Ontogeny, distribution and potential roles of 5-hydroxymethylcytosine in human liver function.

BACKGROUND: Interindividual differences in liver functions such as protein synthesis, lipid and carbohydrate metabolism and drug metabolism are influenced by epigenetic factors. The role of the epigenetic machinery in such processes has, however, been barely investigated. 5-hydroxymethylcytosine (5hmC) is a recently re-discovered epigenetic DNA modification that plays an important role in the control of gene expression. RESULTS: In this study, we investigate 5hmC occurrence and genomic distribution in 8 fetal and 7 adult human liver samples in relation to ontogeny and function. LC-MS analysis shows that in the adult liver samples 5hmC comprises up to 1% of the total cytosine content, whereas in all fetal livers it is below 0.125%. Immunohistostaining of liver sections with a polyclonal anti-5hmC antibody shows that 5hmC is detected in most of the hepatocytes. Genome-wide mapping of the distribution of 5hmC in human liver samples by next-generation sequencing shows significant differences between fetal and adult livers. In adult livers, 5hmC occupancy is overrepresented in genes involved in active catabolic and metabolic processes, whereas 5hmC elements which are found in genes exclusively in fetal livers and disappear in the adult state, are more specific to pathways for differentiation and development. CONCLUSIONS: Our findings suggest that 5-hydroxymethylcytosine plays an important role in the development and function of the human liver and might be an important determinant for development of liver diseases as well as of the interindividual differences in drug metabolism and toxicity.

Extra-hepatic cancer represses hepatic drug metabolism via interleukin (IL)-6 signalling.

PURPOSE: In many cancer patients, the malignancy causes reduced hepatic drug clearance leading to potentially serious complications from the use of anticancer drugs. The mechanisms underlying this phenomenon are poorly understood. We aimed to identify tumor-associated inflammatory pathways that alter drug response and enhance chemotherapy-associated toxicity. METHODS: We studied inflammatory pathways involved in extra-hepatic tumor mediated repression of CYP3A, a major hepatic drug metabolizing cytochrome P450 subfamily, using a murine Engelbreth-Holm-Swarm sarcoma model. Studies in IL-6 knockout mice determined the source of elevated IL-6 in tumor-bearing animals and monoclonal antibodies against IL-6 were used to intervene in this inflammatory pathway. RESULTS: Our studies confirm elevated plasma IL-6 levels and reveal activation of Jak/Stat and Mapk signalling pathways and acute phase proteins in livers of tumor-bearing mice. Circulating IL-6 was predominantly produced by the tumor xenograft, rather than being host derived. Anti IL-6 antibody intervention partially reversed tumor-mediated inflammation and Cyp3a gene repression. CONCLUSIONS: IL-6 is an important player in cancer-related repression of CYP3A-mediated drug metabolism and activation of the acute phase response. Targeting IL-6 in cancer patients may prove an effective approach to alleviating cancer-related phenomena, such as adverse drug-related outcomes commonly associated with cancer chemotherapy.

In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes.

DNA methylation is one of the most important epigenetic alterations involved in the control of gene expression. Bisulfite sequencing of genomic DNA is currently the only method to study DNA methylation patterns at single-nucleotide resolution. Hence, next-generation sequencing of bisulfite-converted DNA is the method of choice to investigate DNA methylation profiles at the genome-wide scale. Nevertheless, whole genome sequencing for analysis of human methylomes is expensive, and a method for targeted gene analysis would provide a good alternative in many cases where the primary interest is restricted to a set of genes. Here, we report the successful use of a custom Agilent SureSelect Target Enrichment system for the hybrid capture of bisulfite-converted DNA. We prepared bisulfite-converted next-generation sequencing libraries, which are enriched for the coding and regulatory regions of 174 ADME genes (i.e. genes involved in the metabolism and distribution of drugs). Sequencing of these libraries on Illumina's HiSeq2000 revealed that the method allows a reliable quantification of methylation levels of CpG sites in the selected genes, and validation of the method using pyrosequencing and the Illumina 450K methylation BeadChips revealed good concordance.

Hepatic fat loss in advanced nonalcoholic steatohepatitis: are alterations in serum adiponectin the cause?

UNLABELLED: Advanced liver fibrosis in nonalcoholic steatohepatitis (NASH) is often accompanied by a reduction in hepatic fat to the point of complete fat loss (burnt-out NASH), but the mechanisms behind this phenomenon have not been elucidated. Adiponectin is raised in cirrhosis of any cause and has potent antisteatotic activity. In this study we examined 65 patients with advanced biopsy-proven NASH (fibrosis stage 3-4) and 54 with mild disease (fibrosis stage 0-1) to determine if disappearance of steatosis correlated with changes in serum adiponectin. All patents had fasting blood tests and anthropometric measures at the time of liver biopsy. Liver fat was accurately quantitated by morphometry. Serum adiponectin was measured by immunoassay. When compared to those with early disease, patients with advanced NASH were more insulin-resistant, viscerally obese, and older, but there was no difference in liver fat content or adiponectin levels. Adiponectin had a significant negative correlation with liver fat percentage in the whole cohort (r = -0.28, P < 0.01), driven by patients with advanced NASH (r = -0.40, P < 0.01). In advanced NASH, for each 4 µg/L increase in adiponectin there was an odds ratio OR of 2.0 (95% confidence interval [CI]: 1.3-3.0, P < 0.01) for a 5% reduction in hepatic fat. Adiponectin was highly and significantly associated with almost complete hepatic fat loss or burnt-out NASH (12.1 versus 7.4 µg/L, P = 0.001) on multivariate analysis. A relationship between adiponectin, bile acids, and adipocyte fexaramine activation was demonstrated in vivo and in vitro, suggestive of hepatocyte-adipocyte crosstalk. CONCLUSION: Serum adiponectin levels in advanced NASH are independently associated with hepatic fat loss. Adiponectin may in part be responsible for the paradox of burnt-out NASH. (HEPATOLOGY 2012).

Epigenetic-dependent regulation of drug transport and metabolism: an update.

The pharmacokinetics of a drug are subject to large interindividual variability, which can result in lack of response or adverse drug reactions. In addition to genetic polymorphisms and drug interactions, key genes involved in the metabolism and transport of drugs are demonstrated to have epigenetic influences that can potentially affect interindividual variability in drug response. Emerging studies have focused on the importance of DNA methylation for ADME gene expression and for drug action and resistance, particularly in cancer. However, the epigenetic and ncRNA-dependent regulation of these genes, as well as the pharmacological consequences, is in need of greater attention. In the current review we provide an update of epigenetic and ncRNA-dependent regulation of ADME genes.

DNA methylation dynamics in the hepatic CYP3A4 gene promoter.

The CYP3A4 gene, encoding the major drug metabolizing enzyme in humans, exhibits a high interindividual variation in hepatic expression that can lead to interindividual differences in drug metabolism and associated adverse drug effects. Much of the interindividual variability in CYP3A4 remains unexplained. In the present study we investigated the role of DNA methylation in influencing the interindividual CYP3A4 expression. Individual CpG methylation within the ∼12 kb CYP3A4 regulatory region was investigated in 72 adult as well as in 7 fetal human livers using bisulfite sequencing. We identified highly variable CpG methylation sites in adult livers, which correspond to important CYP3A4 transcription factor binding sites including the proximal promoter, XREM and CLEM4 as well as in separate C/EBP and HNF4α binding regions. CpG hypermethylation within these regulatory regions was observed in fetal livers when compared to adult livers. This data suggests that dynamic DNA methylation elements are likely associated with key regulatory CYP3A4 promoter regions and may potentially contribute to the commonly observed interindividual expression of CYP3A4 as well as the hepatic developmental shift in its expression. The findings provide novel insight to CYP3A4 regulation with possible implications for understanding interindividual differences in drug response.

Extrahepatic cancer suppresses nuclear receptor-regulated drug metabolism.

PURPOSE: To determine the mechanisms by which tumors situated in extrahepatic sites can cause profound changes in hepatic drug clearance, contributing to altered drug response and chemotherapy resistance. EXPERIMENTAL DESIGN: We studied in wild-type or transgenic CYP3A4 reporter mice implanted with the murine Engelbreth-Holm-Swarm sarcoma changes in nuclear receptor and hepatic transcription factor expression and/or function, particularly related to CYP3A gene regulation. RESULTS: Repression of hepatic CYP3A induction was dramatic and associated with reduced levels of C/EBPß isoforms, impaired pregnane X receptor, and constitutive androstane receptor function. Unexpectedly, extrahepatic tumors strongly reduced nuclear accumulation of retinoid X receptor alpha (RXRα) in hepatocytes, providing a potential explanation for impaired function of nuclear receptors that rely on RXRα dimerization. Profiling revealed 38 nuclear receptors were expressed in liver with 14 showing between 1.5- and four-fold reduction in expression in livers of tumor-bearing animals, including Car, Trß, Lxrß, Pparα, Errα/ß, Reverbα/ß, and Shp. Altered Pparα and γ induction of target genes provided additional evidence of perturbed hepatic metabolic control elicited by extrahepatic tumors. CONCLUSIONS: Extrahepatic malignancy can affect hepatic drug metabolism by nuclear receptor relocalization and decreased receptor expression and function. These findings could aid the design of intervention strategies to normalize drug clearance and metabolic pathways in cancer patients at risk of chemotherapy-induced toxicity or cancer cachexia.

Systemic inflammatory response predicts prognosis in patients with advanced-stage colorectal cancer.

We aim to confirm the prognostic value of an inflammation-based prognostic score (the Glasgow Prognostic Score [GPS]) in advanced colorectal cancer, to explore a predictive pattern of plasma cytokines and their gene polymorphisms for clinical outcome, and to investigate which cytokines contribute to GPS. Inflammatory markers were measured at baseline in 52 patients with stage IV colorectal cancer. Germline DNA was genotyped for interleukin (IL)-1beta-511, IL-1beta +3954, IL-6-174, TNF-alpha-308, IL-10-1082, and IL-10 -592 using Sequenome mass spectrometry-based genotyping technology. Toxicity was graded by the National Cancer Institute Common Toxicity Criteria version 2.0. Response was assessed by the Response Evaluation Criteria in Solid Tumors. Glasgow Prognostic Score, carcinoembryonic antigen and hypoalbuminemia were predictive of overall survival (OS). Hypoalbuminemia (< or = 35 g/L) and GPS were predictive of toxicity; GPS 2 was predictive of increased grade 2/3 toxicity compared with patients with a GPS of 0 or 1 (P < .05). Interleukin-10-592AA and IL-10 -1082CC predicted for OS (P < .05). Elevated levels of circulating IL-4 and soluble glycoprotein 130 (sgp130) were associated with increased grade 2/3 toxicity. Significantly elevated levels of IL-6 and sgp130 were observed in patients with a GPS of 2 (P < .05). In this patient group, inflammatory markers predict for clinical outcome. This could improve prognostication and allow for intervention strategies to reduce tumor-associated inflammation.

Inflammation and CYP3A4-mediated drug metabolism in advanced cancer: impact and implications for chemotherapeutic drug dosing.

BACKGROUND: The inability to accurately predict treatment outcomes for cancer patients in terms of tumour response and anticancer drug toxicity is a severe limitation inherent in current approaches to chemotherapy. Many anticancer drugs are metabolically cleared by cytochrome P450 3A4 (CYP3A4), the predominant CYP expressed in liver. CYP3A4 expression exhibits marked interindividual variation and is repressed in acute inflammatory states. OBJECTIVES: (1) To review the relevance of CYP3A4 variability to drug metabolism in the setting of cancer and to understand how inflammation associated with malignancy contributes to both this variability and to adverse treatment outcomes. (2) To examine the relationship between tumour-induced inflammation and repression of CYP3A4 and to explore methods of dosing of anticancer drugs in the setting of advanced cancer. METHODS: Review of relevant literature covering both human and animal studies as well as in vitro mechanistic studies. RESULTS/CONCLUSIONS: Interindividual variability in CYP3A4 expression is a major confounding factor for effective cancer treatment and methods to predict CYP3A4-mediated drug clearance may have clinical utility in this setting. Although acute inflammation has long been recognised to repress drug metabolism, it is now becoming apparent that cancer patients exhibiting clinical and laboratory features of an inflammatory response have reduced expression of CYP3A4 and possibly other genes relevant to anticancer drug disposition.

Regulation of drug-metabolizing enzymes and transporters in infection, inflammation, and cancer.

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 07 meeting in Washington, DC. The presentations discussed the phenomenology, clinical consequences, and underlying mechanisms of cytochrome P450 and drug transporter regulation by inflammatory and infectious stimuli. Although considerable insights into the links between inflammatory mediators and altered hepatic drug clearance pathways have been gained from previous studies with acute inflammatory stimuli, this symposium highlighted recent advances in understanding how these processes operate in other organs and chronic inflammatory states relevant to human diseases. The development of mouse models of live bacterial infection provides excellent opportunities to explore the impact of infection on drug metabolism beyond the well characterized effects of bacterial endotoxin. Altered levels of cytochromes P450 and especially drug transporters due to inflammation in brain, intestine, and placenta have significant implications for the use of many drugs in diverse clinical settings. The consequences of inflammatory cytokine production by tumors for drug safety and efficacy in cancer patients were outlined. Repression of drug clearance pathways by tumor-derived cytokines may result in extreme toxicity to chemotherapy, compromising treatment of many cancers. It is fitting that, in honoring the career contributions and achievements of Dr. Kenneth W. Renton, this symposium reinforced the clinical relevance of this field.