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Drug adverse reactions predictability: exploring the mechanisms underlying the unexplained interindividual differences in drug metabolism and transport

Final Report Summary - DARMEC (Drug adverse reactions predictability: exploring the mechanisms underlying the unexplained interindividual differences in drug metabolism and transport)


Drug treatment is often inefficient. Only 30–60% of patients respond properly to treatment with e.g. antidepressants, beta-blockers, statins and antipsychotics. Furthermore, adverse drug reactions (ADRs) frequently occur and cause about 7% of all hospital admissions, a frequency that is increased to 30% in elderly subjects above 70 years of age. Epigenetic modifications have been demonstrated to potentially participate in the regulation of more than 90 human genes important for absorption, distribution, metabolism and excretion of drugs (ADME-drugs). However, it has been more and more evidenced that epigenetic modifications act in a concerted way with several other components of an increasingly complex regulatory orchestra. In certain cases, epigenetic modifications resulting from disease progression or drug treatment can be monitored not only in the affected tissue, but also in body fluids by determining DNA elements originating from e.g. tumors. Such circulating DNA elements constitute a novel class of pharmacoepigenetic biomarkers amenable for use to improve and individualize drug therapy. In addition, several drugs with epigenetic actions are currently used or on clinical trials that are given in conjunction with e.g. anticancer therapy in order to decrease drug resistance. The best examples are DNA Methyltransferases (DNMTs) and Histone Deacetylase (HDACs) inhibitors. Apart from epidrugs, other xenobiotics may also possess epigenetic activity. For example, it was demonstrated that exposure of mice to certain drugs or drug receptor ligands during fetal life can trigger epigenetic modifications of specific hepatic genes, causing altered ADME gene expression in adult mice.


In this project, we aim at elucidating the substantial unexplained fraction of interindividual variability in drug response and metabolism using a 3D multi-cell in vitro model of human liver in comparison to transcriptome and phenotype data obtained from a bank of 130 human livers with the following aims:
• AIM1: to unmask the epigenetically regulated ADME genes
• AIM2: to evaluate the epigenetically regulated ADME genes as interindividual variability
biomarkers. A list of epimarks of interindividual variation of drug ADME processes will be
• AIM3: to test the epigenetic influence of candidate drugs. For this aim, the establishment of a stable model for chronic drug exposure is needed.
This approach is extensively multidisciplinary and multisectorial. The experimental design includes tissue bioengineering techniques to develop organotypic cultures, drug metabolism functional profiling, and molecular biology approaches, from the perspective of cutting-edge technology.


We have identified DNA hydroxymethylation as an abundant and functionally important epigenetic mark in the liver, and developed the appropriate technology allowing the base-pair resolution discrimination between DNA methylation and hydroxymethylation (Ivanov et al., 2013). We have also implemented this technological development in protocols for Next Generation Sequencing and Illumina methylation arrays, which are widely used by the scientific community (Ivanov et al., 2016). In collaboration with the Estonian Genome Center, we have defined the combinations of genetic and DNA methylation variations that are liver, muscle, and adipose tissue specific. One of the most striking findings is the better potential of DNA methylation to predict tissue of origin with respect single nucleotide genetic variations ((Bonder et al., 2014))
Secondly, we have characterized liver and muscle organotypic cultures for the long-term exposure to several drugs that affect both the transcriptome and the epigenome (Holmgren et al., 2014; Peric et al., 2015). Of particular interest, we have defined a list of genes subjected to epigenetic regulation that could become biomarkers of drug response and targets for intervention against drug resistance.
The gene expression dynamics show gene-specific patterns of variation under different drugs exposure which is indicative of a context-dependent response to the pharmacological intervention. On the other hand, the memory of the expression variations seems to depend on the specific drug used.
We have also described the genome-wide transcriptomic remodeling of organotypic models of human liver induced by epidrugs, and, for the first time in human liver, we show how these dynamics in gene expression can be associated with global variations in DNA methylation and hydroxymethylation in a panel of genes important for drug metabolism. This sets the basis for the identification of DNA-based biomarkers of gene expression changes associated to pharmacological treatment, which could be used for monitoring drug response and toxicity. Indeed, in the treatment of cancer, several DNA methylation biomarkers of drug response have already been reported (Ivanov et al., 2014)
Finally, when analyzing those DNA methylation and hydroxymethylation variants in the bank of 130 human livers, and their correlation with transcription, we have identified that the global DNA hydroxymethylation levels are highly variable among individual livers and that the genomic distribution of DNA hydroxymethylation is strikingly non-uniform along the chromosomes. Moreover, DNA hydroxymethylation is also in this study associated with active transcription (Ivanov et al., 2016).
This opens a complete new avenue for the identification of the regulatory roles of this recently re-discovered DNA epigenetic modification.

Potential impact and use

The identification of interindividual variability in the levels of both global and gene-specific DNA hydroxymethylation, together with their association to variability in gene expression of drug metabolizing genes, represents a breakthrough in the field of Pharmacogenetics and implies that:
- These two DNA epigenetic marks, DNA methylation and hydroxymethylation, need to be typed independently from each other, and hence not with traditional bisulfite conversion techniques.
- DNA hydroxymethylation in human adult liver, rather than representing a mere intermediate in the cytosine de-methylation pathway, could have a specific role in modulating the transcripts levels of specific genes in response to endogenous or exogenous stimuli.
- DNA methylation and hydroxymethylation variants could be used as predictors of drug response. This is why the new line of research that I am establishing consists of integrating these variants in the prediction of response to anticancer drugs in liver cancer (Kasela et al., 2016).

Moreover, the induction of both transcriptomic and epigenetic perturbations in drug metabolising genes by specific drug treatments sets the basis for the identification of DNA-based biomarkers of gene expression changes associated to pharmacological treatment, which could be used for monitoring drug response and toxicity.

The newly established collaborations in the clinical, technological and academic sector will also contribute not only to the bench-to-bed transition, but to the exploration of a new potential layer of gene expression regulation based on DNA hydroxymethylation.


Bonder, M.J. Kasela, S., Kals, M., Tamm, R., Lokk, K., BARRAGAN, I., Buurman, W.A. Deelen, P., Greve, J.-W. Ivanov, M., et al. (2014). Genetic and epigenetic regulation of gene expression in fetal and adult human livers. BMC Genomics 15, 860.

Holmgren, G., Sjögren, A.-K. BARRAGAN, I., Sabirsh, A., Sartipy, P., Synnergren, J., Björquist, P., Ingelman-Sundberg, M., Andersson, T.B. and Edsbagge, J. (2014). Long-Term Chronic Toxicity Testing Using Human Pluripotent Stem Cell–Derived Hepatocytes. Drug Metab. Dispos. 42, 1401–1406.

Ivanov, M., Kals, M., Kacevska, M., BARRAGAN, I., Kasuga, K., Rane, A., Metspalu, A., Milani, L., and Ingelman-Sundberg, M. (2013). Ontogeny, distribution and potential roles of 5-hydroxymethylcytosine in human liver function. Genome Biol. 14, R83.

Ivanov, M., BARRAGAN, I., and Ingelman-Sundberg, M. (2014). Epigenetic mechanisms of importance for drug treatment. Trends Pharmacol. Sci. 35, 384–396.

Ivanov, M., Kals, M., Lauschke, V., BARRAGAN, I., Ewels, P., Käller, M., Axelsson, T., Lehtiö, J., Milani, L., and Ingelman-Sundberg, M. (2016). Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression. Nucleic Acids Res. 44, 6756–6769.

Kasela, S., Ivanov, M, Sayed, S, Nobre, A, Espino, L, Marabita, F, Milani, L, and BARRAGAN, I (2016). Cross-omics interactions for the identification of new biomarkers in hepatocellular carcinoma. In SEBBM16, (Salamanca), p. 170.

Peric, D., BARRAGAN, I., Giraud-Triboult, K., Egesipe, A.-L. Meyniel-Schicklin, L., Cousin, C., Lotteau, V., Petit, V., Touhami, J., Battini, J.-L. et al. (2015). Cytostatic Effect of Repeated Exposure to Simvastatin: A Mechanism for Chronic Myotoxicity Revealed by the Use of Mesodermal Progenitors Derived from Human Pluripotent Stem Cells. STEM CELLS 33, 2936–2948.