The potential applications of artificial intelligence in drug discovery and development.

Development of a new dug is a very lengthy and highly expensive process since only preclinical, pharmacokinetic, pharmacodynamic and toxicological studies include a multiple of in silico, in vitro, in vivo experimentations that traditionally last several years. In the present review, we briefly report some examples that demonstrate the power of the computer-assisted drug discovery process with some examples that are published and revealing the successful applications of artificial intelligence (AI) technology on this vivid area. Besides, we address the situation of drug repositioning (repurposing) in clinical applications. Yet few success stories in this regard that provide us with a clear evidence that AI will reveal its great potential in accelerating effective new drug finding. AI accelerates drug repurposing and AI approaches are altogether necessary and inevitable tools in new medicine development. In spite of the fact that AI in drug development is still in its infancy, the advancements in AI and machine-learning (ML) algorithms have an unprecedented potential. The AI/ML solutions driven by pharmaceutical scientists, computer scientists, statisticians, physicians and others are increasingly working together in the processes of drug development and are adopting AI-based technologies for the rapid discovery of medicines. AI approaches, coupled with big data, are expected to substantially improve the effectiveness of drug repurposing and finding new drugs for various complex human diseases.

Sirtuin-activating compounds (STACs) alleviate D-galactosamine/lipopolysaccharide-induced hepatotoxicity in rats: involvement of sirtuin 1 and heme oxygenase 1.

Sirtuin activating compounds (STACs) attenuate various type of liver insults through mechanisms which are not fully understood. In the present study, we investigated the ameliorative potential of quercetin (natural polyphenol) and SRT1720 (synthetic SIRT1 activator) against D-galactosamine/lipopolysaccharide-induced hepatotoxicity (an experimental model of acute liver failure). Moreover, we compared and contrasted the roles of stress responsive enzymes, sirtuin 1 (SIRT1) and heme oxygenase 1 (HO-1) in hepatoprotection/ hepatotoxicity. Liver injury was induced in male Wistar rats by intraperitoneal injection of D-galactosamine (400 mg/kg) and lipopolysaccharide (10 microg/kg). Some animals were pretreated with quercetin (50 mg/kg i.p.) or SRT1720 (5 mg/kg i.p.). Twenty-four hours later, the effects of these treatments were evaluated by biochemical studies and Western blot. D-GalN/LPS treatment upregulated HO-1 expression, downregulated SIRT1 expression, decreased AST: ALT ratio and markedly increased bilirubin, catalase and conjugated diene levels. Pretreatment of D-GalN/LPS rats with either quercetin or SRT1720 returned SIRT1 expression, HO-1 expression and all the aforementioned markers towards normal. Collectively, these findings suggest that elevated HO-1 and low SIRT1 expressions are involved in the pathogenesis of D-GalN/LPS-induced hepatotoxicity. Drugs that downregulate HO-1 and/or upregulate SIRT1 seem to have antihepatotoxic effects and need further exploration.

In vitro and in vivo experimental hepatotoxic models in liver research: applications to the assessment of potential hepatoprotective drugs.

This mini-review highlights our and others' experience about in vitro and in vivo models that are being used to follow up events of liver injuries under various hepatotoxic agents and potential hepatoprotective drugs. Due to limitations of the outcomes in each model, we focus primarily on two models. First, a developed perfusion method for isolated immobilized hepatocytes that improves the process of oxygenation and helps in end-product removal is of considerable value in improving cell maintenance. This cellular model is presented as a short-term research-scale laboratory bioreactor with various physiological, biochemical, molecular, toxicological and pharmacological applications. Second, the in vivo model of D-galactosamine and lipopolysaccharide (D-GalN/LPS) combination-induced liver damage is described with some details. Recently, we have revealed that resveratrol and other natural polyphenols attenuate D-GalN/LPS-induced hepatitis. Moreover, we reported that D-GalN/LPS down-regulates sirtuin 1 in rat liver. Therefore, we discuss here the role of sirtuin 1 modulation in hepatoprotection. A successful development of pharmacotherapy for liver diseases depends on the suitability of in vitro and in vivo hepatic injury systems. Several models are available to screen the hepatotoxic or hepatoprotective activity of any substance. It is important to combine different methods for confirmation of the findings.

Comparative effects of Quercetin and SRT1720 against D-galactosamine/lipopolysaccharide-induced hepatotoxicity in rats: biochemical and molecular biological investigations.

OBJECTIVE: Quercetin, a plant flavonoid with potent antioxidant action, has been shown to be ameliorative against different types of liver insults, including D-Galactosamine/Lipopolysaccharide (D-GalN/LPS). The notion that its cytoprotective effects are SIRT1 mediated is still controversial. In this work, we examined whether the synthetic allosteric SIRT1 activator, SRT1720, may similarly attenuate D-GalN/LPS-induced hepatotoxicity. MATERIALS AND METHODS: Male Wistar rats were randomly assigned into 6 groups: (1) Control, (2) Quercetin, (3) SRT1720, (4) D-GalN/LPS, (5) Quercetin + D-GalN/LPS and (6) SRT1720 + D-GalN/LPS. After twenty-four hours, the effects of these treatments were evaluated by biochemical studies, real-time PCR and Western blot. RESULTS: D-GalN/LPS treatment downregulated SIRT1 expression and markedly increased the aminotransferase, bilirubin and conjugated diene levels. Conversely, quercetin and SRT1720 pretreatments upregulated SIRT1 expression and decreased the levels of the aforementioned markers. Quercetin had more profound effect on SIRT1 expression than SRT1720. Moreover, quercetin was more efficacious than SRT1720 in combatting the cytotoxic effects of D-GalN/LPS, as evidenced by lower markers of liver injury. CONCLUSIONS: These results strongly suggest the involvement of SIRT1 in the cytoprotective effects of quercetin and SRT1720 against D-GalN/LPS-induced hepatotoxicity.

Sirtuin 1 modulation in rat model of acetaminophen-induced hepatotoxicity.

Sirtuin 1 (SIRT1) is involved in important biological processes such as energy metabolism and regulatory functions of the cell cycle, apoptosis, and inflammation. Our previous studies have shown hepatoprotective effect of polyphenolic compound resveratrol, which is also an activator of SIRT1. Therefore, the aim of our present study was to clarify the role of SIRT1 in process of hepatoprotection in animal model of drug-induced liver damage. Male Wistar rats were used for both in vivo and in vitro studies. Hepatotoxicity was induced by single dose of acetaminophen (APAP). Some rats and hepatocytes were treated by resveratrol or synthetic selective activator of sirtuin 1 (CAY10591). The degree of hepatotoxicity, the activity and expression of the SIRT1 were determined by biochemical, histological and molecular-biological assessments of gained samples (plasma, liver tissue, culture media and hepatocytes). Resveratrol and CAY attenuated APAP-induced hepatotoxicity in vivo and in vitro. Moreover, both drugs enhanced APAP-reduced SIRT1 activity. Our results show that modulation of the SIRT1 activity plays a role in hepatoprotection. Synthetic activators of SIRT1 would help in understanding the role of SIRT1 and are therefore a major boost towards the search for specific treatment of liver disease.

D-galactosamine/lipopolysaccharide-induced hepatotoxicity downregulates sirtuin 1 in rat liver: role of sirtuin 1 modulation in hepatoprotection.

D-Galactosamine/Lipopolysaccharide (D-GalN/LPS) is a well known model of hepatotoxicity that closely resembles acute liver failure (ALF) seen clinically. The role of sirtuin 1 in this model has not yet been documented. However, there have been a number of studies about the cytoprotective effects of resveratrol, a SIRT1 activator, in the liver. This study was aimed at elucidating the roles of SIRT1 protein expression or catalytic activity in D-GalN/LPS model of hepatotoxicity. ALF was induced in male Wistar rats by intraperitoneal injection of D-GalN and LPS. Some groups of animals were pretreated with resveratrol and/or EX-527 (SIRT1 inhibitor). The effects of these treatments were evaluated by biochemical and Western blot studies. D-GalN/LPS treatment was able to induce hepatotoxicity and significantly increase all markers of liver damage and lipid peroxidation. A dramatic decrease of SIRT1 levels in response to D-GalN/LPS treatment was also documented. Resveratrol pretreatment attenuated D-GalN/LPS-induced hepatotoxicity. EX-527 blocked the cytoprotective effects of resveratrol. However, both resveratrol and EX-527 pretreatments did not exhibit any significant effect on SIRT1 protein expression. Collectively, these results suggest that downregulation of SIRT1 expression is involved in the cytotoxic effects of D-GalN/LPS model and SIRT1 activity contributes to the cytoprotective effects of resveratrol in the liver.

Resveratrol and related compounds as antioxidants with an allosteric mechanism of action in epigenetic drug targets.

The present review is intended to focus on naturally occurring cytoprotective agents such as resveratrol (trans-3,4',5-trihydroxystilbene) and other related compounds, probably with similar molecular mechanisms of action and high capacity to find applications in medical fields. Several physiological aspects have been ascribed to resveratrol and similar compounds. Resveratrol, among others, has been recently described as a silent information regulator T1 (SIRT1) activator that increases AMP-activated protein kinase (AMPK) phosphorylation and reduces the oxidative damage biomarkers during aging in laboratory settings. The reports on resveratrol and other SIRT1 activators from various sources are encouraging. The pharmacological strategies for modulation of sirtuins by small molecules through allosteric mechanisms should gain a greater momentum including human research. Resveratrol and resveratrol-like molecules seem to fulfill the requirement of a new horizon in drug research since these molecules cover a growing research means as antioxidants with allosteric mechanism in epigenetic drug targets. However, one should keep in mind the challenges of extrapolation of basic research into clinical results. Overall, the issue of sirtuins in biology and disease provides an insight on therapeutic potentials of sirtuin-based therapeutics and demonstrates the high complexity of drug-targeting these modalities for human applications.

Differential oxidative stress responses to D-galactosamine-lipopolysaccharide hepatotoxicity based on real time PCR analysis of selected oxidant/antioxidant and apoptotic gene expressions in rat.

Oxidative stress and apoptosis are proposed mechanisms of cellular injury in studies of xenobiotic hepatotoxicity. This study is focused on addressing the mutual relationship and early signals of these mechanisms in the D-galactosamine and lipopolysaccharide (D-GalN/LPS) hepatotoxicity model, with the help of standard liver function and biochemistry tests, histology, and measurement of gene expression by RT-PCR. Intraperitoneal injection of 400 mg/kg D-GalN and 50 µg/kg LPS was able to induce hepatotoxicity in rats, as evidenced by significant increases in liver enzymes (ALT, AST) and raised bilirubin levels in plasma. Heme oxygenase-1 and nitric oxide synthase-2 gene expressions were significantly increased, along with levels of their products, bilirubin and nitrite. The gene expression of glutathione peroxidase 1 remained unchanged, whereas a decrease in superoxide dismutase 1 gene expression was noted. Furthermore, the significant increase in the gene expression of apoptotic genes Bid, Bax and caspase-3 indicate early activation of apoptotic pathways, which was confirmed by histological evaluation. In contrast, the measured caspase-3 activity remained unchanged. Overall, the results have revealed differential oxidative stress and apoptotic responses, which deserves further investigations in this hepatotoxicity model.

[Nitric oxide in patients with obesity and metabolic syndrome]. / Oxid dusnatý u obezity a metabolického syndromu.

Nitric oxide in its pleiotropic role interacts with many diverse systems and beside others acts in pathophysiology of obesity and metabolic syndrome. Our review tends to summarize available basic publications aimed at the impact of NO on mitochondrial respiration, insulin resistance mainly in hepatocyte and the impact of NO on other factors of glucose metabolism. In this review, the authors try to shed light to pathophysiology of impaired NO bioavailability during diabetes and obesity too.

Glucose release as a response to glucagon in rat hepatocyte culture: involvement of NO signaling.

Glucagon and alpha-adrenergic-induced glycogenolysis is realized via the agonist/adenylyl cyclase/cAMP/protein kinase signaling pathway or via the activation of phosphorylase kinase by the mobilized calcium that supports the inhibition of glycogen synthase, respectively. The role of nitric oxide (NO) in this process has not been extensively studied. The present work was directed to the question whether NO is produced during glucagon-induced glycogenolysis in rat hepatocyte in a similar way like alpha-adrenoceptor stimulation. Glycogen-rich hepatocyte cultures were used. NO production (NO(2)(-)) was assessed under the influence of glucagon, dibutyryl cyclic AMP (db-cAMP), forskolin, the nitric oxide synthase (NOS) inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine, and the NO donor S-nitroso-N-acetyl penicillamine (SNAP). Inducible NOS (iNOS) mRNA was examined by reverse transcription-polymerase chain reaction. Glycogenolysis was followed up by estimation of medium glucose levels. The amount of glucose and NO(2)(-) released by glycogen-rich hepatocytes was increased as a result of glucagon, db-cAMP, forskolin and SNAP treatments. iNOS gene expression was upregulated by glucagon. Glycogenolysis that occurs through glucagon receptor stimulation involves NO production downstream of transduction pathways through an isoform of NO synthase. The present and previous studies document possible involvement of NO signaling in glycogenolytic response to glucagon and adrenergic agonists in hepatocytes.

Thapsigargin, a selective inhibitor of sarco-endoplasmic reticulum Ca2+ -ATPases, modulates nitric oxide production and cell death of primary rat hepatocytes in culture.

Increased cytosolic calcium ([Ca2+]i) and nitric oxide (NO) are suggested to be associated with apoptosis that is a main feature of many liver diseases and is characterized by biochemical and morphological features. We sought to investigate the events of increase in [Ca2+]i and endoplasmic reticulum (ER) calcium depletion by thapsigargin (TG), a selective inhibitor of sarco-ER-Ca2+ -ATPases, in relation to NO production and apoptotic and necrotic markers of cell death in primary rat hepatocyte culture. Cultured hepatocytes were treated with TG (1 and 5 micromol/L) for 0-24 or 24-48 h. NO production and inducible NO synthase (iNOS) expression were determined as nitrite levels and by iNOS-specific antibody, respectively. Hepatocyte apoptosis was estimated by caspase-3 activity, cytosolic cytochrome c content and DNA fragmentation, and morphologically using Annexin-V/propidium iodide staining. Hepatocyte viability and mitochondrial activity were evaluated by ALT leakage and MTT test. Increasing basal [Ca2+]i by TG, NO production and apoptotic/necrotic parameters were altered in different ways, depending on TG concentration and incubation time. During 0-24 h, TG dose-dependently decreased iNOS-mediated spontaneous NO production and simultaneously enhanced hepatocyte apoptosis. In addition, TG 5 micromol/L produced secondary necrosis. During 24-48 h, TG dose-dependently enhanced basal NO production and rate of necrosis. TG 5 micromol/L further promoted mitochondrial damage as demonstrated by cytochrome c release. A selective iNOS inhibitor, aminoguanidine, suppressed TG-stimulated NO production and ALT leakage from hepatocytes after 24-48 h. Our data suggest that the extent of the [Ca2+]i increase and the modulation of NO production due to TG treatment contribute to hepatocyte apoptotic and/or necrotic events.

The role of adrenergic agonists on glycogenolysis in rat hepatocyte cultures and possible involvement of NO.

Certain liver metabolic diseases point to the presence of disturbances in glycogen deposition. Epinephrine raises the cAMP level that activates protein kinase A leading to the activation of phosphorylase and glycogen breakdown. In the present report, we sought to investigate whether NO is produced during adrenoceptor agonist-induced glycogenolysis in rat hepatocytes in cultures. Isolated glycogen rich rat hepatocytes in cultures were used. NO production (NO(2)(-)) was assessed under the effect of adrenergic agonists and adrenergic agonist/antagonist pairs, dibutyryl cyclic AMP sodium-potassium salt (db-cAMP), NO synthase (NOS) inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME), aminoguanidine (AG) and the NO donor S-nitroso-N-acetyl penicillamine (SNAP). The inducible NO synthase (iNOS) mRNA was examined by the reverse transcription-polymerase chain reaction (RT-PCR). Glycogenolysis was quantified by glucose levels released into medium. The amount of glucose and NO(2)(-) released by hepatocytes was increased as a result of epinephrine, phenylephrine or db-cAMP treatments. The increase in glucose and NO(2)(-) released by epinephrine or phenylephrine was blocked or reduced by prazosin pretreatment and by NOS inhibitors aminoguanidine and L-NAME. iNOS gene expression was up-regulated by epinephrine. It can be concluded that glycogenolysis occurs through -adrenoceptor stimulation and a signaling cascade may involve NO production.

Effect of certain immunosuppressants on non-specific immunity cells in murine corneal grafts: study on early phases after transplantation.

The aim of our study was to evaluate the efficacy of FK506, mycophenolate mofetil (MM) and aminoguanidine (AMG) on infiltration of macrophages (MPHs), neutrophils (NPHs) and dendritic cells (DC) into corneal grafts during the early phases after transplantation (Tx). Tx was performed in mice (C57BL/10 to BALB/c). Therapy included FK506 (0.2 mg/kg), MM (30 mg/kg) or AMG (0.1 g/kg), started at the day of Tx and was injected i.p. daily. Corneas were excised on the third and seventh day after Tx. Immunohistological evaluation using antibodies against MPHs, NPHs and DC was performed and corneal grafts were assessed in the periphery and in central part of the cornea separately. On the third day after Tx, a massive infiltration of MPHs and NPHs into corneal grafts was revealed; the DC infiltration was lower in all treated groups. Treatment with FK506 and MM led to a significant reduction of NPHs in the centers of the grafts, but not of MPHs. In contrast, AMG significantly reduced MPHs migration into allografts on the third day after Tx, whereas NPHs infiltration has not been attenuated. However, immunosuppressants had no influence on the infiltration of DC during early phases after Tx.

Comparison of FK 506, mycophenolate mofetil, and aminoguanidine effects on delay of corneal allograft rejection in an experimental model of low-risk and high-risk keratoplasty.

The purpose of our study was to compare the effectiveness of immunosuppressive drugs on the prevention of allograft rejection in a murine model of low-risk and high-risk keratoplasty. The therapy included FK 506 (tacrolimus; 0.2 mg/kg), mycophenolate mofetil (30 mg/kg), aminoguanidine (0.1 g/kg), and combination of FK506 + mycophenolate mofetil or FK506 + aminoguanidine. The results obtained from the Gray's survival model stratified according to the type of subjects suggest that a major rejection risk reduction was achieved using FK506; good results also were obtained for mycophenolate mofetil. Although the point estimates of both the survival and relative risk of rejection suggest a deferred effect of the combination FK506 + mycophenolate mofetil, this finding did not prove statistically significant.

Inconsistent role of nitric oxide on lipolysis in isolated rat adipocytes.

Though two isoforms of nitric oxide synthase, iNOS and eNOS, were reported in adipocytes, the role of NO in adipose tissue is still ambiguous. The aims of the present study were 1) to follow the effect of bacterial lipopolysaccharide (LPS), on 24 h-lipolysis in rat epididymal adipocyte culture in relation to iNOS stimulation; 2) to compare LPS-induced NO effects with exogenously NO, delivered as S-nitroso-N-acetylpenicillamine (SNAP), and 3) to examine the possible role of NO signaling agonist in lipolysis mediated by the beta(3)-adrenoreceptor agonist. Lipolysis was measured by glycerol and free fatty acid (FFA) production. The medium nitrite levels were used for the indirect estimation of NOS expression. Adipocyte mitochondrial function was assessed by the MTT test. LPS produced a concentration-dependent increase of NO with a decrease of viability at the highest dose. However, LPS did not affect lipolysis. SNAP did not exhibit significant changes in glycerol, FFA or MTT. BRL-37344 and db-cAMP significantly increased nitrite, glycerol and FFA levels. There was a positive correlation between glycerol release and nitrite production. Moreover, BRL-37344 significantly reduced mitochondrial functions. The pretreatment with bupranolol, beta(3)-antagonist, restored all parameters affected by BRL-37344. These results support a concept that NO fulfils multifaceted role of stimulating lipolysis under physiological conditions (beta-agonistic effect) and modulating the same processes during inflammatory (LPS) processes.

FK 506 and aminoguanidine suppress iNOS induction in orthotopic corneal allografts and prolong graft survival in mice.

The aim of this study was to compare the effectiveness of immunosuppressant FK 506 and the specific inhibitor of inducible nitric oxide synthase (iNOS) aminoguanidine (AG) in prevention of corneal graft rejection and to investigate the iNOS expression in the rejection process. Orthotopic corneal allografting in mice was performed (C57BL/10; H-2(b) to BALB/c; H-2(d)). FK 506 (0.3 mg/kg per day) or AG (100 mg/kg per day) was injected intraperitoneally for 4 weeks. Grafted mice without therapy served as controls. Immunohistological evaluation of iNOS-positive cells and macrophage infiltration in grafts 27th day after grafting was performed. Within 4 weeks FK 506 prevented graft rejection in 71% and AG in 57% of animals compared to 29% of clear grafts in controls. A significant proportion of iNOS-positive cells was detected in the rejected grafts of the control and AG-treated groups. The treatment with FK 506 resulted in the inhibition of iNOS expression to a high degree in the rejected corneas. Non-rejected corneas of all groups and non-transplanted corneas exhibited no iNOS-positive cells. A massive infiltration of macrophages was detected in the rejected grafts, whereas non-rejected grafts exhibited only slight infiltration of macrophages. The presented data suggest that overexpression of iNOS and/or activation of iNOS is one of the several influential factors that contribute to the rejection process and that iNOS suppression delays corneal allograft rejection. FK 506 and AG are effective drugs in preventing corneal allograft rejection. Higher beneficial effect of FK 506 on graft survival could be explained by its well-known selective T-cell immunosuppression.

Urea synthesis and cyclosporin a biotransformation in a laboratory scale hepatocyte bioreactor model.

Inefficient oxygenation and build-up of waste products are inevitable in a conventional cell culture. The development of a perifusion method for isolated hepatocytes improves the process of oxygenation and helps in end-product removal. For the perifusion of cells, they must be immobilized to prepare a bioreactor model. The present work was directed to testing a hepatocyte bioreactor and maintaining tissue metabolizing activity for periods ranging from 24 to 72 h of continuous and intermittent perifusion and to test the ability of this system for cyclosporin A (CsA), biotransformation and urea synthesis as contrasted to hepatocyte in the culture. Hepatocytes were isolated, immobilized and perifused with William's E culture medium containing 1mM NH(4)Cl and CsA (20 microM). Hepatocytes in the culture were treated in the same way. CsA disappearance from the perifusion or culture media was determined by a HPLC method. Higher urea synthesis rate was achieved by cells in the continuously perifused bioreactor for 24 h compared to culture (0.5+/-0.05 mg h(-1) vs 0.33+/-0.03 mg h(-1), respectively). ALT leakage was lower in the bioreactor model (60 Ul(-1)) as compared to hepatocyte culture (125 Ul(-1)). The ability of hepatocytes in the bioreactor to metabolize CsA was very fast compared to hepatocytes in the culture during 24 h (95% vs 50%, respectively). The present data reveal the higher efficiency of hepatocytes in a bioreactor model as compared to hepatocyte culture. Further research is required in relation to better understanding and standardization of the culture conditions for immobilized and perifused hepatocytes. In addition, the cellular model described here inherits economic and ethical potentials.

Effect of nitric oxide donors on isoprenaline-induced lipolysis in rat epididymal adipose tissue: studies in isolated adipose tissues and immobilized perfused adipocytes.

The present investigation was directed to study the effect of in vitro or ex vivo NO donors, sodium nitroprusside and molsidomine, using isolated sliced adipose tissue or in the form of immobilized and perfused adipocytes on the basal and isoprenaline-stimulated lipolysis. The results demonstrated that 1) in vitro application of sodium nitroprusside to perfused adipocytes or molsidomine to sliced adipose tissues affects isoprenaline-induced lipolysis in two ways, an increase in lipolysis at low isoprenaline concentrations (which means the sensitization of adipose tissues to adrenergic effect by NO) and decreased adrenergic agonist-stimulated lipolysis at higher concentration of isoprenaline (a decrease in the maximum lipolytic effect of isoprenaline), 2) low concentrations of molsidomine alone induced lipolysis from adipose tissue which attained more than 60% of that by isoprenaline (pD2 value for molsidomine = 11.2, while pD2 for isoprenaline = 8.17) while sodium nitroprusside did not affect the basal lipolysis significantly, 3) in vivo administration of molsidomine for 2 days reduced the maximum lipolytic effect of isoprenaline and (only non-significantly) increased the sensitivity to low doses of isoprenaline. In conclusion the present data demonstrate that NO plays an important role in adrenergic lipolysis in adipose tissues and further investigations are needed to unravel the exact role of NO in lipolysis.

Potential application of immobilized and perfused hepatocytes in environmental toxicology studies.

Conventional cellular models have contributed significantly to the understanding of many aspects of cell physiology and molecular biology. In these models cells are metabolically less active, due to the inefficient oxygenation and waste product buildup. Therefore perfusion methods for the cells are expected to improve cell activities. Cells have to be fixed in or on an appropriate inert carrier or support, which enables cellular perfusion, maintains integrated cellular functions and makes a bioreactor. Since isolated hepatocytes are extensively used in biomedical studies including those dealing with environmental pollutants or toxins and in xenobiotic biotransformation investigations, an efficient hepatocyte perfusion model has to be available for researchers. This research article is focusing on the value of hepatocyte immobilization as a laboratory bioreactor model and is shedding light on its potentiality in research related to public health. We demonstrate the application of this cellular model as a means to study representative phase I and phase II biotransformation reactions using hexobarbital hydroxylation and 7-ethoxycoumarin deethylation and 4-chloro 2-dinitrobenzene glutathione. Both phase I and phase II drug biotransformation in hepatocytes was demonstrated in this study non-destructively to the cells and in an efficient way. In spite of the aforementioned advantages, immobilized hepatocytes yet have relatively limited applications compared to conventional hepatocyte cellular systems. Reasons for this discrepancy are discussed. This cellular system may become popular due to the better performance of immobilized hepatocytes as compared to conventional hepatocyte culture and due to economic and ethical reasons. Naturally its applicability will cover several biomedical areas including basic research in environmental toxicology and other public health issues.

Modulatory effect of cyclosporin A on tert-butyl hydroperoxide-induced oxidative damage in hepatocytes.

In the present work, we followed an in vitro protective action of cyclosporin A (CsA) against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in hepatocytes. Various parameters (cell viability, cytosolic calcium level, rhodamine 123 accumulation as indicator of mitochondrial membrane potential and alanine-aminotransferase leakage from cells) were measured as an index of cytotoxicity. Tert-butyl hydroperoxide (1 mM) significantly increased cytosolic Ca2+ and affected mitochondrial membrane potential. Pretreatment with cyclosporin A (0.5 microM) reduced t-BHP-induced cytosolic Ca2+ increase and ALT (alanine-aminotransferase) leakage, but had no protective effect on t-BHP-induced changes of mitochondrial membrane potential. Our data thus suggest that the mechanism of cytoprotection of CsA on the cytosolic Ca2+ changes and ALT leakage induced by t-BHP, does not directly correlate with protection of t-BHP-induced changes of mitochondrial membrane potential.