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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. In past years, fungal unspecific peroxygenases (UPOs) have raised to powerful biocatalysts. They can exert the identical selective oxyfunctionalization of organic compounds and drugs as known for CYP enzymes with hydrogen peroxide being used as sole cosubstrate. Herein, we report the efficient enzymatic hydroxylation of CPA using the unspecific peroxygenase from Marasmius rotula (MroUPO) in a simple reaction design. Depending on the conditions used the primary liver metabolite 4-OH-CPA, its tautomer aldophosphamide (APA) and the overoxidized product 4-ketocyclophosphamide (4-keto-CPA) could be obtained. Using a kinetically controlled approach 4-OH-CPA was isolated with a yield of 32% (purity > 97.6%). Two human cancer cell lines (HepG2 and MCF-7) were treated with purified 4-OH-CPA produced by MroUPO (4-OH-CPA UPO). 4-OH-CPA UPO-induced cytotoxicity as measured by a luminescent cell viability assay and its genotoxicity as measured by γH2AX foci formation was not significantly different to the commercially available standard. The high yield of 4-OH-CPA UPO and its biological activity demonstrate that UPOs can be efficiently used to produce CYP-specific drug metabolites for pharmacological assessment.

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BackgroundDamage 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.MethodsT cell clones were derived from healthy 26, 45 and SENIEUR status 80 year old people and the impact of titrated concentrations of Ebselen or N-acetyl cysteine on their proliferation and in vitro lifespan, GSH:GSSG ratio as well as levels of oxidative DNA damage and on MAP kinase signaling pathways was examined.ResultsIn this investigation neither Ebselen nor N-acetyl cysteine supplementation had any impact on the biological endpoints examined in the T cells derived from the SENIEUR status 80 year old donor. This is in contrast to the anti-immunosenescent effects of these antioxidants on T cells from donors of 26 or 45 years of age. The analysis of MAP kinases showed that pro-apoptotic pathways become activated in T cells with increasing in vitro age and that Ebselen or N-acetyl cysteine could decrease activation (phosphorylation) in T cells from 26 or 45 year old donors, but not from the SENIEUR status 80 year old donor.ConclusionsThe results of this investigation demonstrate that the biological phenotype of SENIEUR status derived human T cells negates the anti-immunosenescence effects of Ebselen and also N-acetyl cysteine. The results highlight the importance of pre-antioxidant intervention evaluation to determine risk-benefit.Electronic supplementary materialThe online version of this article (doi:10.1186/s12979-014-0017-5) contains supplementary material, which is available to authorized users.

The liver is the place of biotransformation, where drugs or other substances are metabolized. Cytochrome P450 oxidoreductases (CYPs) play a prominent role in these processes and thus sufficient CYP expression levels are the prerequisite for physiologically relevant liver metabolism or toxicity studies. Human primary hepatocytes, the most popular in vitro liver model for such studies, have several limiting properties: poor availability, rapid dedifferentiation, substantial donor variability and restricted proliferation capacity in vitro. This prompted many research groups to develop alternative models for the investigation of biotransformation-related questions. The hepatoblastoma-derived HepG2 cell line is a highly proliferative and easy to handle in vitro model, but has the disadvantage that expression levels of relevant CYP enzymes are dramatically downregulated. The generation of CYP-overexpressing HepG2 cells is a way to overcome this disadvantage and such cells were used as in vitro alternative to primary hepatocytes. Coding sequences of various CYP isoforms were transiently or stably introduced into HepG2 cells by using viral transduction or transfection reagents. With the frequently used adenoviral transduction, the level of recombinant enzyme activity usually is high within a time window of several days and simultaneous expression of several CYP enzymes is possible. High expression levels can also be achieved with lentiviral transduction which is stable upon virus integration into the host genome. Transient and stable CYP-expressing HepG2 cells serve as a convenient tool for toxicity studies and risk assessment of drugs or other substances undergoing biotransformation, clearance and drug-drug interaction. Furthermore, they can be used for rapid identification of CYP enzymes relevant to a specific reaction or screening for CYP enzyme inhibitors. The use of CYP-overexpressing HepG2 systems also have some disadvantages, such as the cancerous cell origin und their lack of other liver specific functions.The broad spectrum of possible applications of these CYP-expressing HepG2 cells, especially in the early phase of drug development, can quickly and easily provide important information about drug metabolism in the liver and toxicity behaviour of potential metabolites. In this way, unsuitable drug candidates can be excluded at an early stage of pharmacological studies in order to safe costs and to reduce in vivo animal trials.