Cytochrome P450 mRNA expressions along with in vitro differentiation of hepatocyte precursor cells from fetal, young and old rats.

Abstract

Non-differentiated cells are attractive targets for cell therapy. During liver regeneration oval cells intensively proliferate and differentiate extending their metabolic activity. Hepatic cytochromes P450 (CYPs) can be linked either with metabolic activation of toxic compounds or drug metabolism. We investigated the differentiation and biotransformative potential of non-differentiated cells in primary cell cultures isolated from livers of fetuses (16-days-old), young (4-monthsold) and old (20-months-old) rats. Under the conditions of experimental hepatocarcinogenesis, adult rats were fed for three weeks with CDE diet. Liver cells were cultured and precursor cells were differentiated to hepatocytes following induction with sodium butyrate (SB) or dimethyl sulphoxide (DMSO) in culture on MesenCult medium. We identified a number of cells expressing Thy-1, CD34, α-fetoprotein, cytokeratines -CK18 or CK19 and glutathione transferases -GSTπ or GSTα. In vitro differentiation of these cells, isolated from CDE-treated rats begun earlier as compared to non-treated ones. Agedependent changes in the cell differentiation sequence, as well as CYPmRNA expression sequence accompanying precursor cells differentiation, were also observed. mRNA expression of CYP1A2, CYP2B1/2 and CYP3A1 was higher in the cells of young rats, but in the case of CYP2E1 -in the cells of old rats. It was concluded that both proliferation and differentiation potential of oval cells, decreased with age.Key words: cytochromes P450, oval cells, cell differentiation, experimental hepatocarcinogenesis, liver regeneration.Correspondence: P. Czekaj, Dept. of Histology, Medical University of Silesia, Medyków 18, 40-752 Katowice, Poland; tel.: (+4832) 2088374, fax.: (+4832) 2526574, e-mail: pcz@sum.edu.pl Abbreviations: AFP α-fetoprotein; BrdU -bromodeoxyuridin; CDE diet -choline-deficient ethionine-supplemented diet; CK18 and CK19 -cytokeratines CK18 and CK19; DCL(s) -differentiation cell lineage(s); CYP(s) -cytochrome(s) P450; DMSOdimethyl sulphoxide; GSTα -glutathione S transferase α; GSTπ -glutathione S transferase π; L-PK -pyruvate kinase isoform α; M 2 PK -pyruvate kinase; 4m and 20m -4-and 20-month-old rats, respectively; IOD -integrated optical density; SB -sodium butyrate.

Several Important In Vitro Improvements in the Amplification, Differentiation and Tracing of Fetal Liver Stem/Progenitor Cells

“…Since the initial cloning and characterization of HGF as a mitogen for hepatocytes [40], increasing studies have revealed that hepatic differentiation and maturation of cells occurs upon exposure to HGF [18], [19], [41], [42]. In addition to HGF, DMSO can be used to induce hepatic differentiation [20]. However, when separately used, the effects of hepatic induction by these two molecules are not satisfying.…”

“…Although FLSPCs can be largely amplified by soft agar culture, because they are immature and lack function, they have to differentiate into hepatocytes to restore normal liver functioning. Therefore, in this study by combining the hepatic differentiation inducers HGF [17]–[19] with dimethyl sulfoxide (DMSO) [20], [21], we successfully induced the differentiation of FLSPCs into hepatocyte-like cells. Thus, this effective strategy could provide quality assurance for treating liver diseases.…”

Age-associated changes of cytochrome P450 and related phase-2 gene/proteins in livers of rats

“…(Salmin et al 2017) CYP3A1 is higher in cells from young rats than in old rats. (Czekaj et al 2010) CYP3A in the liver are sensitive to aging with 50-70% decreases, while CYP3A in the intestine is unchanged and in the kidney increased. (Warrington et al 2004) Generally speaking, hepatic CYP3A enzymes are decreased with age.…”

“…For example, CYPs expression can be affected by hormones (Daskalopoulos et al 2012), cytokines (Kot & Daujat-Chavanieu 2018), pregnancy, (He et al 2005) sex, (Agrawal & Shapiro 2003;Das et al 2014) and age. CYPs are subjected to agedependent changes in cell differentiation (Czekaj et al 2010) and epigenetic regulation, (Li et al 2009) and age-related metabolic syndrome, (Bondarenko et al 2016) kidney diseases, (Velenosi et al 2012) diabetes, (Park et al 2016) nonalcoholic steatohepatitis, (Li et al 2017) virus hepatitis, and cirrhosis. (Kirby et al 1996) Age of animals greatly affects drug metabolism, (Durnas et al 1990) alters pharmacokinetics of xenobiotics, (Matalova et al 2016;Shi & Klotz 2011) and thus alters the sensitivity to drugs and toxicants such as acetaminophen, (Mach et al 2014) isoniazid, (Mach et al 2016) aflatoxin B1, (Kirby et al 1996;Wang et al 2018) and thioacetamide.…”

What determines ageing of the transplanted liver?

“…Impaired glucose tolerance, however, is neither specific nor sensitive for ageing. Cytochrome p450 (CYP) mRNA expression has been found experimentally to correlate highly with age, with CYP1A2, CYP2B1/2 and CYP3A1 all increased and CYP2E1 decreased in young hepatocyte proliferation in the condition of hepatocarcinogenesis . While this may lead to further advances in the difference in function between young and old livers, it is not yet conceivable that these could be markers to aid in the usability of a potential donor organ and remain firmly as a scientific model.…”