Standardized assessment of cell proliferation: the approach of the RITA-CEPA working group.

Since 1985, quantitative data on cell proliferation increasingly form an integral part in the risk assessment of many pharmaceutical, chemical, and agrochemical compounds. Technical guides for the conduct of cell proliferation studies are not readily available. The detection of cell proliferation relies almost exclusively on immunohistochemistry, which is rarely standardized between laboratories. Against this background pathologists formed the RITA-CEPA working group with the aim of elaborating standard guides for the conduct of cell proliferation studies. We present here, as examples, the general BrdU guide as well as the organ-specific guides for hepatocytes, thyroid gland follicular cells, and renal tubular epithelial cells in the rat. Today, RITA-CEPA has available guides for BrdU cell proliferation studies in 20 organs as well as for studies with PCNA as proliferation marker and for apoptosis studies with the TUNEL technique. The relevant information from more than 500 selected publications is organized in a database structure to make it easily traceable. New hardware and software used for image analysis are being assessed and are included in the information exchange practiced in the CEPA working group. Further fields of major relevance for cell proliferation studies, such as statistics and the validation of image analysis equipment in a GLP environment are investigated. A guide on the use of statistics in cell proliferation studies is in preparation. CEPA-group members meet regularly to exchange information. They use the electronic database for planning and interpretation of cell proliferation studies. CEPA will continue with the preparation of organ guides and expand the series for other proliferation markers, e.g. Ki-67. It promotes standardization of techniques applied in proliferation studies, especially immunohistochemistry and image analysis. Members of the CEPA-group have access to a comprehensive and steadily increasing knowledge base on all aspects of cell proliferation. Membership of the CEPA group is open for all industrial organizations who develop chemical, agrochemical, or pharmaceutical products.

Formic acid excretion in rats and mice exposed to bromodichloromethane: a possible link to renal tubule cell proliferation in long-term studies.

Male F344 rats exposed to bromodichloromethane (BDCM) by gavage at 50 or 100 mg/kg/day for 5 days a week for 28 days excreted large amounts of formic acid in their urine, which was accompanied by a change in urinary pH. Male B6C3F1 mice exposed to BDCM at 25 or 50 mg/kg/day for 5 days a week for 28 days also excreted increased amounts of formic acid in their urine. In rats, formate excretion was dose and time dependant, being markedly elevated after four doses and remaining at that level after 3 weeks of dosing at 100 mg/kg/day BDCM, while at 50 mg/kg/day there was some suggestion of a decline after 3 weeks. In contrast, in mice formate excretion did not start to a major extent until 3 weeks of dosing, with the biggest response at 4 weeks. There was no increase in clinical chemistry markers of liver or kidney injury in either rats or mice following 28-day exposure to BDCM. However, morphological examination of the kidneys showed some mild renal tubule injury in two out of five rats exposed to 100 mg/kg/day BDCM. This was associated with a marked increase in cell proliferation in the renal cortex of all rats exposed to 100 mg/kg/day. No increase in cell proliferation was seen in the renal cortex of rats exposed to BDCM at 50 mg/kg/day, or in mice exposed to 25 or 50 mg/kg/day BDCM for 28 days. Long-term exposure to formic acid is known to cause kidney damage, suggesting that excretion of this acid may be a contributory factor to the increase in cell proliferation and kidney damage seen in the longer-term studies with BDCM.