Cytochrome 1A2 Activity Research Articles

Influence of Antibiotics on Functionality and Viability of Liver Cells In Vitro.

(1) Antibiotics are an important weapon in the fight against serious bacterial infections and are considered a common cause of drug-induced liver injury (DILI). The hepatotoxicity of many drugs, including antibiotics, is poorly analyzed in human in vitro models. (2) A standardized assay with a human hepatoma cell line was used to test the hepatotoxicity of various concentrations (Cmax, 5× Cmax, and 10× Cmax) of antibiotics. In an ICU, the most frequently prescribed antibiotics, ampicillin, cefepime, cefuroxime, levofloxacin, linezolid, meropenem, rifampicin, tigecycline, and vancomycin, were incubated with HepG2/C3A cells for 6 days. Cell viability (XTT assay, LDH release, and vitality), albumin synthesis, and cytochrome 1A2 activity were determined in cells. (3) In vitro, vancomycin, rifampicin, and tigecycline showed moderate hepatotoxic potential. The antibiotics ampicillin, cefepime, cefuroxime, levofloxacin, linezolid, and meropenem were associated with mild hepatotoxic reactions in test cells incubated with the testes Cmax concentration. Rifampicin and cefuroxime showed significantly negative effects on the viability of test cells. (4) Further in vitro studies and global pharmacovigilance reports should be conducted to reveal underlying mechanism of the hepatotoxic action of vancomycin, rifampicin, tigecycline, and cefuroxime, as well as the clinical relevance of these findings.

Impaired Cell Viability and Functionality of Hepatocytes After Incubation With Septic Plasma-Results of a Second Prospective Biosensor Study.

Liver dysfunction (LD) and liver failure are associated with poor outcome in critically ill patients. In patients with severe sepsis or septic shock, LD occurred in nearly 19% of patients. An early diagnosis of LD at time of initial damage of the liver can lead to a better prognosis of these patients because an early start of therapy is possible. We performed a second prospective study with septic patients to test a new cell-based cytotoxicity device (biosensor) to evaluate clinical relevance for early diagnosis of LD and prognostic capacity. In the clinical study, 99 intensive care unit patients were included in two groups. From the patients of the septic group (n = 51, SG), and the control (non-septic) group [n = 49, control group (CG)] were drawn 20 ml blood at inclusion, after 3, and 7 days for testing with the biosensor. Patients’ data were recorded for hospital survival, organ function, and demographic data, illness severity [acute physiology and chronic health evaluation (APACHE) II-, sepsis-related organ failure assessment (SOFA) scores], cytokines, circulating-free deoxyribonucleic acid/neutrophil-derived extracellular traps (cf-DNA/NETs), microbiological results, and pre-morbidity. For the developed cytotoxicity test, the human liver cell line HepG2/C3A was used. Patients’ plasma was incubated in a microtiter plate assay with the test cells and after 6 days incubation the viability (trypan blue staining, XTT-test) and functionality (synthesis of albumin, cytochrome 1A2 activity) was analyzed. An impairment of viability and functionality of test cells was only seen in the SG compared with the CG. The plasma of non-survivors in the SG led to a more pronounced impairment of test cells than the plasma of survivors at inclusion. In addition, the levels of cf-DNA/NETs were significantly higher in the SG at inclusion, after 3, and after 7 days compared with the CG. The SG showed an in-hospital mortality of 24% and the values of bilirubin, APACHE II-, and SOFA scores were markedly higher at inclusion than in the CG. Hepatotoxicity of septic plasma was already detected with the liver cell-based biosensor at inclusion and also in the course of disease. The biosensor may be a tool for early diagnosis of LD in septic patients and may have prognostic relevance.

Hepatotoxicity of Antimycotics Used for Invasive Fungal Infections: In Vitro Results.

Purpose. Drug-induced liver injury (DILI) is the most common cause of liver injury and a serious clinical problem; antimycotics are involved in approximately 3% of all DILI cases. The hepatotoxicity of many drugs, including the antimycotics, is poorly screened in human models. Methods. In a standardized assay the cytotoxicity on hepatocytes of different concentrations (Cmax, 5x Cmax, and 10x Cmax) of the antimycotics used for systemic infections was tested. Anidulafungin (ANI), liposomal amphotericerin B (L-AmB), caspofungin (CASPO), fluconazole (FLUCO), and voriconazole (VORI) were incubated with HepG2/C3A cells. After incubation, the viability of cells (XTT test, LDH release, trypan blue staining), the synthesis of albumin, the cytochrome 1A2 activity, and the cell death (DNA fragmentation) were determined. Kruskal-Wallis and Mann–Whitney tests were used for statistical analyses. Results. L-AmB, ANI, and CASPO showed a mild hepatotoxicity in the Cmax concentrations. Higher concentrations of anidulafungin led to a severe impairment of hepatocyte viability and function. The azoles FLUCO and VORI had a higher hepatotoxic potential in all concentrations. Conclusion. Antimycotics, especially azoles, used for systemic infections should be given with caution in patient with liver insufficiency or liver failure or high risk for this; therefore, therapeutic drug monitoring should be used. Further studies with this approach are encouraged.

Artefacts at the liquid interface and their impact in miniaturized biochemical assay.

Droplet microfluidic technology has the potential to significantly reduce reagent use, and therefore, lower costs of assays employed in drug discovery campaigns. In addition to the reduction in costs, this technology can also reduce evaporation and contamination which are often problems seen in miniaturized microtitre plate formats. Despite these advantages, we currently advise caution in the use of these microfluidic approaches as there remains a lack of understanding of the artefacts of the systems such as reagent partitioning from droplet to carrier oil and interaction of the biological reagents with the water-oil interface. Both types of artefact can lead to inaccurate and misleading data. In this paper, we present a study of the partitioning of a number of drug-like molecules in a range of oils and evidence of protein binding at the water-oil interface which results in reduced activity of a cytochrome P450 enzyme. Data presented show that the drug-like molecules partitioned the least into fluorocarbon oils and the interaction of the 1A2 cytochrome at the water-oil interface resulted in a lower or complete absence of enzyme activity. This loss of activity of cytochrome 1A2 could be restored by the use of secondary blocking proteins although changes in the pharmacology of known 1A2 inhibitors were observed. The artefacts described here due to reagents partitioning into the carrier oil or protein binding at the water-oil interface significantly impact the potential use of these microfluidic systems as a means to carry out miniaturized biological assays, and further work is needed to understand the impact and reduction of these phenomena.

Impaired cell functions of hepatocytes incubated with plasma of septic patients

The development of liver failure is a major problem in septic patients. In this prospective clinical experimental study the hepatotoxicity of plasma from septic and non-septic patients was tested. The basic test components consist of human liver cells (HepG2/C3A) used in a standardized microtiter plate assay. After incubation with patient's plasma viability of cells (XTT-test), the cytochrome 1A2 activity and synthesis of micro albumin were measured. Subjects (28) enrolled comprise the septic shock group (SSG, n=10), the non-septic group (NSG, n=5) and the healthy volunteers group (HVG, n=13). The 28-day mortality was 30% in the SSG. The APACHE II-, SOFA-, and SAPS-scores and the values of bilirubin and prothrombin time as INR were significantly higher in the SSG than in the NSG. The cytochrome 1A2 activity and the release of albumin were significantly reduced in HepG2/C3A cells incubated with plasma of the SSG (p<0.05). The cytochrome 1A2 activities were higher in survivors compared to non-survivors at the time point 0 and were increasing in survivors and decreasing in non-survivors within 54 h in the SSG. In the SSG there was a significant decrease in IL-10 and IL-8 between inclusion and 54 h. Values of IL-6, TNF alpha and IL-10 were significantly lower in the NSG compared with the values of the SSG at inclusion and after 54 h. The plasma of patients with septic shock impaired cellular functions of HepG2/C3A cells.