The concept of “repeated exposure” and possible links to epigenetic regulations. Repeated dose studies introducing baseline responses and transient responses with possible link to epigenetics

Jun Kanno
Head of Cellular and Molecular Toxicology,
NIHS, Japan
The Percellome Project was primarily designed for the comprehensive drawing of gene network(s) in a time- and dose-dependent way after a single oral dosing of a chemical. The dose of each test chemical was determined by the intensive dose-finding study, and the highest dose was set to a level (‘signal dose’) that does not induce morphological changes (macro and micro) and clinical symptoms at 24 or during the first 24 hours post administration. Consequently, ‘phenotypic anchoring’ was not considered as a tool for the transcriptomic data analysis. Along with the adoption of ‘Per cell’ normalisation strategy, use of gene knockout mice were considered for objective analysis of the gene network. It was expected that the gene network located downstream of the knocked-out gene will be highlighted as its ‘shadow’. Indeed, for example, when aryl hydrocarbon receptor knock-out mouse (AhRKO) was challenged with 2,3,7,8 tetrachloro dibenzo-p-dioxin (TCDD) or 3-methyl cholanthrene (3-MC) and compared with wild type mice data, a group of genes including those known to be located downstream of AhR are silenced. The gene list was larger than that of known downstream genes.
During this analysis, Dr Kanno and his team came up with an idea of a new concept of repeated dosing: the ‘chemically-induced transgenic state’. This concept allows us to compare the repeated-dose mice with the KO mice by challenging with a same test chemical. A series of trial studies were performed with a protocol as follows; all 48 mice were given a same amount of chemical A for up to 14 days by oral gavage, and then given, on the next day, a single gavage of the same chemical A or different chemical B at a dose of 0 (vehicle control), low, middle or high dose (in the range of ‘signal dose’) and sampled at 2, 4, 8 and 24 hours thereafter for transcriptomic analysis (designated as [14+1] study).
As a result, compared with mice only receiving single gavage (designated as [0+1] study), we found that repeated dosing induces two types of responses on gene expression, i.e. baseline response and transient response. In general, when the baseline (vehicle control group) goes down (up), the transient response is attenuated (exaggerated). Further analysis on the data, including those from [4+1], [2+1] and [1+1] studies, and using in silico method on the upstream events was discussed to help understanding of the molecular basis of repeated exposure including possible epigenetic mechanisms.