randomly from rat liver tissue. With this information and the methods of quantitative stereology, both the number and volume percentage fraction of AHF in liver are calculated in three dimensions. The recorded data files can be printed graphically or in the format of tabular numerical data. The results of stereologic calculations are stored on floppy disks and can be sorted into different categories and analyzed or displayed with the use of statistics and graphic functions built into the overall program. Results may also be exported into Microsoft Excel for use at a later time. Any IBM-compatible PC capable of utilizing Windows 95 and MS Office can be used with STEREO, which offers inexpensive, easily operated software to obtain three-dimensional information from sections of two dimensions for the identification and relative potency of initiators, promoters, and progressors, and for the establishment of information potentially useful in developing estimations of risk for human cancer.

Multistage Hepatocarcinogenesis in the Rat as a Basis for Models of Risk Assessment of Carcinogenesis

Although biological processes are, in general, far more complex than chemical and physical phenomena, one goal of biology, toxicology, and pathology is the mathematical-statistical formulation of models that describe the mechanisms of normal and disease processes. However, as pointed out by several scientists (Whittemore, 1978; Moolgavkar, 1986; Alavanja et al., 1987), up to this time no mathematical-statistical model of a biological process has faithfully described all of the particulars of the biological phenomenon under consideration. This is especially true in the field of carcinogenesis (cf. Chu, 1987) where many models have been proposed in the past. With any model, however, its formulation is dependent on the level of knowledge of the biological mechanisms controlling the process, i.e., carcinogenesis. Recent advances in our understanding of the mechanisms of carcinogenesis as a multistage process have allowed, at least potentially, for a closer congruence between models representative of carcinogenesis and their pathogenesis.