Stable Chinese Hamster Ovary Suspension Cell Lines Harboring Recombinant Human Cytochrome P450 Oxidoreductase and Human Cytochrome P450 Monooxygenases as Platform for In Vitro Biotransformation Studies

In the liver, phase-1 biotransformation of drugs and other xenobiotics is largely facilitated by enzyme complexes consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs). Generated from human liver-derived cell lines, recombinant in vitro cell systems with overexpression of defined phase-1 enzymes are widely used for pharmacological and toxicological drug assessment and laboratory-scale production of drug-specific reference metabolites. Most, if not all, of these cell lines, however, display some background activity of several CYPs, making it difficult to attribute effects to defined CYPs. The aim of this study was to generate cell lines with stable overexpression of human phase-1 enzymes based on Chinese hamster ovary (CHO) suspension cells. Cells were sequentially modified with cDNAs for human CPR in combination with CYP1A2, CYP2B6, or CYP3A4, using lentiviral gene transfer. In parallel, CYP-overexpressing cell lines without recombinant CPR were generated. Successful recombinant expression was demonstrated by mRNA and protein analyses. Using prototypical CYP-substrates, generated cell lines proved to display specific enzyme activities of each overexpressed CYP while we did not find any endogenous activity of those CYPs in parental CHO cells. Interestingly, cell lines revealed some evidence that the dependence of CYP activity on CPR could vary between CYPs. This needs to be confirmed in further studies. Recombinant expression of CPR was also shown to enhance CYP3A4-independent metabolisation of testosterone to androstenedione in CHO cells. We propose the novel serum-free CHO suspension cell lines with enhanced CPR and/or defined CYP activity as a promising “humanised” in vitro model to study the specific effects of those human CYPs. This could be relevant for toxicology and/or pharmacology studies in the pharmaceutical industry or medicine

Estragole: DNA adduct formation in primary rat hepatocytes and genotoxic potential in HepG2-CYP1A2 cells

Estragole is a natural constituent in herbs and spices and in products thereof such as essential oils or herbal teas. After cytochrome P450-catalyzed hydroxylation and subsequent sulfation, estragole acts as a genotoxic hepatocarcinogen forming DNA adducts in rodent liver. Because of the genotoxic mode of action and the widespread occurrence in food and phytomedicines a refined risk assessment for estragole is needed. We analyzed the time- and concentration-dependent levels of the DNA adducts N2-(isoestragole-3‘-yl)-2‘-desoxyguanosine (E3′N2dG) and N6-(isoestragole-3‘-yl)-desoxyadenosine (E3′N6dA), reported to be the major adducts formed in rat liver, in rat hepatocytes (pRH) in primary culture after incubation with estragole. DNA adduct levels were measured via UHPLC-ESI-MS/MS using stable isotope dilution analysis. Both adducts were formed in pRH and could already be quantified after an incubation time of 1 h (E3′N6dA at 10 μM, E3′N2dG at 1μM estragole). E3′N2dG, the main adduct at all incubation times and concentrations, could be detected at estragole concentrations < 0.1 μM after 24 h and < 0.5 μM after 48 h. Adduct levels were highest after 6 h and showed a downward trend at later time-points, possibly due to DNA repair and/or apoptosis. While the concentration-response characteristics of adduct formation were apparently linear over the whole concentration range, strong indication for marked hypo-linearity was obtained when the modeling was based on concentrations < 1 μM only. In the micronucleus assay no mutagenic potential of estragole was found in HepG2 cells whereas in HepG2-CYP1A2 cells 1 μM estragole led to a 3.2 fold and 300 μM to a 7.1 fold increase in micronuclei counts. Our findings suggest the existence of a ‘practical threshold’ dose for DNA adduct formation as an initiating key event of the carcinogenicity of estragole indicating that the default assumption of concentration-response-linearity is questionable, at least for the two major adducts studied here

Structure-dependent genotoxic potencies of selected pyrrolizidine alkaloids in metabolically competent HepG2 cells

1,2-unsaturated pyrrolizidine alkaloids (PAs) are natural plant constituents comprising more than 600 different structures. A major source of human exposure is thought to be cross-contamination of food, feed and phytomedicines with PA plants. In humans, laboratory and farm animals, certain PAs exert pronounced liver toxicity and can induce malignant liver tumors in rodents. Here, we investigated the cytotoxicity and genotoxicity of eleven PAs belonging to different structural classes. Although all PAs were negative in the fluctuation Ames test in Salmonella, they were cytotoxic and induced micronuclei in human HepG2 hepatoblastoma cells over-expressing human cytochrome P450 3A4. Lasiocarpine and cyclic diesters except monocrotaline were the most potent congeners both in cytotoxicity and micronucleus assays with concentrations below 3 μM inducing a doubling in micronuclei counts. Other open di-esters and all monoesters exhibited weaker or much weaker geno- and cytotoxicity. The findings were in agreement with recently suggested interim Relative Potency (iREP) factors with the exceptions of europine and monocrotaline. A more detailed micronuclei analysis at low concentrations of lasiocarpine, retrorsine or senecionine indicated that pronounced hypolinearity of the concentration-response curves was evident for retrorsine and senecionine but not for lasiocarpine. Our findings show that the genotoxic and cytotoxic potencies of PAs in a human hepatic cell line vary in a structure-dependent manner. Both the low potency of monoesters and the shape of prototype concentration-response relationships warrant a substance- and structure-specific approach in the risk assessment of PAs

Spirulina platensis, a super food?

Spirulina platensis, a multicelluar, photosynthetic prokaryote (algae) contains a high amount of proteins, vitamins and minerals superior to many foods as e.g. soybeans. Thus, Spirulina platensis was recognized as nutritious food by the United Nations World Food Conference. Due to the high amount of nutritive ingredients Spirulina has a long history as dietary supplement. In addition, spirulina platensis is also efficiently used as forage with known effects on flesh, egg and plumage color, milk yield and fertility. The versatile utilization of the alga can be explained on the one hand with the nutrient levels and on the other hand with recognized effects as anti-viral, anti-bacterial, anti-oxidant, anti-diabetic, anti-cancer and anti-inflammatory substance. Therefore, this alga is named as “superfood”. Beyond, these algae convert carbon dioxide into organic substances and produce oxygen during their growth in alkaline and saline water thereby not wasting fresh water allowing the production in barren areas. Despite this diverse use of Spirulina platensis due to its beneficial properties, many basic mechanisms on a molecular and cellular level are not well understood and should be explored in future studies

Potential Effects of Nonadherent on Adherent Human Umbilical Venous Endothelial Cells in Cell Culture

The adherence and shear-resistance of human umbilical venous endothelial cells (HUVEC) on polymers is determined in vitro in order to qualify cardiovascular implant materials. In these tests, variable fractions of HUVEC do not adhere to the material but remain suspended in the culture medium. Nonadherent HUVEC usually stop growing, rapidly lose their viability and can release mediators able to influence the growth and function of the adherent HUVEC. The aim of this study was the investigation of the time dependent behaviour of HUVEC under controlled nonadherent conditions, in order to gain insights into potential influences of these cells on their surrounding environment in particular adherent HUVEC in the context of in vitro biofunctionality assessment of cardiovascular implant materials. Data from adherent or nonadherent HUVEC growing on polystyrene-based cell adhesive tissue culture plates (TCP) or nonadhesive low attachment plates (LAP) allow to calculate the number of mediators released into the culture medium either from adherent or nonadherent cells. Thus, the source of the inflammatory mediators can be identified. For nonadherent HUVEC, a time-dependent aggregation without further proliferation was observed. The rate of apoptotic/dead HUVEC progressively increased over 90% within two days. Concomitant with distinct blebbing and loss of membrane integrity over time, augmented releases of prostacyclin (PGI2, up to 2.91 ± 0.62 fg/cell) and platelet-derived growth factor BB (PDGF-BB, up to 1.46 ± 0.42 fg/cell) were detected. The study revealed that nonadherent, dying HUVEC released mediators, which can influence the surrounding microenvironment and thereby the results of in vitro biofunctionality assessment of cardiovascular implant materials. Neglecting nonadherent HUVEC bears the risk for under- or overestimation of the materials endothelialization potential, which could lead to the loss of relevant candidates or to uncertainty with regard to their suitability for cardiac applications. One approach to minimize the influence from nonadherent endothelial cells could be their removal shortly after observing initial cell adhesion. However, this would require an individual adaptation of the study design, depending on the properties of the biomaterial used

Phycocyanin from <i>Arthrospira platensis</i> as Potential Anti-Cancer Drug: Review of In Vitro and In Vivo Studies

The application of cytostatic drugs or natural substances to inhibit cancer growth and progression is an important and evolving subject of cancer research. There has been a surge of interest in marine bioresources, particularly algae, as well as cyanobacteria and their bioactive ingredients. Dried biomass products of Arthrospira and Chlorella have been categorized as “generally recognized as safe” (GRAS) by the US Food and Drug Administration (FDA). Of particular importance is an ingredient of Arthrospira: phycocyanin, a blue-red fluorescent, water-soluble and non-toxic biliprotein pigment. It is reported to be the main active ingredient of Arthrospira and was shown to have therapeutic properties, including anti-oxidant, anti-inflammatory, immune-modulatory and anti-cancer activities. In the present review, in vitro and in vivo data on the effects of phycocyanin on various tumor cells and on cells from healthy tissues are summarized. The existing knowledge of underlying molecular mechanisms, and strategies to improve the efficiency of potential phycocyanin-based anti-cancer therapies are discussed

Factors influencing the growth of Spirulina platensis in closed photobioreactors under CO2 - O2 conversion

Since there is growing interest throughout the world in photosynthetic microbes as a potential source of food or food supplements, an assessment of factors which influence the biomass obtained in bioreactors, protein contents and constituents is important. This work reviews the autotrophic cultivation conditions of Spirulina platensis especially the dependency on the strain, the composition of the nutrient solution, pH, temperature of the medium, light intensity and color as well as exposure rhythm, the flow rate and composition of the aerating gas mixture and the bubble size, the content of oxygen, CO2 and HCO3 in the medium and last but not least from the optical density of the spirulina suspension during growth

Investigation of Radiotracer Metabolic Stability In Vitro with CYP-Overexpressing Hepatoma Cell Lines

The characterization of novel radiotracers toward their metabolic stability is an essential part of their development. While in vitro methods such as liver microsome assays or ex vivo blood or tissue samples provide information on overall stability, little or no information is obtained on cytochrome P450 (CYP) enzyme and isoform-specific contribution to the metabolic fate of individual radiotracers. Herein, we investigated recently established CYP-overexpressing hepatoblastoma cell lines (HepG2) for their suitability to study the metabolic stability of radiotracers in general and to gain insight into CYP isoform specificity. Wildtype HepG2 and CYP1A2-, CYP2C19-, and CYP3A4-overexpressing HepG2 cells were incubated with radiotracers, and metabolic turnover was analyzed. The optimized protocol, covering cell seeding in 96-well plates and analysis of supernatant by radio thin-layer-chromatography for higher throughput, was transferred to the evaluation of three 18F-labeled celecoxib-derived cyclooxygenase-2 inhibitors (coxibs). These investigations revealed time-dependent degradation of the intact radiotracers, as well as CYP isoform- and substrate-specific differences in their metabolic profiles. HepG2 CYP2C19 proved to be the cell line showing the highest metabolic turnover for each radiotracer studied here. Comparison with human and murine liver microsome assays showed good agreement with the human metabolite profile obtained by the HepG2 cell lines. Therefore, CYP-overexpressing HepG2 cells provide a good complement for assessing the metabolic stability of radiotracers and allow the analysis of the CYP isoform-specific contribution to the overall radiotracer metabolism

Influence of Different Light-Emitting Diode Colors on Growth and Phycobiliprotein Generation of Arthrospira platensis

Light-emitting diodes (LED) can be utilized as tailorable artificial light sources for the cultivation of cyanobacteria such as Arthrospira platensis (AP). To study the influence of different LED light colors on phototrophic growth and biomass composition, AP was cultured in closed bioreactors and exposed to red, green, blue, or white LED lights. The illumination with red LED light resulted in the highest cell growth and highest cell densities compared to all other light sources (order of cell densities: red &gt; white &gt; green &gt; blue LED light). In contrast, the highest phycocyanin concentrations were found when AP was cultured under blue LED light (e.g., order of concentrations: blue &gt; white &gt; red &gt; green LED light). LED-blue light stimulated the accumulation of nitrogen compounds in the form of phycobiliproteins at the expense of cell growth. The results of the study revealed that exposure to different LED light colors can improve the quality and quantity of the biomass gained in AP cultures