Synthesis of cyclophosphamide metabolites by a peroxygenase from Marasmius rotula for toxicological studies on human cancer cells.

Cyclophosphamide (CPA) represents a widely used anti-cancer prodrug that is converted by liver cytochrome P450 (CYP) enzymes into the primary metabolite 4-hydroxycyclophosphamide (4-OH-CPA), followed by non-enzymatic generation of the bioactive metabolites phosphoramide mustard and acrolein. The use of human drug metabolites as authentic standards to evaluate their toxicity is essential for drug development. However, the chemical synthesis of 4-OH-CPA is complex and leads to only low yields and undesired side products.

HepG2-1A2 C2 and C7: Lentivirus vector-mediated stable and functional overexpression of cytochrome P450 1A2 in human hepatoblastoma cells.

Novel HepG2 cell clones 1A2 C2 and 1A2 C7 were independently generated by lentiviral transduction to functionally overexpress cytochrome P450 1A2 (CYP1A2). We found similar and stable CYP1A2 transcript and protein levels in both cell clones leading to specific enzyme activities of about 370 pmol paracetamol x min x mg protein analyzed by phenacetin conversion. Both clones showed dramatically increased sensitivity to the hepatotoxic compound aflatoxin B (EC < 100 nM) when compared to parental HepG2 cells (EC5 μM).

Metabolic activity testing can underestimate acute drug cytotoxicity as revealed by HepG2 cell clones overexpressing cytochrome P450 2C19 and 3A4.

Preclinical drug safety assessment includes in vitro studies with physiologically relevant cell cultures. As an in vitro system for hepatic toxicology testing, we have been generating cell clones of human hepatoblastoma cell line HepG2 by lentiviral transduction of phase I cytochrome P450 (CYP) enzymes. Here, we present a stable CYP2C19-overexpressing HepG2 cell clone (HepG2-2C19 C1) showing an enzyme activity of approximately 82 pmol x min x mg total cellular protein.

SENIEUR status of the originating cell donor negates certain 'anti-immunosenescence' effects of ebselen and N-acetyl cysteine in human T cell clone cultures.

Background: Damage to T cells of the immune system by reactive oxygen species may result in altered cell function or cell death and thereby potentially impact upon the efficacy of a subsequent immune response. Here, we assess the impact of the antioxidants Ebselen and N-acetyl cysteine on a range of biological markers in human T cells derived from a SENIEUR status donor. In addition, the impact of these antioxidants on different MAP kinase pathways in T cells from donors of different ages was also examined.