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Genotoxic stress causes a variety of cellular and molecular responses in mammalian cells, including cell cycle arrest, DNA repair, and apoptosis. These responses result from the interplay between the genotoxic events themselves, and the biological context in which they occur. To better understand this interplay, we investigated cytotoxicty, mutagenesis, cell cycle profile, and global gene expression in the human TK6 lymphoblastoid cell line exposed to six genotoxicants. The six compounds have broad structural diversity and cause genotoxic stress by many different mechanisms, including covalent modification (methyl methanesulfonate, mitomycin C), reactive oxygen species (hydrogen peroxide, bleomycin), and topoisomerase II inhibition (etoposide and doxorubicin). Cell cycle analysis was performed 4 and 20 h following a 4 h chemical exposure. Cells exposed to all compounds experienced S-phase arrest at the 8h time point, but by 24 h had markedly different cell cycle responses. Cells exposed to compounds that cause covalent modification had a strong G2/M arrest at 24 h. These cells also had a robust (>25-fold) increase in mutant frequency, and had a moderate but sustained p53 response at 4, 8, and 24h, detectable as approximately 2-5-fold increases in transcript levels for p21WAF1/CIP1, GADD45alpha, BTG2, and cyclin G1. In contrast, cells exposed to the reactive oxygen compounds had little or no G2/M arrest at 24 h and no increase in mutant frequency. In addition, these compounds caused a strong but transient induction of the p53 pathway, detectable as 15-25-fold increases in p21WAF1/CIP1 transcription at 4 h that decreased dramatically by 8h and was near control levels at 24 h. Thus, the mutagenic effect of compounds was consistent with G2/M arrest and sustained kinetics of p53 pathway activation. Global gene expression data were also consistent with the mutagenesis data. Activation of genes associated with cell cycle arrest, the p53 and TNF-related pathways, and chemokines and chemokine receptors, were particularly evident for the reactive oxygen compounds. In contrast, the most mutagenic compounds caused fewer and less robust changes in global gene expression. There was therefore an inverse relationship between global gene expression and mutagenic potency.

The purpose of this study was to examine the predictive value of humoral and cellular immune parameters in determining the immunotoxic effects of the oral administration of azathioprine (AZA), cyclophosphamide (CY), or cyclosporin A (CsA) at doses of 25/17, 10, or 25 mg/kg per day, respectively, for 30 days in F344 female rats. The effect of these known immunosuppressive compounds on the immune response was assessed in a humoral model that consisted of the administration of nitrophenyl-chicken gamma globulin (NP-CgammaG) and keyhole limpet hemocyanin (KLH) antigens during immunosuppressive treatment and the measurement of resulting rat antigen-specific IgG and IgM, as well as total IgG, levels. Cellular assessment parameters were collected from the same groups of animals as the humoral parameters and included organ weights and cellularity, hematology, lymphocyte phenotype characteristics, spleen cell mitogen stimulation (T and B cell-dependent), splenic natural killer (NK) cell cytotoxicity, and bone marrow cellularity and lymphocyte phenotype differential. Although decreases in several of the cellular assay parameters were observed, the only functional assays to demonstrate a statistically significant immunosuppressive effect by all three immunosuppressive agents were the antigen-specific serum IgG levels. The primary (day 10; 15 days post-immunization) and secondary (day 25; 5 days post-rechallenge) nitrophenyl (NP) responses were significantly suppressed by > or =60%. The use of NP hapten provided consistent responses when analyzed with a sensitive, well developed, ELISA methodology. Absolute lymphocyte phenotyping and lymphocyte hematology were also predictive of T cell immunosuppression for all three compounds. The data presented herein suggests that these two parameters, NP-IgG humoral response and lymphocyte phenotyping, are sufficient for identifying immunosuppressive compounds.