Progression of Repair and Injury in Human Liver Slices.

Human liver slice function was stressed by daily dosing of acetaminophen (APAP) or diclofenac (DCF) to investigate injury and repair. Initially, untreated human liver and kidney slices were evaluated with the global human U133A array to assess the extended culture conditions. Then, drug induced injury and signals of repair in human liver slices exposed to APAP or DCF (1 mM) were evaluated via specific gene expression arrays. In culture, the untreated human liver and kidney slices remained differentiated and gene expression indicated that repair pathways were activated in both tissues. Morphologically the human liver slices exhibited evidence of repair and regeneration, while kidney slices did not. APAP and DCF exposure caused a direct multi-factorial response. APAP and DCF induced gene expression changes in transporters, oxidative stress and mitochondria energy. DCF caused a greater effect on heat shock and endoplasmic reticulum (ER) stress gene expression. Concerning wound repair, APAP caused a mild repression of gene expression; DCF suppressed the expression of matrix collagen genes, the remodeling metalloproteases, cell adhesion integrins, indicating a greater hinderance to wound repair than APAP. Thus, human liver slices are a relevant model to investigate the mechanisms of drug-induced injury and repair.

Liver Effects of Clinical Drugs Differentiated in Human Liver Slices.

Drugs with clinical adverse effects are compared in an ex vivo 3-dimensional multi-cellular human liver slice model. Functional markers of oxidative stress and mitochondrial function, glutathione GSH and ATP levels, were affected by acetaminophen (APAP, 1 mM), diclofenac (DCF, 1 mM) and etomoxir (ETM, 100 µM). Drugs targeting mitochondria more than GSH were dantrolene (DTL, 10 µM) and cyclosporin A (CSA, 10 µM), while GSH was affected more than ATP by methimazole (MMI, 500 µM), terbinafine (TBF, 100 µM), and carbamazepine (CBZ 100 µM). Oxidative stress genes were affected by TBF (18%), CBZ, APAP, and ETM (12%-11%), and mitochondrial genes were altered by CBZ, APAP, MMI, and ETM (8%-6%). Apoptosis genes were affected by DCF (14%), while apoptosis plus necrosis were altered by APAP and ETM (15%). Activation of oxidative stress, mitochondrial energy, heat shock, ER stress, apoptosis, necrosis, DNA damage, immune and inflammation genes ranked CSA (75%), ETM (66%), DCF, TBF, MMI (61%-60%), APAP, CBZ (57%-56%), and DTL (48%). Gene changes in fatty acid metabolism, cholestasis, immune and inflammation were affected by DTL (51%), CBZ and ETM (44%-43%), APAP and DCF (40%-38%), MMI, TBF and CSA (37%-35%). This model advances multiple dosing in a human ex vivo model, plus functional markers and gene profile markers of drug induced human liver side-effects.

Isoproterenol effects evaluated in heart slices of human and rat in comparison to rat heart in vivo.

Human response to isoproterenol induced cardiac injury was evaluated by gene and protein pathway changes in human heart slices, and compared to rat heart slices and rat heart in vivo. Isoproterenol (10 and 100µM) altered human and rat heart slice markers of oxidative stress (ATP and GSH) at 24h. In this in vivo rat study (0.5mg/kg), serum troponin concentrations increased with lesion severity, minimal to mild necrosis at 24 and 48h. In the rat and the human heart, isoproterenol altered pathways for apoptosis/necrosis, stress/energy, inflammation, and remodeling/fibrosis. The rat and human heart slices were in an apoptotic phase, while the in vivo rat heart exhibited necrosis histologically and further progression of tissue remodeling. In human heart slices genes for several heat shock 70kD members were altered, indicative of stress to mitigate apoptosis. The stress response included alterations in energy utilization, fatty acid processing, and the up-regulation of inducible nitric oxide synthase, a marker of increased oxidative stress in both species. Inflammation markers linked with remodeling included IL-1α, Il-1ß, IL-6 and TNFα in both species. Tissue remodeling changes in both species included increases in the TIMP proteins, inhibitors of matrix degradation, the gene/protein of IL-4 linked with cardiac fibrosis, and the gene Ccl7 a chemokine that induces collagen synthesis, and Reg3b a growth factor for cardiac repair. This study demonstrates that the initial human heart slice response to isoproterenol cardiac injury results in apoptosis, stress/energy status, inflammation and tissue remodeling at concentrations similar to that in rat heart slices.

Preparation and culture of precision-cut organ slices from human and animal.

1.Human and animal precision-cut organ slices are being widely used to obtain drug metabolism and toxicity profiles in vitro. These data are then used to predict what might be seen in human patients. The accuracy of this prediction and extrapolation of the findings based on human or animal in vitro systems to the findings that occur in vivo is dependent on both the quality of the tissue itself and the quality of the in vitro system. 2.The quality of human organs used in research is dependent on procurement methods, warm ischaemia time, preservation solutions, cold ischaemia time, and donor-specific factors. It is important to confirm that the organs being used are highly viable and fully functional before using them in scientific studies. 3.The optimal preparation and incubation of organ slices is also essential in maintaining slice viability and function. It is important to prepare the slices in a cold preservation solution, to prepare the slices at a correct thickness, and to incubate the slices in a system where the slice rotates in out of the oxygen atmosphere and medium. 4.Meeting the criteria outlined here will lead to successful organ slice cultures for investigating drug-induced mechanisms and organ-specific toxicity.

Evaluation of drug-induced injury and human response in precision-cut tissue slices.

1.Drug induced organ injury is multifaceted, encompassing a spectrum of cell types and numerous networks reflecting cell-cell and cell-matrix interactions. Characterization of drug induced side effects and human response can be addressed in organ slice models. 2.The application of human tissue to various organ slice models including liver, intestine, kidney, liver-blood co-cultures and thyroid enhances our ability to focus on the clinical relevance of side effects identified in animal studies for human, and to evaluate potential biomarkers of the side effects. Dose-response relationships can help discern drug concentrations which alter organ function or affect morphology, to identify drug concentrationswhich could pose a risk for humans. 3.Insight into pathways of organ injury, by incorporating gene and protein expression profiling, with functional measurements and morphology, aid to define species differences and sensitivity. 4.Human organ slice studies are valuable for bridging the extrapolation of animal derived data and for identifying mechanisms relevant for humans, thereby expanding the scope of translational research for drug safety assessment.

Thyroid organotypic rat and human cultures used to investigate drug effects on thyroid function, hormone synthesis and release pathways.

Drug induced thyroid effects were evaluated in organotypic models utilizing either a rat thyroid lobe or human thyroid slices to compare rodent and human response. An inhibition of thyroid peroxidase (TPO) function led to a perturbation in the expression of key genes in thyroid hormone synthesis and release pathways. The clinically used thiourea drugs, methimazole (MMI) and 6-n-propyl-2-thioruacil (PTU), were used to evaluate thyroid drug response in these models. Inhibition of TPO occurred early as shown in rat thyroid lobes (2 h) and was sustained in both rat (24-48 h) and human (24 h) with ≥ 10 µM MMI. Thyroid from rats treated with single doses of MMI (30-1000 mg/kg) exhibited sustained TPO inhibition at 48 h. The MMI in vivo thyroid concentrations were comparable to the culture concentrations (~15-84 µM), thus demonstrating a close correlation between in vivo and ex vivo thyroid effects. A compensatory response to TPO inhibition was demonstrated in the rat thyroid lobe with significant up-regulation of genes involved in the pathway of thyroid hormone synthesis (Tpo, Dio1, Slc5a5, Tg, Tshr) and the megalin release pathway (Lrp2) by 24h with MMI (≥ 10 µM) and PTU (100 µM). Similarly, thyroid from the rat in vivo study exhibited an up-regulation of Dio1, Slc5a5, Lrp2, and Tshr. In human thyroid slices, there were few gene expression changes (Slc5a5, ~2-fold) and only at higher MMI concentrations (≥ 1500 µM, 24h). Extended exposure (48 h) resulted in up-regulation of Tpo, Dio1 and Lrp2, along with Slc5a5 and Tshr. In summary, TPO was inhibited by similar MMI concentrations in rat and human tissue, however an increased sensitivity to drug treatment in rat is indicated by the up-regulation of thyroid hormone synthesis and release gene pathways at concentrations found not to affect human tissue.

Glutathione modulation and oxidative stress in human liver slices.

Glutathione (GSH) levels are modulated in human liver slices to evaluate if drug induced liver injury is enhanced by a poor liver GSH status. Liver slice GSH levels were decreased by: 1) BSO (L-buthionine-S-sulfoximine) to inhibit GSH synthesis, and by 2) APAP (acetaminophen) which consumes GSH via conjugation to a metabolite. In this study, methimazole (MMI) liver injury was evaluated in the presence of a poor GSH status. MMI was selected because its structural thione moiety is linked with hepatotoxicity and during metabolism GSH is co-oxidized. MMI (500-1000 µM) affected oxidative stress pathways and mitochondrial function, resulting in lower liver slice GSH and ATP levels. Co-incubation of MMI with BSO or APAP led to further decreases of GSH and ATP levels in some human livers, at time points and concentrations not detected with MMI alone. Variation in human response was evident and demonstrated that some subjects with a poor liver GSH status could be further compromised with high MMI concentrations. MMI induced an up-regulation of gene expression linked with the GSH pathway, mitochondrial GSH and inflammation. Co-treatment of MMI with BSO induced a mixed response of oxidative stress related genes and an up-regulation of heat shock genes. The combination of MMI with APAP increased the expression of genes involved with oxidative stress and anti-oxidant defense, likely to protect the cells from mitochondrial injury. In summary, MMI induces oxidative stress at high concentrations, which can be augmented when liver GSH levels are decreased by the co-administration of some drugs or health status.

Blood cell oxidative stress precedes hemolysis in whole blood-liver slice co-cultures of rat, dog, and human tissues.

A novel in vitro model to investigate time-dependent and concentration-dependent responses in blood cells and hemolytic events is studied for rat, dog, and human tissues. Whole blood is co-cultured with a precision-cut liver slice. Methimazole (MMI) was selected as a reference compound, since metabolism of its imidazole thione moiety is linked with hematologic disorders and hepatotoxicity. An oxidative stress response occurred in all three species, marked by a decline in blood GSH levels by 24 h that progressed, and preceded hemolysis, which occurred at high MMI concentrations in the presence of a liver slice with rat (>or=1000 microM at 48 h) and human tissues (>or=1000 microM at 48 h, >or=750 microM at 72 h) but not dog. Human blood-only cultures exhibited a decline of GSH levels but minimal to no hemolysis. The up-regulation of liver genes for heme degradation (Hmox1 and Prdx1), iron cellular transport (Slc40a1), and GSH synthesis and utilization (mGST1 and Gclc) were early markers of the oxidative stress response. The up-regulation of the Kupffer cell lectin Lgals3 gene expression indicated a response to damaged red blood cells, and Hp (haptoglobin) up-regulation is indicative of increased hemoglobin uptake. Up-regulation of liver IL-6 and IL-8 gene expression suggested an activation of an inflammatory response by liver endothelial cells. In summary, MMI exposure led to an oxidative stress response in blood cells, and an up-regulation of liver genes involved with oxidative stress and heme homeostasis, which was clearly separate and preceded frank hemolysis.

Precision-cut organ slices to investigate target organ injury.

Drug-induced organ injury is a multifaceted process, involving numerous cell types and mediators, and remains a significant safety issue in pharmaceutical development and clinical therapy. Organ slices, an in vitro model representing the multicellular, structural and functional features of in vivo tissue, is a promising model for elucidating mechanisms of drug-induced organ injury and for characterising species susceptibilities. Time- and concentration-dependent drug-induced effects on organ slice gene expression, function and morphology are providing insight into the molecular and biochemical pathways leading to organ dysfunction, an altered morphology and the induction of repair pathways. Human organ slice studies are valuable for bridging the extrapolation of animal-derived data and for identifying mechanisms relevant for humans. The liver is the major organ used in organ slice studies; however, the utility of extrahepatic-derived slices, as well as cocultures for investigating multiple organ involvement in tissue injury is increasing. Organ slice investigations can further our understanding of the cell types and cell interactions involved in drug-induced injury and the consequences of drug-induced off-target effects for identifying compound liabilities that will impact safety.

Organ slices for the evaluation of human drug toxicity.

Human organ slices, an in vitro model representing the multicellular and functional features of in vivo tissue, is a promising model for characterizing mechanisms of drug-induced organ injury and for identifying biomarkers of organ injury. Target organ injury is a significant clinical issue. In vitro models, which compare human and animal tissue to improve the extrapolation of animal in vivo studies for predicting human outcome, will contribute to improving drug candidate selection and to defining species susceptibilities in drug discovery and development programs. A critical aspect to the performance and outcome of human organ slice studies is the use of high quality tissue, and the use of culture conditions that support optimum organ slice survivability, in order to accurately reproduce mechanisms of organ injury in vitro. The attribute of organ slices possessing various cell types and interactions contributes to the overall biotransformation, inflammatory response and assessment of injury. Regional differences and changes in morphology can be readily evaluated by histology and special stains, similar to tissue obtained from in vivo studies. The liver is the major organ of which slice studies have been performed, however the utility of extra-hepatic derived slices, as well as co-cultures is increasing. Recent application of integrating gene expression, with human organ slice function and morphology demonstrate the increased potential of this model for defining the molecular and biochemical pathways leading to drug-induced tissue changes. By gaining a more detailed understanding of the mechanisms of drug-induced organ injury, and by correlating clinical measurements with drug-induced effects in the in vitro models, the vision of human in vitro models to identify more sensitive and discriminating markers of organ damage is attainable.

Cold and cryopreservation of monkey liver slices.

Both cynomolgus and rhesus monkeys are utilized in chemical toxicity screening and drug development studies by industry and government laboratories. Along with better utilization of their tissue for in vivo studies, it would be advantageous if the tissue could be cold-preserved or cryopreserved for future in vitro experimentation. Therefore, livers were excised from control monkeys and precision-cut tissue slices were prepared. The objective of this study was twofold: to compare cold-preservation solutions (V-7 and Viaspan) and to compare controlled-rate and vitrification cryopreservation protocols. Monkey liver slices were cold-stored in V-7 or Viaspan preservation solutions for 7 days. V-7 maintained slice viability for 5 days whereas Viaspan maintained slice viability for 1 day. In the controlled-rate freezing procedure, slices were exposed to 10% dimethyl sulfoxide and 90% fetal calf serum (FCS); cooled at the rate of 0.5 degrees C, 1 degrees C, or 12 degrees C per min to -70 degrees C; then placed into liquid nitrogen. Vitrification was accomplished by exposing slices stepwise to increasing concentrations of 1,2-propanediol (1.2, 2.4, and 4 M) in FCS with direct submersion into liquid nitrogen. In both protocols, slices were rewarmed quickly to 37 degrees C and then incubated in FCS for 4 h. Three viability parameters were used to measure slice viability - retention of potassium, leakage of lactate dehydrogenase, and synthesis of protein. Liver slices cryopreserved at a rate of 0.5 degrees C per min and vitrified successfully retained 80 to 90% of their viability. These results confirm the feasibility of functional cold preservation and cryopreservation protocols for monkey liver slices that would allow for a more efficient use of monkey tissue.