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Drug-induced liver injury (DILI) is a major factor influencing new drug withdrawal; therefore, an appropriate toxicity assessment at the preclinical stage is required. Previous in silico models have been established using compound information listed in large data sources, thereby limiting the DILI risk prediction for new drugs. Herein, we first constructed a model to predict DILI risk based on a molecular initiating event (MIE) predicted by quantitative structure–activity relationships, admetSAR parameters (e.g. cytochrome P450 reactivity, plasma protein binding, and water-solubility), and clinical information (maximum daily dose [MDD] and reactive metabolite [RM]) for 186 compounds. The accuracy of the models using MIE, MDD, RM, and admetSAR alone were 43.2%, 47.3%, 77.0%, and 68.9%, while the “predicted MIE + admetSAR + MDD + RM” model’s accuracy was 75.7%. The contribution of MIE to the overall prediction accuracy was little effect or rather worsening it. However, it was considered that MIE was a valuable parameter and that it contributed to detect high DILI risk compounds in the early development stage. We next examined the effect of stepwise changes in MDD on altering the DILI risk and estimating the maximum safety dose (MSD) for clinical use based on structural information, admetSAR, and MIE parameters because it is important to estimate the dose that could prevent the DILI onset in clinical conditions. Low-MSD compounds might increase the DILI risk, as these compounds were classified as “most-DILI concern” at low doses. In conclusion, MIE parameters were especially useful to check the DILI concern compounds and to prevent the underestimation of DILI risk in the early stage of drug development.

Cancer cells rely on glycolysis to generate ATP for survival. However, inhibiting glycolysis is insufficient for the eradication of cancer cells because glycolysis-suppressed cells undergo metabolic reprogramming toward mitochondrial oxidative phosphorylation. We previously described that upon glycolytic suppression in pancreatic cancer cells, intracellular glycometabolism is shifted toward mitochondrial oxidative phosphorylation in an autophagy-dependent manner for cellular survival. Here, we hypothesized that mitophagy, which selectively degrades mitochondria via autophagy, is involved in mitochondrial activation under metabolic reprogramming. We revealed that glycolytic suppression notably increased mitochondrial membrane potential and mitophagy in a pancreatic cancer cell model (PANC-1). PTEN-induced kinase 1 (PINK1), a ubiquitin kinase that regulates mitophagy in healthy cells, regulated mitochondrial activation through mitophagy by glycolytic suppression. However, Parkin, a ubiquitin ligase regulated by PINK1 in healthy cells to induce mitophagy, was not involved in the PINK1-dependent mitophagy of the cancer glycometabolism. These results imply that cancer cells and healthy cells have different regulatory pieces of machinery for mitophagy, and inhibition of cancer-specific mechanisms may be a potential strategy for cancer therapy targeting metabolic reprogramming.

Despite progress in the use of hyperthermia in clinical practice, the thermosensitivity of cancer cells is poorly understood. In a previous study, we found that sensitivity to hyperthermia varied between ovarian and uterine cancer cell lines. Upon hyperthermia, glycolytic enzymes decreased in hyperthermia-resistant SKOV3 cells. However, the mechanisms of glycolysis inhibition and their relationship with thermoresistance remain to be explored. In this study, metabolomic analysis indicated the downregulation of glycolytic metabolites in SKOV3 cells after hyperthermia. Proteomic and pathway analyses predicted that the ubiquitin pathway was explicitly activated in resistant SKOV3 cells, compared with hyperthermia-sensitive A2780 cells, and STUB1, a ubiquitin ligase, potentially targeted PKM, a glycolytic rate-limiting enzyme. PKM is degraded via ubiquitination upon hyperthermia. Although glycolysis is inactivated by hyperthermia, ATP production is maintained. We observed that oxygen consumption and mitochondrial membrane potential were activated in SKOV3 cells but suppressed in A2780 cells. The activation of mitochondria could compensate for the loss of ATP production due to the suppression of glycolysis by hyperthermia. Although the physiological significance has not yet been elucidated, our results demonstrated that metabolomic adaptation from the Warburg effect to mitochondrial oxidative phosphorylation could contribute to thermoresistance in ovarian and uterine cancer cells.

Systemically administered lipid nanoparticles (LNPs) are complexed with Apolipoprotein E (ApoE) in the bloodstream, and the complex is subsequently largely taken up by hepatocytes. Based on a previous report showing that, like blood, lymph fluid also contains ApoE, and that LECs, in turn, expresses a low density-lipoprotein receptor (LDLR), which is the receptor responsible for the ApoE-bound LNP, we hypothesized that subcutaneously administered LNPs would be taken up by LECs via an ApoE-LDLR pathway. Our in vitro studies using immortal LECs that we established in a previous study showed that LEC indeed took up LNPs in an ApoE-dependent manner. We then reported on the development of LNPs that target the lymphatic endothelium for in vivo siRNA delivery after subcutaneous administration. The key to success for in vivo LEC targeting is that the surface needs to be modified with a high density of polyethylene glycol (PEG)-conjugated lipids with short acyl chains (C14). The LNPs were drained into the lymphatic system, and then accumulated in lymphatic endothelial cells in an ApoE-dependent manner, most likely after the release of the PEG-lipid. Subcutaneous administration of optimized LNPs containing encapsulated siRNA against VEGFR3, a marker of LECs, significantly inhibited the expression of VEGFR3. These findings are the first report of a simple straightforward strategy for targeting lymphatic endothelial cells by using ionizable lipid-formulated LNPs.

Inhibition of bile acid excretion by drugs is a significant factor in the development of drug-induced cholestatic liver injury. We constructed a new in vitro assay system to detect bile acid-dependent cytotoxicity in hepatocytes. This cell-based system can assess the toxicity of the parent compound, as well as the contribution of metabolite(s). In addition, this system can utilize several types of hepatocytes (primary hepatocytes, hepatoma cell line, and induced pluripotent stem cell-induced hepatocytes). In this chapter, a method to detect drug-induced bile acid-dependent toxicity in hepatocytes is described.

In drug development, a system for predicting drug metabolism and drug‐induced toxicity is necessary to ensure drug safety. Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) is an important drug‐metabolizing enzyme expressed in the liver and small intestine, and predicting CYP3A4‐mediated drug metabolism and drug‐induced toxicity is essential. We previously developed procedures to differentiate human induced pluripotent stem (iPS) cells into hepatocyte‐like cells (HLCs) or intestinal epithelial‐like cells (IECs) with a fetal phenotype as well as a highly efficient genome editing technology that could enhance the homologous recombination efficiency at any locus, including CYP3A4. By using human iPS cells and our genome editing technology, we generated CYP3A4‐knockout (KO) iPS cell‐derived HLCs and IECs for the evaluation of CYP3A4‐mediated drug metabolism and drug‐induced toxicity. CYP3A4 deficiency did not affect pluripotency and hepatic and intestinal differentiation capacities, and CYP3A4 activity was entirely eradicated by CYP3A4 KO. Off‐target effects (e.g., inhibition of bile acid excretion) were hardly observed in CYP3A4‐KO cells but were observed in CYP3A4 inhibitor‐treated (e.g., ketoconazole) cells. To evaluate whether drug‐induced hepatotoxicity and enterotoxicity could be predicted using our model, we exposed CYP3A4‐KO HLCs and IECs to acetaminophen, amiodarone, desipramine, leflunomide, tacrine, and tolcapone and confirmed that these cells could predict CYP3A4‐mediated toxicity. Finally, we examined whether the therapeutic effects of an anti‐hepatitis C virus (HCV) drug metabolized by CYP3A4 would be predicted using our model. CYP3A4‐KO HLCs were treated with asunaprevir (antiviral drug metabolized by CYP3A4) after HCV infection, and the anti‐viral effect was indeed strengthened by CYP3A4 KO. Conclusion: We succeeded in generating a novel evaluation system for prediction of CYP3A4‐mediated drug metabolism and drug‐induced toxicity., To evaluate CYP3A4‐mediated drug metabolism and drug‐induced toxicity accurately, we generated CYP3A4‐knockout (KO) iPS cell‐derived hepatocyte‐like cells and intestinal epithelial‐like cells. By using our model, CYP3A4‐mediated drug‐induced toxicity and the therapeutic effects of an anti‐HCV drug could be predicted.

Despite increased aerobic glycolysis is generally seen in human cancers, pancreatic ductal adenocarcinoma (PDAC) cells keep alive in glycolysis-suppressed conditions (1). Here, we sought there are potential therapeutic targets in glycolysis-suppressed PDAC cells. By screening anti-cancer metabolism compounds, we identified SP2509, a selective lysine specific demethylase 1 (LSD1) inhibitor (2). We also tested the effect of another three LSD1 inhibitors (Iadademstat, OG-L002 and T-3775440) on PDAC cells viability. The results showed that OG-L002, but not Iadademstat or T-3775440, lowered glycolysis-suppressed PDAC cells viability, like SP2509. Importantly, only knockdown of LSD1 or LSD2 failed to lower the viability of glycolysis suppressed PDAC cells. In our previous study, we proved that glucose-starvation make PDAC cells yield survival to mitochondrial oxidative phosphorylation (1). In this study, we cleared fatty acids metabolism played a crucial role for glycolysis suppressed PDAC cells survival. Then, we confirmed that SP2509 and OG-L002 can lead lipid droplets accumulation in glycolysis-suppressed conditions. In conclusion, our results showed that SP2509 and OG-L002 may involve regulatory effects on lipid droplets homeostasis, which control storage and release of fatty acids, not only by targeting LSD1 or LSD2. Moreover, SP2509 and OG-L002 significantly lowered viability of glycolysis-suppressed PDAC cells, supposing a novel therapeutic potential of approaches to override resistance of glucose-starvation in PDAC cells. 1.Shiratori R, Furuichi K, Yamaguchi M, Miyazaki N, Aoki H, Chibana H, et al. Glycolytic suppression dramatically changes the intracellular metabolic profile of multiple cancer cell lines in a mitochondrial metabolism-dependent manner. Sci Rep. 2019;9(1):18699. 2.Fiskus W, Sharma S, Shah B, Portier BP, Devaraj SGT, Liu K, et al. Highly effective combination of LSD1 (KDM1A) antagonist and pan-histone deacetylase inhibitor against human AML cells. Leukemia. 2017;31(7):1658. Citation Format: Zhiheng Zhang, Haruna Aoki, Keitaro Umezawa, Taichi Oshima, Takuya Hirao, Yuri Miura, Kousei Ito, Shigeki Aoki. Several LSD1 inhibitors have a potential to disturb fatty acid metabolism necessary &It glycolysis-inhibited pancreatic cancers to survive [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2673.

[Purpose] Tumors with low T cell infiltrates (poor immunogenicity) display lower probability of clinical response to immune checkpoint inhibitors. To achieve higher immunotherapy efficacy, it is imperative to establish a novel strategy for improving immunogenicity. Abacavir (ABC), an anti-HIV drug, binds to the specific allele of human leukocyte antigen (HLA-B*57:01) and activate CD8+ T cells by presenting altered abnormal peptides. In this study, we considered the immune interaction between ABC and HLA-B*57:01 improving immunogenicity in poorly immunogenic tumors leading to a potent anti-cancer effect. [Methods] Mice were inoculated subcutaneously with tumor cells into the right flanks and interperitoneally treated with either ABC 5 mg or vehicle (water) for consecutive 9 days (B16F10) or 13 days (3LL) after tumor inoculation. Each tumor size was measured using vernier calipers on every 2 days. Activation, proliferation, and IFN-γ secretion of tumor infiltrating CD8+ T cells were analyzed by FACS. [Results and Discussion] ABC treatment showed significant tumor growth inhibition associated with CD8+ T cell infiltration into tumor in mice inoculated with HLA-B*57:01 expressing B16F10 melanoma or Lewis lung carcinoma (3LL) cells, but neither in HLA-B*57:03 (negative allele)-expressing nor control tumors inoculated mice. Moreover, the tumor infiltrating CD8+ T cell were immune-activated and proliferated, with secreting IFN-γ. In contrast, ABC-induced anti-tumor effect was abrogated in CD8+ T cell-depleted mice, IFN-γ KO mice, and CXCR3-blocked mice. The combination therapy with anti-PD-1 antibody treatment induced better inhibition of tumor growth than ABC monotherapy in HLA-B*57:01 expressing B16F10 inoculated mice. These results suggested that ABC/HLA-B*57:01 interaction could improve tumor immunogenicity associated with CD8+ T cell-mediated anti-tumor effect in poorly immunogenic tumors. This finding lays a foundation to develop a novel drug-induced cancer immunotherapy targeting immune resistant tumors. Citation Format: Takeshi Susukida, So-ichiro Sasaki, Tomohiro Shirayanagi, Shigeki Aoki, Kousei Ito, Yoshihiro Hayakawa. Development of a novel strategy for cancer immunotherapy based on tumor immunogenicity improvement by HLA-drug interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6391.

Bone homeostasis is maintained by bone remodeling, which involves continuous bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation of bone turnover, caused by osteoclast overactivation, causes destructive bone diseases. However, the mechanisms underlying the maintenance of osteoclast differentiation and activation are unclear. Herein, we examined the role of autophagy in the maintenance of osteoclast differentiation and maturation. We used in vitro and in vivo assays to evaluate relationships between mitochondrial activity and autophagy during osteoclast differentiation and maturation. Our results indicate that autophagy was enhanced during osteoclast differentiation and maturation, and autophagic activity was positively correlated with osteoclast activity and survival. Maintenance of mitochondrial function, which is critical during osteoclast differentiation and maturation, was controlled by autophagy. Continuous exposure of osteoclasts to glucocorticoids upregulated autophagic processes. Treatment with the autophagic inhibitor chloroquine suppressed prolonged survival of activated osteoclasts and attenuated excessive osteoclast activity. Our study shows that autophagy-dependent mitochondrial function plays an important role in osteoclast differentiation and maturation. Elucidating the mechanisms regulating autophagic activity in osteoclasts, and developing bone-tissue—specific inhibitors of autophagy, will lead to improved understanding of the pathologies involved in destructive bone diseases.

The interaction of human leukocyte antigen (HLA) with specific drugs is associated with delayed-type hypersensitivity reactions, which cause severe cutaneous toxicity. Such interactions induce structural alterations in HLA complexes via several different mechanisms such as the hapten theory, p-i concept, and altered peptide repertoire model, leading to the activation of cytotoxic T cells. To date, comprehensive detection of such structural alterations in preclinical studies has been difficult. Here, we evaluated structural alterations in HLA complexes focusing on the interaction between the HLA-B*57 : 01 allele and abacavir (an anti-human immunodeficiency virus drug), representing a model of abacavir hypersensitivity syndrome induced by changes in the peptide repertoire on the HLA molecule. We employed a phage display method using a commercially available antibody library to screen specific phage antibodies able to recognize HLA-B*57 : 01. The affinity of selected phage antibodies increased because of structural alterations in HLA-B*57 : 01 following exposure to abacavir, indicating that specific phage antibodies can identify drug-mediated structural changes in HLA complexes. We also identified an unreported structural change in HLA-B*57 : 01 using the phage display method, whereby abacavir increased the expression of peptide-deficient HLA-B*57 : 01 on the cell surface. These results suggest that phage display technology is a useful method for detecting structural changes in HLA complexes. This technology represents a potential novel strategy for predicting HLA-associated hypersensitivity reactions by drugs in pre-clinical studies.

The combination of certain human leukocyte antigen (HLA) polymorphisms with administration of certain drugs shows a strong correlation with developing drug hypersensitivity. Examples of typical combinations are HLA-B*57:01 with abacavir and HLA-B*15:02 with carbamazepine. However, despite belonging to the same serotype, HLA-B*57:03 and HLA-B*15:01 are not associated with drug hypersensitivity. Recent studies have shown that several HLA polymorphisms are associated with multiple drugs rather than a single drug, all resulting in drug hypersensitivity. In this study, we compared the molecular structures and intracellular localization of HLA-B*57:01, HLA-B*58:01, and HLA-B*15:02, which pose risks for developing drug hypersensitivity, as well as HLA-B*57:03 and HLA-B*15:01 that do not present such risks. We found that HLA molecules posing risks have a low affinity for the subunit β

Flucloxacillin (FLX) induces adverse liver reactions, which has been reported to be related to human leukocyte antigen (HLA)-B*57:01. In a previous study, abacavir-induced hypersensitivity was induced in HLA-B*57:01-transgenic mice (B*57:01-Tg), originally constructed by our group (Susukida et al., 2021). In this study, B*57:01-Tg mice were used to reproduce FLX-induced liver injury. However, treatment of B*57:01-Tg mice with FLX alone did not increase serum ALT levels. Immune-deficient B*57:01-Tg/PD-1

The liver microphysiological system (MPS) model is an in-vitro culture method that mimics physiological blood flow, which enhances basal cellular functions. However, the liver MPS model has not been tested in the preclinical stage because of its obscure utility. It can overcome the major problem of conventional systems-rapid loss of mitochondrial activity in cultured hepatocytes due to limited oxygen supply-by supplying oxygen to cultured hepatocytes using a perfusion device. In this study, we developed a new perfusion culture system that can detect mitochondrial toxicity. Primary mouse hepatocytes were cultured under perfusion condition for 48 hr. The hepatocytes showed increased oxygen consumption and reduced lactate release. These results indicated that the ATP-production pathway was switched from glycolysis to mitochondrial oxidative phosphorylation in the perfusion culture system. Furthermore, ATP levels were considerably reduced in the perfusion culture system after exposure to phenformin, a mitochondrial complex I inhibitor. To summarize, the perfusion culture system could improve the mitochondrial activity in primary mouse hepatocytes, and thus, has potential implications in the detection of mitochondrial toxicity.

Based on recent genome-wide association studies, abacavir-induced hypersensitivity is highly associated with human leukocyte antigen (HLA)-B*57:01 allele. However, the underlying mechanism of this occurrence is unclear. To investigate the underlying mechanism, we developed HLA-B*57:01 transgenic mice and found that application of abacavir could cause CD8 T cell activation with elevation in PD1 expression; however, severe skin hypersensitivity was not observed. To eliminate the immunosuppressive effect of PD1, HLA-B*57:01 transgenic/PD1 knockout (01Tg/PD1) mice were generated by mating HLA-B*57:01 transgenic mice and PD1 knockout mice. Thereafter, 01Tg/PD1 mice were treated with abacavir. Similar to the above results, severe skin hypersensitivity was not observed. Therefore, we treated 01Tg/PD1 mice with an anti-CD4 antibody to deplete CD4 T cells, followed by abacavir topically and orally. Severe abacavir-induced skin hypersensitivity was observed in 01Tg/PD1 mice after depletion of CD4 T cells, in addition to significant CD8 T cell activation and dendritic cell maturation. Taken together, we succeeded in reproducing severe skin hypersensitivity in a mouse model. And we found that through the combined depletion of PD1 and CD4 T cells, CD8 T cells could be activated and could proceed to clonal proliferation, which is promoted by mature dendritic cells, thereby eventually inducing severe skin hypersensitivity.

Idiosyncratic drug toxicity (IDT) associated with specific human leukocyte antigen (HLA) allotype is a rare and unpredictable life-threatening adverse drug reaction for which prospective mechanistic studies in humans are difficult. Here, we show the importance of immune tolerance for IDT onset and determine whether it is susceptible to a common IDT, HLA-B*57:01-mediated abacavir (ABC)-induced hypersensitivity (AHS), using CD4+ T cell-depleted programmed death-1 receptor (PD-1)-deficient HLA-B*57:01 transgenic mice (B*57:01-Tg/PD-1−/−). Although AHS is not observed in B*57:01-Tg mice, ABC treatment increases the proportion of cytokine- and cytolytic granule-secreting effector memory CD8+ T cells in CD4+ T cell-depleted B*57:01-Tg/PD-1−/− mice, thereby inducing skin toxicity with CD8+ T cell infiltration, mimicking AHS. Our results demonstrate that individual differences in the immune tolerance system, including PD-1highCD8+ T cells and regulatory CD4+ T cells, may affect the susceptibility of humans to HLA-mediated IDT in humans., Using a transgenic mouse model that recapitulates abacavir hypersensitivity syndrome, an idiosyncratic adverse drug reaction, Susukida et al show that individual differences in the immune tolerance system affect the susceptibility to idiosyncratic drug toxicity.

Cholestatic drug-induced liver injury (DILI) is a type of hepatotoxicity. Its underlying mechanisms are dysfunction of bile salt export pump (BSEP) and multidrug resistance-associated protein 2/3/4 (MRP2/3/4), which play major roles in bile acid (BA) excretion into the bile canaliculi and blood, resulting in accumulation of BAs in hepatocytes. The sandwich-cultured hepatocyte (SCH) model can simultaneously analyze hepatic uptake and biliary excretion. Therefore, we investigated whether sandwich-cultured human induced pluripotent stem cell (iPS cell)-derived hepatocytes (SCHiHs) are suitable for evaluating cholestatic DILI. Fluorescent N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2'-aminoethanesulfonate (tauro-nor-THCA-24-DBD, a BSEP substrate) was accumulated in bile canaliculi, which supports the presence of a functional bile canaliculi lumen. MRP2 was highly expressed in the Western blot analysis, whereas the mRNA expression of BSEP was hardly detectable. MRP3/4 mRNA levels were maintained. Of the 22 compounds known to cause DILI with BAs, 7 showed significant cytotoxicity. Most high-risk drugs were detected using the developed SCHiH system. However, a shortcoming was the considerably low expression level of BSEP, which prevented the detection of some relevant drugs whose risks should be detected in primary human hepatocytes.

Early hepatocyte death occurs in most liver injury cases and triggers liver inflammation, which in combination with other risk factors leads to the development of liver disease. However, the pathogenesis of early phase hepatocyte death remains poorly understood. Here, C57BL/6J mice were treated with the hepatotoxic drug flucloxacillin (FLUX) and the toll-like receptor 9 agonist CpG oligodeoxynucleotide (ODN) to reproduce the early phase of drug-induced hepatotoxicity and investigate its pathogenesis. C57BL/6J mice were treated with FLUX (100 mg/kg, gavage) alone or in combination with ODN (40 μg/mouse, intraperitoneally). Plasma alanine aminotransferase (ALT) level was measured as a marker of hepatotoxicity. FLUX or ODN alone was insufficient to induce ALT elevation, whereas combination treatment with FLUX and ODN increased ALT levels 24 h after FLUX treatment and upregulated Fas ligand in natural killer T (NKT) cells and Fas in hepatocytes. FLUX induced mitochondrial permeability transition (MPT), and pretreatment with ODN sensitized mitochondria to FLUX-induced MPT. The increase in ALT levels induced by ODN and FLUX co-treatment was suppressed in Fas ligand (gld/gld)-deficient mice and in mice deficient in a component of MPT pore opening (cyclophilin D-knockout mice). These results suggested that ODN activated the Fas/Fas ligand-mediated pathway in NKT cells and hepatocytes, which may predispose to FLUX-induced mitochondrial dysfunction and lead to early phase hepatocyte apoptosis. Taken together, these findings elucidate a potentially novel mechanism underlying drug-induced early phase hepatocyte death related to the Fas/Fas ligand death receptor pathway and mitochondrial dysfunction.

Troglitazone, the first peroxisome proliferator-associated receptor γ agonist developed as an antidiabetic drug, was withdrawn from the market due to idiosyncratic severe liver toxicity. One proposed mechanism by which troglitazone causes liver injury is induction of mitochondrial membrane permeability transition (MPT), which occurs in a calcium-independent phospholipase A2 (iPLA2)-dependent manner at a concentration of 10 µM. MPT, induced by opening of the MPT pore, leads to the release of cytochrome c and consequent apoptosis or necrosis. In the present study, we aimed to clarify the mechanism of troglitazone-induced MPT in more detail using isolated rat liver mitochondria. We focused on extra-mitochondrial Ca2+ and membrane potential as triggers of iPLA2 activation or MPT induction. As a link between iPLA2 and MPT, we focused on cardiolipin (CL), a unique, mitochondria-specific phospholipid with four acyl chains that affects respiration, the morphology, and other mitochondrial functions. We found that (1) Ca2+ release from the mitochondrial matrix was induced prior to troglitazone-induced onset of MPT, (2) released Ca2+ was involved in troglitazone-induced MPT, (3) mild depolarization (approximately 10%) may be a trigger of troglitazone-induced MPT and (4) enhanced decomposition of CL following mitochondrial iPLA2 activation might mediate troglitazone-induced MPT.

Drug-induced liver injury is not readily detectable using conventional animal studies during pre-clinical drug development. To address this problem, other researchers have proposed the use of co-administration of lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, and a drug. Using this approach, liver injury that is otherwise not detected following drug administration alone can be successfully identified. Previous studies have demonstrated that such injury is suppressed by heparin; therefore, the mechanism may involve coagulation-dependent ischemia. However, it has not been established how LPS-induced ischemia might sensitize hepatocytes to a potentially hepatotoxic drug. In the present study, we aimed to determine the effect of LPS-induced ischemia on liver mitochondrial function and downstream toxicologic responses. Consistent with previous findings, plasma alanine transaminase (ALT) activity was higher in rats co-administered with LPS (1 mg/kg) and diclofenac (100 mg/kg), but reduced by heparin. Liver mRNA expression of Hmox1, encoding heme oxygenase-1, an oxidative stress indicator, was three times higher at 2 hr after LPS administration. Furthermore, respiratory activity via mitochondrial complex II, lipid peroxidation in mitochondria, and the susceptibility to mitochondrial permeability transition pore opening in response to diclofenac administration were significantly increased by LPS administration. The increase in plasma ALT activity and the sensitization to mitochondrial permeability transition pore opening were reduced by the co-administration of heparin. In conclusion, LPS-induced transient ischemia disrupts respiratory chain complex activities, enhances reactive oxygen species production, especially in mitochondria, and sensitizes mitochondria to permeability transition pore opening when testing a potentially hepatotoxic drug in vivo.

Mitochondrial injury contributes to severe drug-induced liver injury. Particularly, mitochondrial permeability transition (MPT) is thought to be relevant to cytolytic hepatitis. However, the mechanism of drug-induced MPT is unclear and prediction of MPT is not adequately evaluated in the preclinical stage. In a previous study, we found that troglitazone, a drug withdrawn due to liver injury, induced MPT via mild depolarization probably resulting from uncoupling. Herein, we investigated whether other drugs that induce MPT share similar properties as troglitazone, using isolated mitochondria from rat liver. Of the 22 test drugs examined, six drugs, including troglitazone, induced MPT and showed an uncoupling effect. Additionally, receiver operating characteristic analysis was conducted to predict the MPT potential from the respiratory control ratio, an indicator of uncoupling intensity. Results showed that 2.5 was the best threshold that exhibited high sensitivity (1.00) and high specificity (0.81), indicating that uncoupling was correlated with MPT potential. Activation of calcium-independent phospholipase A2 appeared to be involved in uncoupling-induced MPT. Furthermore, a strong relationship between MPT intensity and the uncoupling effect among similar compounds was confirmed. These results may help in predicting MPT potential using cultured cells and modifying the chemical structures of the drugs to reduce MPT risk.

Tyrosine kinase inhibitors (TKIs) are widely utilized in clinical practice to treat carcinomas, but secondary tumor resistance during chronic treatment can be problematic. AKR1B1 and AKR1B10 of the aldo-keto reductase (AKR) superfamily are highly expressed in cancer cells and are believed to be involved in drug resistance. The aim of this study was to understand how TKI treatment of chronic myelogenous leukemia (CML) cells changes their glucose metabolism and if inhibition of AKRs can sensitize CML cells to TKIs. K562 cells were treated with the TKIs imatinib, nilotinib, or bosutinib, and the effects on glucose metabolism, cell death, glutathione levels, and AKR levels were assessed. To assess glucose dependence, cells were cultured in normal and low-glucose media. Pretreatment with AKR inhibitors, including epalrestat, were used to determine AKR-dependence. Treatment with TKIs increased intracellular glucose, AKR1B1/10 levels, glutathione oxidation, and nuclear translocation of nuclear factor erythroid 2-related factor 2, but with minimal cell death. These effects were dependent on intracellular glucose accumulation. Pretreatment with epalrestat, or a selective inhibitor of AKR1B10, exacerbated TKI-induced cell death, suggesting that especially AKR1B10 was involved in protection against TKIs. Thus, by disrupting cell protective mechanisms, AKR inhibitors may render CML more susceptible to TKI treatments.

Drug-induced liver injury (DILI) is the major reason for the discontinuation of new drug development and the withdrawal of drugs from the market. Hence, the evaluation systems which predict the onset of DILI in the pre-clinical stage are needed. To date, many researchers have conducted the mechanism of DILI, but the DILI prediction is poor because of the complexity of DILI. In this regard, based on the information obtained from basic research and clinical case, several pharmaceutical companies have been developed DILI prediction methods with high sensitivity and specificity by combining multiple targets. Another reason for low predictability is derived from the conventional culture method which causes a rapid decrease in hepatocyte function. To overcome these problems, the construction of a high-level in vitro evaluation system has been developed and applied to DILI evaluation. On the other hand, these in vitro evaluation methods require a lot of labor and cost so, in silico prediction methods have also been constructed in recent years. Based on this point, this article reviews the trends in DILI prediction systems in the non-clinical stage.

Various types of transgenic mice carrying either class I or II human leukocyte antigen (HLA) molecules are readily available, and reports describing their use in a variety of studies have been published for more than 30 years. Examples of their use include the discovery of HLA-specific antigens against viral infection as well as the reproduction of HLA-mediated autoimmune diseases for the development of therapeutic strategies. Recently, HLA transgenic mice have been used to reproduce HLA-mediated idiosyncratic drug toxicity (IDT), a rare and unpredictable adverse drug reaction that can result in death. For example, abacavir-induced IDT has successfully been reproduced in HLA-B*57:01 transgenic mice. Several reports using HLA transgenic mice for IDT have proven the utility of this concept for the evaluation of IDT using various HLA allele combinations and drugs. It has become apparent that such models may be a valuable tool to investigate the mechanisms underlying HLA-mediated IDT. This review summarizes the latest findings in the area of HLA transgenic mouse models and discusses the current challenges that must be overcome to maximize the potential of this unique animal model.

Cancer-associated fibroblasts (CAFs) have recently gained attention as potent targets in cancer therapy because they are a crucial component of the tumor microenvironment and promote the growth and invasion of cancer cells. CAFs differentiate from fibroblasts, mesenchymal stem cells (MSCs), epithelial cells, and other cell types in response to transforming growth factor β (TGFβ) stimulation. The drugs tranilast, imatinib, and pirfenidone reportedly inhibit the differentiation of such cells into CAFs; however, it is unclear how they regulate TGFβ signaling. Here, we differentiated MSCs into CAFs in vitro and investigated which drugs suppressed this differentiation. Based on these results, we focused on platelet-derived growth factor (PDGF) receptor β (PDGFRβ) as a key molecule in the initiation of TGFβ signaling. PDGFRβ transmitted TGFβ signaling in MSCs by forming a complex with TGFβ receptor (TGFβR) independently of stimulation with its well-known ligand PDGF. Inhibitors of the differentiation of MSCs into CAFs attenuated complex formation between PDGFRβ and TGFβR. Moreover, PDGF stimulated PDGFRβ to a lesser extent in CAFs than in MSCs. This study indicates that PDGFRβ and TGFβ-TGFβR signaling cooperatively promote the differentiation of MSCs into CAFs in tumor microenvironments independently of canonical PDGF-PDGFR signaling. We propose that blockade of the interaction between PDGFRβ and TGFβR is a potential strategy to prevent TGFβ-mediated differentiation of MSCs into CAFs.

Tyrosine kinase inhibitors (TKI), including imatinib (IM), improve the outcome of CML therapy. However, TKI treatment is long-term and can induce resistance to TKI, which often leads to a poor clinical outcome in CML patients. Here, we examined the effect of continuous IM exposure on intracellular energy metabolism in K562 cells, a human Philadelphia chromosome-positive CML cell line, and its subsequent sensitivity to anti-cancer agents. Contrary to our expectations, we found that continuous IM exposure increased sensitivity to TKI. Cancer energy metabolism, characterized by abnormal glycolysis, is linked to cancer cell survival. Interestingly, glycolytic activity was suppressed by continuous exposure to IM, and autophagy increased to maintain cell viability by compensating for glycolytic suppression. Notably, increased sensitivity to TKI was not caused by glycolytic inhibition but by altered intracellular signaling, causing glycolytic suppression and increased autophagy, as evidenced by suppression of p70 S6 kinase 1 (S6K1) and activation of AMP-activated protein kinase (AMPK). Using another human CML cell line (KCL22 cells) and BCR/ABL+ Ba/F3 cells (mimicking Philadelphia chromosome-positive CML cells) confirmed that suppressing S6K1 and activating AMPK increased sensitivity to TKI. Furthermore, suppressing S6K1 and activating AMPK had a synergistic anti-cancer effect by inhibiting autophagy in the presence of TKI. The present study provides new insight into the importance of signaling pathways that affect cellular energy metabolism, and suggests that co-treatment with agents that disrupt energy metabolic signaling (using S6K1 suppressors and AMPK activators) plus blockade of autophagy may be strategies for TKI-based CML therapy.

Immune-mediated idiosyncratic drug toxicity (IDT) is a rare adverse drug reaction, potentially resulting in death. Although genome-wide association studies suggest that the occurrence of immune-mediated IDT is strongly associated with specific human leukocyte antigen (HLA) allotypes, these associations have not yet been prospectively demonstrated. In this study, we focused on HLA-B*57:01 and abacavir (ABC)-induced immune-mediated IDT, and constructed transgenic mice carrying chimeric HLA-B*57:01 (B*57:01-Tg) to determine if this in vivo model may be useful for evaluating immune-mediated IDT. Local lymph node assay (LLNA) results demonstrated that percentages of BrdU+, IL-2+, and IFN-γ+ in CD8+ T cells of ABC (50 mg/kg/day)-applied B*57:01-Tg mice were significantly higher than those in littermates (LMs), resulting in the infiltration of inflammatory cells into the ear. These immune responses were not observed in B*57:03-Tg mice (negative control). Furthermore, oral administration of 1% (v/v) ABC significantly increased the percentage of CD44highCD62Llow CD8+ memory T cells in lymph nodes and spleen derived from B*57:01-Tg mice, but not in those from B*57:03-Tg mice and LMs. These results suggest that B*57:01-Tg mice potentially enable the reproduction and evaluation of HLA-B*57:01 and ABC-induced immune-mediated IDT.

// Shigeki Aoki 1, * , Michie Morita 1, * , Takuya Hirao 1 , Masashi Yamaguchi 2 , Reika Shiratori 1 , Megumi Kikuya 1 , Hiroji Chibana 2 and Kousei Ito 1 1 Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba-city, Chiba 260-8675, Japan 2 Medical Mycology Research Center, Chiba University, Chiba-city, Chiba 260-8673, Japan * These authors have contributed equally to this work Correspondence to: Shigeki Aoki, email: aokishigeki@chiba-u.jp Keywords: ALL, autophagy, glucocorticoids, glycolysis, oxidative phosphorylation Received: June 16, 2017 Accepted: September 21, 2017 Published: October 09, 2017 ABSTRACT Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Treatments include glucocorticoids (GCs) such as dexamethasone (Dex) and prednisolone, which may be of value when used alongside cytotoxic anti-cancer drugs. To predict therapeutic efficacy of GCs, their activity against ALL cells is usually examined prior to chemotherapy; however, few studies have examined their effects when used in combination with other drugs. The paradox is that cytotoxic anti-cancer drugs that are effective against proliferating cancer cells show synergistic effects when used with GCs that prevent cell proliferation. To address this point, we investigated intracellular energy metabolism in ALL CCRF-CEM cell clones classified according to their sensitivity to Dex and cytotoxic anti-cancer drugs in bulk cultures of mixed cells. We found that Dex suppressed glycolysis, the most important metabolic system in cancer cells, in cells that were damaged by etoposide (a cytotoxic anti-cancer drug), and the cells showed a concomitant increase in mitochondrial oxidative phosphorylation. Furthermore, autophagy, an intracellular bulk degradation system, regulated mitochondrial viability. We also found that mitochondria, whose function is enhanced by Dex, were susceptible to anti-cancer drugs that inhibit respiratory complexes (e.g., etoposide and daunorubicin), resulting in increased production of reactive oxygen species and subsequent cytotoxicity. Taken together, the present study points the way toward a more accurate prediction of the sensitivity of ALL cells to the combined action of anti-cancer drugs and GCs, by taking into consideration the shift in intracellular energy metabolism caused by GCs: namely, from glycolysis to mitochondrial oxidative phosphorylation mediated by autophagy.

We previously reported a cell-based toxicity assay using sandwich-cultured hepatocytes in combination with a titrated amount of human bile acid (BA) species. In this assay, test compound-induced inhibition of BA efflux from sandwich-cultured hepatocytes leads to BA-dependent cell toxicity (BAtox, i.e., cell death due to the accumulation of BAs). Using this assay, we investigated whether 1-aminobenzotriazole (1-ABT; a nonselective cytochrome P450 inhibitor) enhanced or suppressed test compound-induced BAtox. There was a tendency that BAtox of many compounds was enhanced by 1-ABT in human hepatocytes; in contrast, such a tendency was not observed in rat hepatocytes. In particular, 1-ABT tended to enhance BAtox of several compounds (clopidogrel, ticlopidine, everolimus, etc.) in human, whereas 1-ABT tended to enhance BAtox of only ticlopidine in rat. These results indicate that this system can be used to evaluate BAtox while taking into account drug metabolism and the existence of an interspecies difference in the effect of 1-ABT treatment on BAtox.

Inhibition of bile salt export pump (BSEP) causes hepatic accumulation of toxic bile acid (BA), leading to hepatocyte death. We reported a sandwich-cultured hepatocyte (SCH)-based model that can estimate potential cholestatic compounds by assessing their ability to induce hepatotoxicity in combination with a titrated amount of human 12 BA species. However, there is little information about the specific BAs responsible for hepatotoxicity, when BSEP is inhibited. This study measured the accumulation of each BA in rat SCHs in the presence of 10 μM cyclosporine A (CsA), which only inhibits BSEP, and 50 μM CsA, which further inhibits basolateral BA efflux transporters. The accumulation of all BAs (not significant for deoxycholic acid [DCA]) was observed in the presence of 10 μM CsA. In particular, 3 BAs (chenodeoxycholic acid [CDCA], DCA, and glyco-DCA [GDCA]) showed increased toxicity in the presence of 10 μM CsA, whereas the other BAs did not. In addition to these BAs, taurolithocholic acid, glyco-CDCA, and glycocholic acid showed increased toxicity in the presence of 50 μM CsA, but additional accumulation of these BAs could not be observed. These results indicate the inhibiting BSEP results in the accumulation of CDCA, GDCA, and partially DCA, thereby resulting in hepatotoxicity.

Troglitazone and pioglitazone were developed as thiazolidinedione-type antidiabetes drugs, but only troglitazone was withdrawn from the markets due to severe liver injury. As both troglitazone and its sulfate metabolite are strong inhibitors of the bile salt export pump (BSEP), troglitazone-induced bile acid (BA) retention is thought to be one of the underlying mechanisms of liver injury. However, pioglitazone is also a strong BSEP inhibitor, indicating other mechanisms may also be involved in troglitazone-induced BA retention. Although retention of hydrophobic BAs (eg, chenodeoxycholic acid [CDCA]: a nonamidated BA) is known to cause hepatocyte injury, little is known about the hepatic conversion of nonamidated, hydrophobic BA species into less toxic hydrophilic BAs (eg, glycochenodeoxycholic acid: amidated BA) as a mechanism of drug-induced liver injury. In this study, we, therefore, investigated the effects of troglitazone and pioglitazone on BA amidation and the role of amidated BAs in troglitazone-associated BA-mediated hepatotoxicity. We also evaluated the intracellular BA composition of human hepatocytes treated with nonamidated BA species (CDCA or deoxycholic acid [DCA]) in the presence of troglitazone or pioglitazone. Amidation of CDCA and DCA was significantly inhibited by troglitazone (IC50: 5 and 3 µmol/l, respectively), but not pioglitazone. Moreover, treatment with troglitazone led to the retention of CDCA and DCA and decrease of glycine-amidation in hepatocytes. From these results, we suggest that troglitazone-induced liver injury might be caused by the accumulation of nonamidated BAs in hepatocytes due to inhibition of BA amidation.

Drug-induced liver injury (DILI) can cause hepatic failure and result in drug withdrawal from the market. It has host-related and compound-dependent mechanisms. Preclinical prediction of DILI risk is very challenging and safety assessments based on animals inadequately forecast human DILI risk. In contrast, human-derived in vitro cell culture-based models could improve DILI risk prediction accuracy. Here, we developed and validated an innovative method to assess DILI risk associated with various compounds. Fifty-four marketed and withdrawn drugs classified as DILI risks of "most concern", "less concern", and "no concern" were tested using a combination of four assays addressing mitochondrial injury, intrahepatic lipid accumulation, inhibition of bile canalicular network formation, and bile acid accumulation. Using the inhibitory potencies of the drugs evaluated in these in vitro tests, an algorithm with the highest available DILI risk prediction power was built by artificial neural network (ANN) analysis. It had an overall forecasting accuracy of 73%. We excluded the intrahepatic lipid accumulation assay to avoid overfitting. The accuracy of the algorithm in terms of predicting DILI risks was 62% when it was constructed by ANN but only 49% when it was built by the point-added scoring method. The final algorithm based on three assays made no DILI risk prediction errors such as "most concern " instead of "no concern" and vice-versa. Our mechanistic approach may accurately predict DILI risks associated with numerous candidate drugs.

Most cancer cells rely on glycolysis to generate ATP, even when oxygen is available. However, merely inhibiting the glycolysis is insufficient for the eradication of cancer cells. One main reason for this is that cancer cells have the potential to adapt their metabolism to their environmental conditions. In this study, we investigated how cancer cells modify their intracellular metabolism when glycolysis is suppressed, using PANC-1 pancreatic cancer cells and two other solid tumor cell lines, A549 and HeLa. Our study revealed that glycolytically suppressed cells upregulated mitochondrial function and relied on oxidative phosphorylation (OXPHOS) to obtain the ATP necessary for their survival. Dynamic changes in intracellular metabolic profiles were also observed, reflected by the reduced levels of TCA cycle intermediates and elevated levels of most amino acids. Glutamine and glutamate were important for this metabolic reprogramming, as these were largely consumed by influx into the TCA cycle when the glycolytic pathway was suppressed. During the reprogramming process, activated autophagy was involved in modulating mitochondrial function. We conclude that upon glycolytic suppression in multiple types of tumor cells, intracellular energy metabolism is reprogrammed toward mitochondrial OXPHOS in an autophagy-dependent manner to ensure cellular survival.

Troglitazone, the first peroxisome proliferator-associated receptor γ agonist developed as an antidiabetic drug, was withdrawn from the market due to idiosyncratic severe liver toxicity. One proposed mechanism by which troglitazone causes liver injury is induction of mitochondrial membrane permeability transition (MPT), which occurs in a calcium-independent phospholipase A2 (iPLA

Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and in hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity.

Bile acid (BA) retention within hepatocytes is an underlying mechanism of cholestatic drug-induced liver injury (DILI). We previously developed an assay using sandwich-cultured human hepatocytes (SCHHs) to evaluate drug-induced hepatocyte toxicity accompanying intracellular BA accumulation. However, due to shortcomings commonly associated with the use of primary human hepatocytes (e.g., limited availability, lot-to-lot variability, and high cost), we examined if the human hepatic stem cell line, HepaRG, might also be applicable to our assay system. Consequently, mRNA expression levels of human BA efflux and uptake transporters were lower in HepaRG cells than in SCHHs but higher than in HepG2 human hepatoma cells. Nevertheless, HepaRG cells and SCHHs showed similar toxicity responses to 22 selected drugs, including cyclosporine A (CsA). CsA (10 μM) was cytotoxic toward HepaRG cells in the presence of BAs and also reduced the biliary efflux rate of [(3)H]taurocholic acid from 38.5% to 19.2%. Therefore, HepaRG cells are useful for the evaluation of BA-dependent drug toxicity caused by biliary BA efflux inhibition. Regardless, the prediction accuracy for cholestatic DILI risk was poor for HepaRG cells versus SCHHs, suggesting that our DILI model system requires further improvements to increase the utility of HepaRG cells as a preclinical screening tool.

Recently, mitochondrial dysfunction is thought of as an important factor leading to a drug-induced liver injury. Our previous reports show that mitochondria-related toxicity, including respiratory chain inhibition (RCI) and reactive oxygen species (ROS) induction, can be detected by the modification of sugar resource substitution and high oxygen condition. However, this in vitro model does not detect mitochondrial permeability transition (MPT)-induced toxicity. Another study with a lipopolysaccharide-pre-administered rodent model showed that ischemia/reperfusion induced ROS, sensitized the susceptibility of MPT pore opening and, finally developed drug-induced liver toxicity. Based on this result, the present study investigated the effect of hypoxia/reoxygenation (H/R) treatment mimicking the ischemia/reperfusion on MPT-dependent toxicity, aiming to construct a system that can evaluate MPT by drugs in hepatocytes. Mitochondrial ROS were enhanced by H/R treatment only in the galactose culture condition. Amiodarone, benzbromarone, flutamide and troglitazone which induced MPT pore opening led to hepatocyte death only in combination with H/R and galactose. Moreover, this alteration was significantly suppressed in hepatocytes lacking cyclophilin D. In conclusion, MPT-induced cytotoxicity can be detected by activating mitochondrial function and H/R. This cell-based assay system could evaluate MPT induced-cytotoxicity by drugs, besides RCI and ROS induction.

Mitochondrial toxicity is a factor of drug-induced liver injury. Previously, we reported an in vitro rat hepatocyte assay where mitochondrial toxicity was more sensitively evaluated, using sugar resource substitution and increased oxygen supply. Although this method could be applicable to human cell-based assay, cryopreserved human hepatocyte (CHH) has some disadvantages/uncertainty, including unstable same donor supply and potential organelle damage due to cryopreservation. Herein, we compared the mitochondrial functions of freshly-isolated hepatocytes from humanized chimeric mice liver (PXB-cells) and three CHH lots to determine the better cell source for mitochondrial toxicity assay. Two CHH lots declined after replacing glucose with galactose. To confirm the shift in energy production from glycolysis to oxidative phosphorylation, lactate and oxygen consumption rate (indicators of glycolytic activity and mitochondrial oxidative phosphorylation, respectively) were measured. In PXB-cells, lactate amount decreased, while oxygen consumption in 100 min increased. These effects were less evident in CHH. The cytotoxicity of the select respiratory chain inhibitors was enhanced in PXB-cells upon sugar replacement, but no change occurred with negative control drugs (bicalutamide and metformin). Altogether, CHH was more vulnerable to sugar resource substitution than PXB-cells. The substitution activated mitochondrial function and enhanced cytotoxicity of respiratory chain inhibitors in PXB-cells.

The liver performs a variety of essential functions; hence drug-induced liver injury (DILI) is a serious concern that can ultimately lead to the withdrawal of a drug from the market or discontinuation of drug development. However, the mechanisms of drug-induced liver injury are not always clear. We hypothesized that drugs may inhibit the liver recovery process, especially bile canalicular (BC) network reformation, leading to persistent liver injury and deterioration, and tested this hypothesis in the present work. The BC structure disappeared in mice following treatment with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or thioacetamide (TAA) for 4 weeks, then reappeared after 4 weeks of receiving a normal diet. By contrast, reconstruction of the BC structure was suppressed in mice fed a diet containing 0.3% benzbromarone (BBR; which can induce fatal liver injury in clinical settings) after liver injury. Plasma ALT levels were increased significantly in mice treated with BBR after DDC or TAA treatment, compared with BBR alone. To confirm whether BBR has a direct inhibitory effect on hepatocytes, we also examined BC reformation in primary cultured mouse hepatocytes with a sandwich configuration. Under these culture conditions, the BC network rapidly reformed from days 2 and 3 after seeding. During the reformation period, BBR inhibited BC reformation significantly. These results suggest that BBR inhibits BC reconstruction and delays recovery from pre-existing liver injury.

Genome-wide association studies indicate that several idiosyncratic adverse drug reactions are highly associated with specific human leukocyte antigen (HLA) alleles. For instance, abacavir, a human immunodeficiency virus reverse transcriptase inhibitor, induces multiorgan toxicity exclusively in patients carrying the HLA-B*57:01 allele. However, the underlying mechanism is unclear due to a lack of appropriate animal models. Previously, we developed HLA-B*57:01 transgenic mice and found that topical application of abacavir to the ears induced proliferation of CD8+ lymphocytes in local lymph nodes. Here, we attempted to reproduce abacavir-induced liver injury in these mice. However, oral administration of abacavir alone to HLA-B*57:01 transgenic mice did not increase levels of the liver injury marker alanine aminotransferase. Considering the importance of innate immune activation in mouse liver, we treated mice with CpG oligodeoxynucleotide, a toll-like receptor 9 agonist, plus abacavir. This resulted in a marked increase in alanine aminotransferase, pathological changes in liver, increased numbers of activated CD8+ T cells, and tissue infiltration by immune cells exclusively in HLA-B*57:01 transgenic mice. These results indicate that CpG oligodeoxynucleotide-induced inflammatory reactions and/or innate immune activation are necessary for abacavir-induced HLA-mediated liver injury characterized by infiltration of CD8+ T cells. Thus, we developed the first mouse model of HLA-mediated abacavir-induced idiosyncratic liver injury. Further investigation will show that the proposed HLA-mediated liver injury model can be applied to other combinations of drugs and HLA types, thereby improving drug development and contributing to the development of personalized medicine.

The bile salt export pump (BSEP or Bsep) functions as an apical transporter to eliminate bile acids (BAs) from hepatocytes into the bile. BSEP or Bsep inhibitors engender BA retention, suggested as an underlying mechanism of cholestatic drug-induced liver injury. We previously reported a method to evaluate BSEP-mediated BA-dependent hepatocyte toxicity by using sandwich-cultured hepatocytes (SCHs). However, basal efflux transporters, including multidrug resistance-associated proteins (MRP or Mrp) 3 and 4, also participate in BA efflux. This study examined the contribution of basal efflux transporters to BA-dependent hepatocyte toxicity in rat SCHs. The apical efflux of [(3)H]taurocholic acid (TC) was potently inhibited by 10 μM cyclosporine A (CsA), with later inhibition of basal [(3)H]TC efflux, while MK571 simultaneously inhibited both apical and basal [(3)H]TC efflux. CsA-induced BA-dependent hepatocyte toxicity was 30% at most at 10 μM CsA and ∼60% at 50 μM, while MK571 exacerbated hepatocyte toxicity at concentrations of ≥50 μM. Quinidine inhibited only basal [(3)H]TC efflux and showed BA-dependent hepatocyte toxicity in rat SCHs. Hence, inhibition of basal efflux transporters as well as Bsep may precipitate BA-dependent hepatocyte toxicity in rat SCHs.