Histologic and Genomic Evaluation of Liver Cell Proliferation in the Chicken Egg Genotoxicity Assay (CEGA)
A variety of genotoxic carcinogens tested in the Chicken Egg Genotoxicity Assay (CEGA), which assesses liver DNA strand breaks and adducts, also interfered with fetal liver proliferation, differentiation and migration, leading to hepatocellular dysplasia and distortion of trabecular pattern. The present study assesses cell proliferation and gene expression profile in fetal chicken livers, since these processes are involved in embryo-fetal development and their dysregulation can lead to neoplastic development. Groups of at least 12 white leghorn chicken eggs were administered the vehicles for CEGA, deionized water (DW) and 20% aqueous solution of Solutol HS15 (20% HS15), in 3 daily injections on days 9 - 11 of incubation. The group used as control did not receive any injections. Three hours after the last injection, half of the livers were collected for gene expression analysis. One day (day 12 of incubation) and one week (day 18) after dosing was discontinued, the remaining livers were collected for histologic analysis. At day 12, in sections stained with hematoxylin and eosin (H&E), in all groups, nascent trabecular hepatocellular pattern was established and normal liver cells and extracellular matrix elements were present. At day 18, the hepatocellular trabecular pattern was completed. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) showed no significant differences in cell proliferation between the groups, the percentage of proliferating cells at 12 and 18 days was in the range of 61% to 65%. Thus, cellular proliferation in fetal chicken livers was not affected by DW and HS 15. Gene expression analysis using chicken 44K Agilent microarray, revealed that DW had minimal effect on the expression of genes involved in the regulation of cell cycle and proliferation, while downregulation of two tumor suppressor genes p53 and APC was observed. HS15 significantly deregulated multiple genes involved in cell cycle and proliferation pathways. Among genes upregulated were Rad21, p300, Mdm2, Mad1. Downregulated genes included APC, CycA, CycB, TGFβ, p53, GADD45 and PCNA. Despite alterations in the expression of genes involved in cell cycle and proliferation produced by HS15, fetal chicken hepatocytes evidently maintained their proliferative status. A 30% aqueous solution of ethanol (30% ETOH) was tested in CEGA for its effects on gene expression as a possible substitution for 20% HS15. Similar to DW, 30% ETOH did not cause significant alterations in gene expression profile. Thus, it can be used as a vehicle in CEGA.
Kobets, Tetyana; Iatropoulos, Michael J.; Duan, Jian Dong; Brunnemann, Klaus D.; Iacobas, Dumitru A.; Iacobas, Sanda; and Williams, Gary M., "Histologic and Genomic Evaluation of Liver Cell Proliferation in the Chicken Egg Genotoxicity Assay (CEGA)" (2017). NYMC Faculty Posters. 9.