The reference liver-CYP450 and UGT enzymes in healthy donor and metastatic livers: the impact of genotype.

BACKGROUND: Hepatic enzymes involved in drug metabolism vary markedly in expression, abundance and activity, which affects individual susceptibility to drugs and toxicants. The present study aimed to compare mRNA expression and protein abundance of the most pharmacologically relevant drug-metabolizing enzymes in two main sources of the control liver samples that are used as the reference, i.e. organ donor livers and non-tumorous tissue from metastatic livers. An association analysis of the most common genetic variants with mRNA and protein levels was also performed. METHODS: The CYP450 and UGT enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, UGT1A1, UGT1A3, UGT2B7 and UGT2B15) were analyzed for mRNA (qPCR) and protein abundance (LC-MS/MS) in healthy donors (n = 11) and metastatic (n = 13) livers. Genotyping was performed by means of TaqMan assays and pyrosequencing. RESULTS: Significantly higher protein abundance in the metastatic livers was observed in case of CYP2C9, CYP2D6, and UGT2B7, and for UGT1A3 the difference was only significant at mRNA level. For all the enzymes except CYP2E1 some significant correlation between mRNA and protein content was observed, and for UGT1A1 an inverse correlation with age was noted. CYP2C19, CYP3A5 and CYP2D6 were significantly affected by genotype. CONCLUSION: The selection of a control group for the study on drug-metabolizing enzymes (e.g. in pathological states) may possibly affect its conclusions on differences in mRNA and protein content. Genotyping for common functional variants of CYP450 enzymes should be performed in all studies on drug-metabolizing enzymes.

Late Recurrence of Hepatocellular Carcinoma in a Patient 10 Years after Liver Transplantation Unrelated to Transplanted Organ.

Liver transplantation (LTx) is an accepted method of hepatocellular carcinoma (HCC) treatment in cirrhotic patients; however, it has many limitations, and there is a substantial risk of recurrence. Most relapses occur within the first 2 posttransplant years. We aimed to present a late extrahepatic recurrence of HCC 10 years after LTx, and we discuss the possible risk factors and ways to improve transplantation results. A 68-year-old patient with liver cirrhosis and HCC on the background of chronic HCV and past HBV infection was transplanted urgently due to the rapid decompensation. Anti-HCV treatment before surgery was unsuccessful. Pretransplant computed tomography showed 1 focal 4.5 cm lesion consistent with HCC. Histopathology of the explanted organ showed 2 nodules outside the Milan criteria. Angioinvasion was not found. The patient achieved a sustained viral response to pegylated interferon and ribavirin 2 years post-LTx. Eight years were uneventful. CT of the abdomen performed occasionally was normal. Ten years after LTx, the patient unexpectedly presented with shortness of breath, fatigue, and weight loss. Two metastatic nodules of HCC in the lungs and pelvis were found. Although late HCC recurrence post-LTx is rare, it should be always considered, especially when risk factors such as viral infections and underestimation of tumor advancement were identified. We advocate that oncological surveillance of HCC relapse has to be continued during the whole posttransplant period. High AFP levels, the unfavorable neutrophil to lymphocyte ratio, and better estimation of primary tumor size seem to be useful in the identification of good candidates for transplantation.

Protein Abundance of Hepatic Drug Transporters in Patients With Different Forms of Liver Damage.

Hepatocellular transporter levels were quantified using quantitative reverse transcription polymerase chain reaction and liquid chromatography-tandem mass spectrometry methods. Liver function deterioration (Child-Pugh class C) produced significant protein abundance (mean values) increase (to healthy livers) in P-gp (to 260% (CV (coefficient of variation) 82%)) and MRP4 (CV 230%) (not detected in healthy livers), decrease in MRP2 (to 30% (CV 126%)), NTCP (to 34% (CV 112%)), OCT1 (to 35% (CV 153%)), OATP1B1 (to 46% (CV 73%)), and OATP2B1 (to 27% (CV 230%)), whereas BSEP (CV 99%), MRP3 (CV 106%), OAT2 (CV 97%), OCT3 (CV 113%), and OATP1B3 (CV 144%) remained unchanged. Alcoholic liver disease produced significant protein downregulation of MRP2 (to 30% (CV 134%)), NTCP (to 76% (CV 78%)), OAT2 (to 26% (CV 117%)), OATP1B1 (to 61% (CV 76%)), OATP1B3 (to 79% (CV 160%)), and OATP2B1 (to 73% (CV 90%)) of healthy tissue values. Hepatitis C produced BSEP (to 47% (CV 99%)) and OATP2B1 (to 74% (CV 91%)) protein reduction. Primary biliary cholangitis and primary sclerosing cholangitis demonstrated P-gp and MRP4 protein upregulation (to 350% (CV 47%) and 287% (CV 38%), respectively). Autoimmune hepatitis revealed P-gp (to 410% (CV 49%)) and MRP4 (CV 96%) increase, and MRP2 (to 18% (CV 259%)) protein decrease. Drug transporters' protein abundance depends on liver pathology type and its functional state.

The reference liver - ABC and SLC drug transporters in healthy donor and metastatic livers.

BACKGROUND: Analysis of results and conclusions in studies dedicated to pathology of the liver are usually based on comparison of pathological liver specimens and control/reference (considered as healthy) tissues. There are two main sources of the control liver samples used as the reference livers, i.e. deceased organ donor livers and non-tumorous tissue from metastatic livers, which are also applied for drug transporter investigations. However, no information has yet been published on drug transporters in these two major types of reference livers. METHODS: We explored ABC (P-gp, MRP1, MRP2, MRP3, MRP4, BCRP, BSEP) and SLC (NTCP, MCT1, OCT1, OCT3, OAT2, OATP1B1, OATP1B3, OATP2B1) family transporters expression (qPCR) and protein abundance (LC-MS/MS) in healthy donors (n = 9) and metastatic (n = 13) livers. RESULTS: The analysis of mRNA content revealed significant differences in ABCB11, ABCC1, ABCG2, SLC10A1, SLC16A1, SLCO1B1 and SLCO2B1 gene expression between livers from organ donors and patients who underwent surgical resection of metastatic tumors. The protein abundance of NTCP was significantly higher, whereas of P-gp significantly lower in non-tumorous tissues from metastatic livers. Greater inter-individual variability in protein abundance of all studied transporters in subjects with metastatic colon cancer was also observed. CONCLUSIONS: The results suggest that final conclusions in liver pathology studies may depend on the reference liver tissue used, especially in gene expression studies.