There were significant differences in mRNA

There were significant differences in mRNA expression levels observed for 7 out of 15 analyzed genes. This observation suggests, that selection of a reference/control group may influence interpretation of gene expression studies performed in liver pathologies. This study directly evidenced that analysis of ABCB11, ABCC1, ABCG2, SLC10A1, SLC16A1, SLCO1B1 and SLCO2B1 expression studies may be affected by the type of the reference liver used. As for protein abundance, with structure being more stable than mRNA structures, significant differences were noted for only two studied transporters, namely P-gp (MDR1, ABCB1), one of the most important drug transporters (higher values in donors) as well as NTCP (SLC10A1), a sodium-dependent uptake transporter expressed in the basolateral membrane of human hepatocytes, primarily responsible for the uptake of bile acids from the sinusoids (higher values in metastatic liver tissue) [15,16]. Combining the changes in mRNA expression and protein abundance data of SLC10A1/NTCP and ABCB1/P-gp it can be observed that mRNA levels paralleled protein information for NTCP in both types of the studied livers (significantly lower levels in donor livers), whereas levels of mRNA were comparable in donor and metastatic liver tissues, but the protein abundance was significantly different (higher in donor livers). The latter observation suggests that analysis of ABCB1 mRNA expression results and P-gp protein (especially when quantitative methods are used) may lead to different conclusions depending on whether donor livers or metastatic livers are taken as reference tissues. There are several factors that may be responsible for the observed findings. Organ donors are usually administered emergency drugs, including cardiovascular agents (dobutamine, dopamine, ephedrine, lidocaine, nitroprusside, procainamide, phenylephrine, vasopressin), 2179 (cephalosporins, clindamycin, ampicillin/sulbactam, gentamicin, vancomycin), insulin and mannitol. In the case of subjects with brain injury, they can additionally receive dexamethasone and phenytoin, drugs with proven influence on drug metabolizing enzyme and transporter gene expression (influence depends on duration of drug treatment) [17,18]. Some donors, especially those deceased in accidents, experience multiorgan trauma (bone fractures and muscle injuries), which may result in release of not yet defined factors affecting transporter gene expression (more prominent changes at gene expression levels were observed in the present studies). Additionally, donor liver samples used in several reports might present some abnormalities, like hepatic steatosis or fibrotic changes (this is not the case of the current study), which could further influence the reported conclusions [10,11,19]. Metastatic patients are administered general anesthesia drugs (in the present study: sevoflurane, propofol, rocuronium, fentanyl), and in the current study colon cancer patients were significantly older than organ donors. However, there is no direct data available on age- related NCTP and P-gp abundance changes in the analyzed samples. The P-gp quantification through the age interval from 7 to 70 years revealed that age and P-gp protein expression were not correlated [11]. Based on the latter observation it might be suggested that age should not be a factor determining the observed differences in P-gp levels in the donor and nontumoral metastatic livers. As for liver samples obtained from patients undergoing metastatic colon cancer resection, malignancy might be considered as a factor with potential systemic effects, including hepatic transporter expression. The decrease in ABCB1 gene expression in normal intestinal tissue in patients with colorectal cancer (when compared with healthy individuals) was also documented [20]. Evidence exists that serum profile of circulating cytokines is significantly altered in colorectal cancer [21] and it may probably affect functions of peripheral tissues. Some inflammatory mediators (e.g. interleukin-6, IL-6) were demonstrated to suppress ABCB1 expression in primary hepatocytes, as well as in HepaRG cell line, and significant elevation of IL-6 colon cancer patients was reported [[21], [22], [23]]. We also documented significant reduction of ABCB1 transcription in another general inflammatory state, i.e. in mucosal biopsy specimens from patients with ulcerative colitis, and significant reduction of P-gp protein levels in Caco-2 cells under IFN-γ exposure [24]. Hence, reduced abundance of P-gp in normal liver tissue, observed in the current study may potentially result from generalized cancerous process and systemic changes in immune system. Unfortunately, plasma samples from patients involved in the current study were not available, which may be considered as one of the study limitations. Analysis of IL1B, IL6, IL10 and TNF relative expression was performed in the same liver samples that had been investigated for drug transporters gene expression. One should be aware of fact that measurement of in situ cytokine gene expression does not necessary reflect systemic changes in protein levels. However, we observed significantly increased expression of TNF in liver tissue from metastatic patients (242% of mean value for organ donor group). Since TNF is mainly produced by immune cells, acting as a potent mediator of inflammation, its altered expression observed in the current study may be related to immunological processes, as consequence of metastatic liver disease (due to colon cancer).