Research Report 1: The role of glutathione in idiosyncratic and non-idiosyncratic drug induced liver injury

Introduction: The probability of idiosyncratic drug-induced liver injury (IDILI) occurrence is defined as a rare combination of several events at the same time, such as chemical properties of the drug, toxicant exposure, environmental and genetic factors. In the present study, we addressed the interaction of drugs and an environmental factor. It is known that different environmental factors can enhance the probability of IDILI: tobacco, alcohol consumption, diet, pre-existing diseases, viral or bacterial infections, etc. Accordingly, the aim of this study was to investigate the effect of an inflammatory agent, lipopolysaccharide (LPS), on the redox cellular homeostasis and to discover the role of glutathione in IDILI manifestation. Material and Methods: Mouse and human precision-cut liver slices (PCLS) were incubated for 24 h with acetaminophen (APAP) and its isomer 3’-hydroxyacetanilide (AMAP), different IDILI-related drugs: diclofenac (DF), ketoconazole (KT), clozapine (CZ), carbamazepine (CBZ), troglitazone (TGZ); and two non-toxic comparator drugs of KT and CZ, voriconazole (VC) and olanzapine (OZ) respectively, in the absence or presence of LPS. Furthermore, drugs, that did not reveal synergistic toxicity with LPS following 24 h incubation of mouse PCLS, were tested for 48 h. Viability of PCLS was assessed by means of total and reduced (GSH) glutathione levels, GSH/GSSG ratio, ATP content and LDH leakage from PCLS to incubation media. Results: AMAP was shown to be less toxic in mouse PCLS than APAP, while in human PCLS, AMAP revealed higher toxicity in comparison with APAP. It was shown that LPS by itself noticeably but not significantly increased total glutathione and GSH levels in mouse PCLS after 24 h incubation, which was not found after 48 h. Statistically significant synergistic toxicity was observed when mouse PCLS were incubated for 24 h with KT+LPS and CZ+LPS with a decline in glutathione level, and for 48 h with TGZ+LPS without changes in glutathione level. Similar results were demonstrated with human PCLS after 24 h incubation: supra-additive toxicity with a decline in glutathione level was shown in KT+LPS-/CZ+LPS-treated groups, though it was not found to be statistically significant. Discussion: The maintenance of a glutathione level under conditions of mild liver injury after LPS exposure is possibly caused by the up-regulated production of reducing power, NADPH, and/or induction of glutathione synthesis de novo. Different molecular mechanisms seem to be responsible for IDILI development. Accordingly, glutathione was shown to play a role in KT and CZ synergistic hepatotoxicity, while TGZ-induced toxicity appears to be independent of the antioxidant status and glutathione level. The present study demonstrated once more, that inflammation can play a vital role in IDILI development. Additionally, it was shown, that glutathione content may be regarded as a potential novel biomarker for certain IDILI prediction and prevention.

Research Report 2: The role of Pgp in zebrafish cardiac glycosides chronotoxicity

Introduction: Circadian rhythms influence susceptibility and responses of organisms to xenobiotic exposure. ATP binding cassette transporters have a major influence on bio-availability, metabolism and excretion of drugs. Recently it has been shown that the gene expression of P-glycoprotein (Pgp), is under circadian transcriptional regulation. It has been hypothesized that Pgp expression might contribute to the observed diurnal variation in cardiac glycosides toxicity in human. Accordingly, the aims of this study were to establish a cardiac glycosides chronotoxicity model using zebrafish and to investigate the role of Pgp expression in observed diurnal changes in toxicity. Materials and Methods: Three or eight days post fertilization (dpf) zebrafish were incubated for 3 hours with digitoxin (DT) at two different time points: at the beginning (Zeitgeber, ZT 1-4) and at the end (ZT 10-13) of the light cycle. In the case of Pgp inhibitory studies, 3dpf or 8 dpf fish at two different circadian time points were pre-soaked for 1 hour with verapamil (VP) or ketoconazole (KT) before 3h incubation with DT. Toxicity was assessed using toxicity scoring systems. Furthermore, qRT-PCR was performed to measure gene expression levels of the gene for Pgp MDR1. Results: Diurnal variation in DT toxicity was demonstrated on 3 and 8 dpf zebrafish: significantly higher toxicity occurred at ZT 10-13 versus ZT 1-4. Additionally, a trend toward reduced expression levels of MDR1 in 3 and 8dpf fish ZT 13 in comparison with ZT 4 was observed. Increase in toxicity was observed when zebrafish were pre-soaked with KT before incubation with DT at ZT 1-4. VP in the tested concentration did not cause an increase in DT toxicity. Discussion: The present study highlights the suitability of zebrafish as a model to perform cardiac glycosides chronotoxicity studies, since a significant diurnal difference in DT toxicity has been demonstrated on 3 and 8 dpf zebrafish. The observed augmented toxicity of DT with KT could be explained by inhibition of both Pgp and CYP3A, which cannot be differentiated in the present study. Although the diurnal difference in MDR1 expression between ZT 4 and ZT 13 was not found to be significant, the general fluctuation trend in MDR1 expression levels corresponded well with variation in toxicity manifestation.