Unspecific peroxygenases (UPO, EC 1.11.2.1) secreted by fungi open an efficient way to selectively oxyfunctionalize diverse organic substrates, including less‐activated hydrocarbons, by transferring peroxide‐borne oxygen. We investigated a cell‐free approach to incorporate epoxy and hydroxyl functionalities directly into the bulky molecule testosterone by a novel unspecific peroxygenase (UPO) that is produced by the ascomycetous fungus Chaetomium globosum in a complex medium rich in carbon and nitrogen. Purification by fast protein liquid chromatography revealed two enzyme fractions with the same molecular mass (36 kDa) and with specific activity of 4.4 to 12 U mg−1. Although the well‐known UPOs of Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) failed to convert testosterone in a comparative study, the UPO of C. globosum (CglUPO) accepted testosterone as substrate and converted it with total turnover number (TTN) of up to 7000 into two oxygenated products: the 4,5‐epoxide of testosterone in β‐configuration and 16α‐hydroxytestosterone. The reaction performed on a 100 mg scale resulted in the formation of about 90 % of the epoxide and 10 % of the hydroxylation product, both of which could be isolated with purities above 96 %. Thus, CglUPO is a promising biocatalyst for the oxyfunctionalization of bulky steroids and it will be a useful tool for the synthesis of pharmaceutically relevant steroidal molecules.

PURPOSE. We investigated the response of retinal vessel diameters and oxygen saturation to flicker light stimulation of neuronal activity in patients with diabetic retinopathy. METHODS.We included 18 patients with nonproliferative diabetic retinopathy (mean age 62.2 6 8.3 years, diabetes type 1 in 4 patients and type 2 in 14, hemoglobin A1c 7.7 6 0.9%, duration of diabetes 24.1 6 9.3 years) and 20 agematched healthy controls (age 66.7 6 10.3 years). Dual wavelength (548 and 610 nm) fundus images were taken before and during luminance flicker stimulation (12.5 Hz, modulation depth > 1:25) for 90 seconds. Diameters (central retinal arterial [CRAE] and venous [CRVE] equivalents) and oxygen saturation (SO 2 ) were determined, and averaged for all arterioles and venules in an annular area centered at the optic disk.RESULTS. Flicker light increased CRAE, CRVE, and venous SO 2 by 0.6 6 6.6%, 2.7 6 6.1%, and 2.0 6 2.4% (P < 0.05), respectively, in the patients as well as 4.7 6 8.4% (P < 0.05), 8.7 6 5.2% (P < 0.05), and 4.2 6 3.5% (P < 0.05), respectively, in the controls. The arterial SO 2 remained unchanged in both groups. The increase of the venous SO2 correlated significantly (P ¼ 0.027) with that of the CRAE. There was a trend (P ¼ 0.06) for lower increase of the venous SO 2 with higher body mass index. CONCLUSIONS.Our results support the thesis of an impaired regulation of oxygen supply to the diabetic retina. Whereas in healthy subjects the stimulation of neuronal activity increases the vascular diameters and, subsequently, the oxygen supply, this increase is reduced in diabetic retinopathy. This may hint at the role of endothelial dysfunction in the etiology of the disease. (Invest Ophthalmol Vis Sci.

Detoxification of xenobiotics including drugs is catalyzed by liver phase I and phase II enzymes. There are three main families of phase I cytochrome P450 (CYP450) monoxygenases that introduce polar groups on drugs. These phase I metabolites can then be further conjugated by transferases during phase II reaction. Liver biotransformation can also lead to toxic drug metabolites, the most common cause of drug failure during clinical investigation. CYP3A4 is considered to be the most important enzyme in drug metabolism. Drug development relies on the use of human liver cells in order to investigate drug metabolism and potential toxicity. With primary human liver cells as the gold standard several problems have to be solved, i.e. scarcity of functional human liver tissue, donor variation of CYP activity and rapid dedifferentiation processes during primary cell cultivation. These features make it difficult to use primary human liver cells as standard to measure CYP activity. To avoid problems with primary human liver cells, many attempts have been undertaken to establish liver carcinoma cell lines, non-transformed proliferating human liver cell systems and induced pluripotent stem cell-derived hepatocytes. Due to different problems with these surrogate systems, the one cell line that could be used as convenient standard cell system to benchmark CYP3A4 enzyme activity has not been established yet. Based on the widely used hepatocellular carcinoma cell line HepG2 and a lentiviral vector system, we generated cell clones for stable CYP3A4 overexpression. Here we present data on a new HepG2 cell clone (clone 9) showing higher than 10,000-fold overexpression of CYP3A4 compared to HepG2 parental cells. As measured by conversion of testosterone into 6␤-hydroxytestosterone, we found an enzyme activity of about 600 pmol per minute per mg total cellular protein, which ranges at the upper end reported for primary human liver cells. This enzyme activity appeared to be kept stable in clone 9 cells, because there was no influence detectable when cells were treated with 5-azacytidine, a drug that interferes with epigenetic silencing processes. Prototypic CYP3A4 inducer rifampicin led to significant increase of CYP3A4 testosterone hydroxylase activity in HepG2 clone 9 cells. Altogether, HepG2 clone 9 strongly and stably overexpressed CYP3A4 leading to a physiological enzyme activity, which apparently was unaffected by epigenetic processes. Thus, HepG2 clone 9 could be a useful reference cell clone for CYP3A4 enzyme activity.

Primary human hepatocytes are in great demand during drug development and in hepatology. However, both scarcity of tissue supply and donor variability of primary cells create a need for the development of alternative hepatocyte systems. By using a lentivirus vector system to transfer coding sequences of Upcyte® proliferation genes, we generated non-transformed stable hepatocyte cultures from human liver tissue samples. Here, we show data on newly generated proliferation-competent HepaFH3 cells investigated as conventional two-dimensional monolayer and as organotypical three-dimensional (3D) spheroid culture. In monolayer culture, HepaFH3 cells show typical cobblestone-like hepatocyte morphology and anchorage-dependent growth for at least 20 passages. Immunofluorescence staining revealed that characteristic hepatocyte marker proteins cytokeratin 8, human serum albumin, and cytochrome P450 (CYP) 3A4 were expressed. Quantitative real-time PCR analyses showed that expression levels of analyzed phase I CYP enzymes were at similar levels compared to those of cultured primary human hepatocytes and considerably higher than in the liver carcinoma cell line HepG2. Additionally, transcripts for phase II liver enzymes and transporter proteins OATP-C, MRP2, Oct1, and BSEP were present in HepaFH3. The cells produced urea and converted model compounds such as testosterone, diclofenac, and 7-OH-coumarin into phases I and II metabolites. Interestingly, phases I and II enzymes were expressed at about the same levels in convenient monolayer cultures and complex 3D spheroids. In conclusion, HepaFH3 cells and related primary-like hepatocyte lines seem to be promising tools for in vitro research of liver functions and as test system in drug development and toxicology analysis.

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