Relationships between genomic, cell cycle, and mutagenic responses of TK6 cells exposed to DNA damaging chemicals.

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.

Administration of a PPARalpha agonist increases serum apolipoprotein A-V levels and the apolipoprotein A-V/apolipoprotein C-III ratio.

Apolipoprotein A-V (apoA-V) first gained attention as a regulator of triglycerides through transgenic mouse studies. Furthermore, peroxisome proliferator-activated receptor alpha (PPARalpha) agonists such as fenofibrate increase apoA-V mRNA expression. Our group recently developed the first assay to quantitate serum apoA-V levels. Therefore, we sought to determine whether administration of a PPARalpha agonist would increase circulating apoA-V. Cynomolgus monkeys were dosed for 14 days with 0.3 mg/kg/day LY570977 L-lysine, a potent and selective PPARalpha agonist. Blood samples were drawn throughout the treatment period and after a 2 week washout. Administration of the PPARalpha agonist caused a 50% decrease in triglycerides that reversed at washout. Serum apoA-V concentrations increased 2-fold, correlated inversely with triglycerides, and were reversible at washout. The apoA-V/apoC-III ratio increased >2-fold, with this increase also reversible at washout. These data demonstrate for the first time that a PPARalpha agonist increases circulating apoA-V protein levels and the apoA-V/apoC-III ratio.

Comparison of gene expression changes induced in mouse and human cells treated with direct-acting mutagens.

Exposure to DNA-damaging agents can elicit a variety of stress-related responses that may alter the gene expression of numerous biological pathways. We used Affymetrix microarrays to detect gene expression changes in mouse lymphoma (L5178Y) and human lymphoblastoid (TK6) cells in response to methyl methanesulfonate (MMS; a prototypical alkylating agent) and bleomycin (a prototypical oxidative mutagen). Cells were treated for 4 hr, and RNA was isolated either at the end of the treatment or after a 20-hr recovery period. Two concentrations of each agent were used based on cytotoxicity levels and Tk mutant frequencies. Our microarray data analysis indicated that MMS and bleomycin gene expression responses were considerably different in mouse cells versus human cells. The results also suggested that more comprehensive cellular responses to MMS and bleomycin occurred in TK6 cells than in L5178Y cells. In contrast to L5178Y cells, the response of TK6 cells to MMS and bleomycin was characterized by the induction of p53-dependent genes that are involved in DNA repair, cell cycle regulation, and apoptosis. It appears that the induction of DNA damage by MMS in human TK6 cells mediated cytotoxicity and led to decreased cell survival. This may explain the greater sensitivity of TK6 cells to cytotoxic effects of MMS compared to L5178Y cells. Bleomycin exerted comparable cytotoxic effects in the two cell lines. Overall, these studies were unable to identify distinctive gene expression changes that differentiated bleomycin from MMS in either TK6 cells or mouse lymphoma cells.

The utility of DNA microarrays for characterizing genotoxicity.

Microarrays provide an unprecedented opportunity for comprehensive concurrent analysis of thousands of genes. The global analysis of the response of genes to a toxic insult (toxicogenomics), as opposed to the historical method of examining a few select genes, provides a more complete picture of toxicologically significant events. Here we examine the utility of microarrays for providing mechanistic insights into the response of cells to DNA damage. Our data indicate that the value of the technology is in its potential to provide mechanistic insight into the mode of action of a genotoxic compound. Array-based expression profiling may be useful for differentiating compounds that interact directly with DNA from those compounds that are genotoxic via a secondary mechanism. As such, genomic microarrays may serve as a valuable alternative methodology that helps discriminate between these two classes of compounds. Key words: biomarkers, gene expression profile, genetic toxicology, mechanism of action, toxicogenomics.