Identification of transcriptome profiles for the DNA‐damaging agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma cells

Abstract

It is believed that some aspects of genotoxicity are associated with changes in the transcription levels of certain genes, especially those involved in DNA repair and cell cycle control. Additionally, it is hypothesized that chemicals sharing a common mode of genotoxicity should exhibit similar changes in gene expression. We have evaluated these hypotheses by analyzing transcriptome profiles of mouse lymphoma L5178Y/TK(+/-) cells treated with bleomycin and hydrogen peroxide, two mutagens that produce genotoxicity by generating reactive free radicals. The cells were treated for 4 hr and RNA was isolated at the end of the treatment and after a 20 hr recovery. Transcriptome analyses were performed using the Clontech Mouse 1.2K cDNA microarray (1,185 genes) and hybridization with a (32)[P]-labeled probe. Of the genes examined, each mutagen altered the expression (1.5-fold or greater) of only two genes after the 4 hr treatment. In cells allowed to recover for 20 hr after treatment, bleomycin and hydrogen peroxide altered the expression of 8 and 5 genes, respectively. Many of the altered genes have some association with apoptosis. Of these genes, three (the genes encoding granzyme A, integrin beta 7, and 45 kDa calcium-binding protein precursor) were in common between chemical treatments. The expression of DNA repair and cell cycle controlling genes present on the array was not affected by the treatments. These results show that bleomycin and hydrogen peroxide both have unique and commonly regulated genes that have the potential to serve as biomarkers of exposure to agents causing DNA damage by free radical mechanisms.

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

“…In contrast to another study published from the ILSI genotoxicity working group [Seidel et al, 2003], our group did see changes in DNA repair and cell cycle control genes with mouse lymphoma cells. This other study on the treatment of L5178Y mouse lymphoma cells with bleomycin used a different platform (the Clontech Atlas array) with a different gene content and demonstrated effects only for genes associated with apoptosis.…”

“…Our studies did confirm that bleomycin induced many apoptosis-related genes in human cells (Table X). However, we did not observe the identical genes that were Seidel et al [2003] study. This highlights a potential for variability in results when using different platforms for measuring gene expression changes.…”

“…In the follow-up study, upregulated genes were noted from treatment with bleomycin, although the magnitude of change remained low (data not shown). The lack of reproducibility of data between experiments for a specific treatment using DNA microarrays has been observed by other investigators [Seidel et al, 2003]. These studies imply that there may be variability in DNA microarray data for genes that undergo minimal change, and a method of confirmation of gene expression change is desirable.…”

Genomic Approaches for Investigating Mechanisms of Genotoxicity

“…Therefore, the pathway analysis is crucial for evaluating genotoxic mechanism. This has also become apparent in studies performed by participating laboratories in HESI Genomic consortium (Akerman et al 2004;Dickinson et al 2004;Hu et al 2004;Islaih et al 2004;Seidel et al 2003Seidel et al , 2004 and others (reviewed in Aubrecht and Caba 2005). Although the HESI Genomic consortium studies were performed under similar conditions in p53 deficient L5189Y and p53 proficient TK-6 cell lines, considerable differences were evident at the individual gene level.…”

The Utility of Dna Microarrays for Characterizing Genotoxicity

“…Aardema and colleagues are analyzing additional data to help validate the utility of this approach. In addition, Gollapudi and colleagues reported certain gene expression changes that could serve as biomarkers of exposure to free radical-generating agents on the (Seidel et al 2003). Some preliminary results indicate that even within a category, namely, indirect-acting genotoxicants (hydroxyurea and taxol), differences exist in the gene expression patterns generated by these two compounds.…”