Defining the impact of epigenetics on the expression of human reductases in heart and lung
Hoefer, Carrie Catherine
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Carbonyl reductases ( CBR s) and aldo-keto reductases ( AKR s) catalyze the two-electron reduction of many pharmacological substrates such as the anthracyclines daunorubicin (DAUN) and doxorubicin (DOX). The CBR s and AKR s are generally monomeric, cytosolic and ubiquitous in nature. Although DAUN and DOX have proven to be extremely beneficial anti-cancer therapies with up to 80% survival rates, their use is hampered by the development of anthracycline related cardiotoxicity. Despite the many well characterized genetic factors such as Down syndrome (DS), and genetic variations, such as single nucleotide polymorphisms (SNPs), in the CBR s and AKR s, a substantial amount of interpatient variability in mRNA and protein expression. remains unaccounted for. The variability of the protein expression in these reductases can range up to 10 fold in cardiac donors. The CBR s and AKR s have a distinct mechanism of action in reducing many pharmacological substrates; however exposure to many chemicals such as benzo[a]pyrene (B[a]P) can impact the expression of these genes. Therefore, instead of selectively looking at genetic factors, exploring other components of gene expression, such as epigenetics, may help elucidate the unexplained variability of expression observed in these reductases. We have demonstrated AKR7A2 to be the most abundant anthracycline reductase at the protein level in cardiac tissue from patients with and without DS. The abundance of AKR7A2 in DS is of interest because AKR7A2 lies on chromosome 1, whereas CBR1 lies within the DS critical region on chromosome 21. One may assume CBR1 to be the most abundant anthracycline reductase in donors with Down syndrome due to the unifying hypothesis of the 'gene dosage effect'. However, AKR7A2 protein content is also known to be one of the most significant factors in basal DAUN reductase activity. With the wide range of interpatient variability, and known genomic factors we decided to first examine the DNA methylation profiles of the anthracycline reductases, specifically AKR7A2. We found a DNA methylation cluster ∼900 base pairs (bp) upstream of the ATG start codon of AKR7A2 that contributes to AKR7A2 protein expression. To further expand our work we then examined lymphoblastoid cell lines which can be considered a 'proxy' tissue to our cardiac tissue. We found a unique methylation site located 232 bp upstream of the ATG start codon in AKR7A2 in DS donors that exhibits a unique pattern not found in donors without DS. Next, we compiled our methylation data with past data uncovered in our lab (genomic, demographic, mitochondrial) to develop a classification and regression tree (CART) model. CART models are widely used for medical decision making due to their flexibility and ease of interpretation. Our developed model was used to classify cardiac donors into high, intermediate or low metabolizers of DAUN activity. The CART model suggests that mitochondrial DNA (mtDNA) content, and the common mitochondrial deletion mtDNA 4977 are two of the most important determinants in assessing the metabolism of DAUN, followed by AKR7A2 protein content. Finally, we addressed the impact of a toxic carcinogen (B[a]P) found in cigarette smoke on the methylation profile of CBR1 in lung donors. It has recently been demonstrated that B[a]P can induce CBR1 mRNA expression; therefore we sought to explore the epigenetic characteristics of this phenomena. We found a distinct methylation site 1297 bp upstream of the ATG start codon that exhibited a 20% difference in methylation levels between smokers and never-smokers. This site also demonstrated to be a non-verified xenobiotic response element (XRE) binding motif. We then developed several unique plasmids (both methylated and un-methylated) that explored the CBR1 promoter region, and exposed these regions to B[a]P. This revealed that methylation can completely inhibit CBR1 promoter activity, and with exposure of B[a]P, promoter activity begins to increase towards normal levels in the methylated plasmids. Our work represents a very robust set of data that elucidates the interpatient variability within the anthracycline metabolizing enzymes.