CONCEPTS, LABELING PROCEDURES, AND DESIGN OF CELL-PROLIFERATION STUDIES RELATING TO CARCINOGENESIS

Chemicals may induce cell proliferation directly as mitogens or indirectly via cell death with subsequent proliferation to replace lost cells. Chemically induced proliferation has been demonstrated to play a role in the carcinogenic process. A wide range of procedures and techniques are currently being used to define the quantitative relationship between the extent and duration of chemically induced cell proliferation and carcinogenic potential in different species and target organs. However, a limited database and nonstandard protocols and procedures for measuring cell proliferation have made it difficult to compare results between laboratories. Comparison of frequencies of S phase between control and treated animals is the most commonly used end point in cell proliferation studies and may be regarded as an indirect indication of a proliferative response. This response can be ascertained as labeling indexes (LI; percentage of cells in S phase) after the administration of the DNA precursor labels (tritiated thymidine; H-3-TdR; bromodeoxyuridine, BrdU) or through immunostaining of the endogenous cell replication marker, proliferating cell nuclear antigen (PCNA). Both approaches are applicable to tissue sections. An important issue in the design of experimental studies for measuring LI is determining how and when to investigate proliferative responses in relation to the chemical treatment regimen. Variables to consider when designing cell proliferation studies include the animal's age, chemical dose and method of treatment, choice and dose of label, time and length that the label is administered, and methods of quantitation. Study design considerations depend on the experimental objective. A common approach to characterize the complex relationship of cell proliferation and carcinogenic activity has been to focus on relatively early (less than 90 days) proliferative responses in the target tissue. However, a larger database on the duration and nature of the chemically induced proliferative response under bioassay conditions in the target cell population is required to more clearly establish the role of this end point in the cancer process. in addition, studies must also investigate mitogenic versus cytotoxic induction of cell proliferation in normal and preneoplastic cells and differential toxicity that may provide a preferential growth advantage to spontaneous or chemically induced intermediate or malignant cells.