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Updated: Aug 24, 2026
Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform
Published on: January 13, 2016
Identification of a gene expression profile that discriminates indirect-acting genotoxins from direct-acting
Ting Hu1, David P Gibson, Gregory J Carr
1The Procter and Gamble Company, Miami Valley Laboratories, P.O. Box 538707 09, Cincinnati, OH 45253-8707, USA.
Abstract:
During the safety evaluation process of new drugs and chemicals, a battery of genotoxicity tests is conducted starting with in vitro genotoxicity assays. Obtaining positive results in in vitro genotoxicity tests is not uncommon. Follow-up studies to determine the biological relevance of positive genotoxicity results are costly, time consuming, and utilize animals. More efficient methods, especially for identifying a putative mode of action like an indirect mechanism of genotoxicity (where DNA molecules are not the initial primary targets), would greatly improve the risk assessment for genotoxins. To this end, we are participating in an International Life Sciences Institute (ILSI) project involving studies of gene expression changes caused by model genotoxins. The purpose of the work is to evaluate gene expression tools in general, and specifically for discriminating genotoxins that are direct-acting from indirect-acting. Our lab has evaluated gene expression changes as well as micronuclei (MN) in L5178Y TK(+/-) mouse lymphoma cells treated with six compounds. Direct-acting genotoxins (where DNA is the initial primary target) that were evaluated included the DNA crosslinking agents, mitomycin C (MMC) and cisplatin (CIS), and an alkylating agent, methyl methanesulfonate (MMS). Indirect-acting genotoxins included hydroxyurea (HU), a ribonucleotide reductase inhibitor, taxol (TXL), a microtubule inhibitor, and etoposide (ETOP), a DNA topoisomerase II inhibitor. Microarray gene expression analysis was conducted using Affymetrix mouse oligonucleotide arrays on RNA samples derived from cells which were harvested immediately after the 4 h chemical treatment, and 20 h after the 4 h chemical treatment. The evaluation of these experimental results yields evidence of differentially regulated genes at both 4 and 24 h time points that appear to have discriminating power for direct versus indirect genotoxins, and therefore may serve as a fingerprint for classifying chemicals when their mechanism of action is unknown.
Insights
Identifying genotoxic mechanisms is crucial for drug safety. This study uses gene expression analysis to differentiate direct-acting genotoxins from indirect-acting ones, offering a more efficient risk assessment method.
Area of Science:
- Toxicology
- Molecular Biology
- Genetics
Background:
- In vitro genotoxicity assays are standard for new drug safety evaluations.
- Positive results necessitate costly, time-consuming, animal-utilizing follow-up studies.
- Efficient methods to identify genotoxin mechanisms, particularly indirect ones, are needed for improved risk assessment.
Purpose of the Study:
- To evaluate gene expression tools for discriminating between direct-acting and indirect-acting genotoxins.
- To identify gene expression patterns that serve as a fingerprint for genotoxin classification.
- To improve the efficiency and reduce animal use in genotoxicity risk assessment.
Main Methods:
- Gene expression changes and micronuclei formation were analyzed in L5178Y mouse lymphoma cells.
- Cells were treated with six model genotoxins: direct-acting (MMC, CIS, MMS) and indirect-acting (HU, TXL, ETOP).
- Microarray analysis (Affymetrix) was performed on RNA samples at 4 and 24 hours post-treatment.
Main Results:
- Differentially regulated genes were identified at both 4 and 24-hour time points.
- These gene expression changes showed discriminating power between direct and indirect genotoxins.
- The findings suggest a potential gene expression fingerprint for classifying genotoxins based on their mechanism of action.
Conclusions:
- Gene expression profiling can effectively distinguish between direct and indirect genotoxins.
- This approach offers a more efficient alternative to traditional follow-up studies.
- The identified gene expression patterns may serve as a valuable tool for genotoxin classification and risk assessment.
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