You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology (MP54)1 Apr 2020MP54-16 CHARACTERIZATION OF FUNCTIONAL AND HISTOLOGICAL CHANGES OF THE URETHRA IN RAT MODELS OF STRESS URINARY INCONTINENCE (SUI) INDUCED BY SIMULATED BIRTH TRAUMA OR ESTROGEN DEFICIENCY Masahiro Kurobe*, Ei-ichiro Takaoka, Nailong Cao, Takahisa Suzuki, Tetsuichi Saito, Daisuke Gotoh, Eduardo C. Alexandre, Taro Igarashi, Jun Miyazaki, Hiroyuki Nishiyama, and Naoki Yoshimura Masahiro Kurobe*Masahiro Kurobe* More articles by this author , Ei-ichiro TakaokaEi-ichiro Takaoka More articles by this author , Nailong CaoNailong Cao More articles by this author , Takahisa SuzukiTakahisa Suzuki More articles by this author , Tetsuichi SaitoTetsuichi Saito More articles by this author , Daisuke GotohDaisuke Gotoh More articles by this author , Eduardo C. AlexandreEduardo C. Alexandre More articles by this author , Taro IgarashiTaro Igarashi More articles by this author , Jun MiyazakiJun Miyazaki More articles by this author , Hiroyuki NishiyamaHiroyuki Nishiyama More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000916.016AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Multiple vaginal parities and estrogen deficiency (ED) are considered to be major causes of SUI in women. In rats, bilateral ovariectomy (OVX) reportedly induces the morphological changes in the urethral epithelium. It has also been reported that serotonergic (5HT+) paraneurons uniquely located in the urethra modulate urethral sensory function and their numbers decrease in aged or diabetes rats. We therefore evaluated the urethral histological changes of the epithelium and 5HT+ cell expression, and urethral continence function in rats with OVX-induced ED or vaginal distention (VD)-induced simulated birth trauma. METHODS: Virgin SD rats were divided into: (1) sham group; (2) OVX 3-weeks group; (3) OVX 6-weeks group; (4) VD-1 group (1 time VD 2 weeks before assays); (5) VD-3 group (3 times VDs every 2 weeks). VD was induced by balloon catheter inflation in vagina. Urethral function was evaluated by the amplitude of urethral pressure responses during sneezing (A-URS) and urethral baseline pressure (UBP), followed by histological analyses of mid-urethra (n=6, each group). Urethral mRNA levels of Tryptophan hydroxylase-1 (TPH1), a key enzyme of 5HT synthesis, were quantified by RT-PCR (n=6, each group). RESULTS: OVX 6-weeks and VD-3 rats exhibited significant decreases in UBP and A-URS vs. sham (Fig. 1A). The mean epithelial thickness was significantly decreased in OVX 3, 6-weeks, VD-1 and 3 rats (Fig 1B,C) vs. sham. The number of urethral 5HT+ paraneurons was significantly decreased in OVX 3, 6-weeks, VD-1 and 3 rats (Fig. 2A,B) vs. sham. TPH-1 mRNA levels were significantly decreased in OVX 3, 6-weeks and VD-3 rats (Fig. 2C) vs. sham. CONCLUSIONS: These results indicate that urethral epithelial atrophy and loss of 5HT+ paraneurons occur prior to urethral dysfunction inducing SUI in both models, with the changes being rapid and greater in OVX compared to VD. These histological changes in the urethra may contribute to the establishment of SUI in ED and multiple birth trauma. Source of Funding: NIH R01DK107450, Uehara Memorial Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e797-e798 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Masahiro Kurobe* More articles by this author Ei-ichiro Takaoka More articles by this author Nailong Cao More articles by this author Takahisa Suzuki More articles by this author Tetsuichi Saito More articles by this author Daisuke Gotoh More articles by this author Eduardo C. Alexandre More articles by this author Taro Igarashi More articles by this author Jun Miyazaki More articles by this author Hiroyuki Nishiyama More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement PDF downloadLoading ...

Assessing the potential of a new drug to cause drug-induced liver injury (DILI) is a challenge for the pharmaceutical industry. We therefore determined whether cell models currently used in safety assessment (HepG2, HepaRG, Upcyte and primary human hepatocytes in conjunction with basic but commonly used endpoints) are actually able to distinguish between novel chemical entities (NCEs) with respect to their potential to cause DILI. A panel of thirteen compounds (nine DILI implicated and four non-DILI implicated in man) were selected for our study, which was conducted, for the first time, across multiple laboratories. None of the cell models could distinguish faithfully between DILI and non-DILI compounds. Only when nominal in vitro concentrations were adjusted for in vivo exposure levels were primary human hepatocytes (PHH) found to be the most accurate cell model, closely followed by HepG2. From a practical perspective, this study revealed significant inter-laboratory variation in the response of PHH, HepG2 and Upcyte cells, but not HepaRG cells. This variation was also observed to be compound dependent. Interestingly, differences between donors (hepatocytes), clones (HepG2) and the effect of cryopreservation (HepaRG and hepatocytes) were less important than differences between the cell models per se. In summary, these results demonstrate that basic cell health endpoints will not predict hepatotoxic risk in simple hepatic cells in the absence of pharmacokinetic data and that a multicenter assessment of more sophisticated signals of molecular initiating events is required to determine whether these cells can be incorporated in early safety assessment.

In vitro preclinical models for the assessment of drug-induced liver injury (DILI) are usually based on cryopreserved primary human hepatocytes (cPHH) or human hepatic tumor-derived cell lines; however, it is unclear how well such cell models reflect the normal function of liver cells. The physiological, pharmacological, and toxicological phenotyping of available cell-based systems is necessary in order to decide the testing purpose for which they are fit. We have therefore undertaken a global proteomic analysis of 3 human-derived hepatic cell lines (HepG2, Upcyte, and HepaRG) in comparison with cPHH with a focus on drug metabolizing enzymes and transport proteins (DMETs), as well as Nrf2-regulated proteins. In total, 4946 proteins were identified, of which 2722 proteins were common across all cell models, including 128 DMETs. Approximately 90% reduction in expression of cytochromes P450 was observed in HepG2 and Upcyte cells, and approximately 60% in HepaRG cells relative to cPHH. Drug transporter expression was also lower compared with cPHH with the exception of MRP3 and P-gp (MDR1) which appeared to be significantly expressed in HepaRG cells. In contrast, a high proportion of Nrf2-regulated proteins were more highly expressed in the cell lines compared with cPHH. The proteomic database derived here will provide a rational basis for the context-specific selection of the most appropriate 'hepatocyte-like' cell for the evaluation of particular cellular functions associated with DILI and, at the same time, assist in the construction of a testing paradigm which takes into account the in vivo disposition of a new drug.

In this paper we report quantitative structure-activity models linking in vivo Drug-Induced Liver Injury (DILI) of organic molecules with some parameters both measured experimentally in vitro and calculated theoretically from the molecular structure. At the first step, a small database containing information of DILI in humans was created and annotated by experimentally observed information concerning hepatotoxic effects. Thus, for each compound a binary annotation "yes/no" was applied to DILI and seven endpoints causing different liver pathologies in humans: Cholestasis (CH), Oxidative Stress (OS), Mitochondrial injury (MT), Cirrhosis and Steatosis (CS), Hepatitis (HS), Hepatocellular (HC), and Reactive Metabolite (RM). Different machine-learning methods were used to build classification models linking DILI with molecular structure: Support Vector Machines, Artificial Neural Networks and Random Forests. Three types of models were developed: (i) involving molecular descriptors calculated directly from chemical structure, (ii) involving selected endpoints as "biological" descriptors, and (iii) involving both types of descriptors. It has been found that the models based solely on molecular descriptors have much weaker prediction performance than those involving in vivo measured endpoints. Taking into account difficulties in obtaining of in vivo data, at the validation stage we used instead five endpoints (CH, CS, HC, MT and OS) measured in vitro in human hepatocyte cultures. The models involving either some of experimental in vitro endpoints or their combination with theoretically calculated ones correctly predict DILI for 9 out of 10 reference compounds of the external test set. This opens an interesting perspective to use for DILI predictions a combination of theoretically calculated parameters and measured in vitro biological data.

Since drug induced liver injury is difficult to predict in animal models, more representative tests are needed to better evaluate these effects in humans. Existing in vitro systems hold great potential to detect hepatotoxicity of pharmaceuticals. In this study, the in vitro biokinetics of the model hepatotoxicant chlorpromazine (CPZ) were evaluated in three different liver cell systems after repeated exposure in order to incorporate repeated-dose testing into an in vitro assay. Primary rat and human hepatocytes, cultured in sandwich configuration and the human HepaRG cell line were treated daily with CPZ for 14 days. Samples were taken from medium, cells and well plastic at specific time points after the first and last exposure. The samples were analysed by HPLC-UV to determine the amount of CPZ in these samples. Based on cytotoxicity assays, the three models were tested at 1-2 μM CPZ, while the primary rat hepatocytes and the HepaRG cell line were in addition exposed to a higher concentration of 15-20 μM. Overall, the mass balance of CPZ decreased in the course of 24 h, indicating the metabolism of the compound within the cells. The largest decrease in parent compound was seen in the primary cultures; in the HepaRG cell cultures the mass balance only decreased to 50%. CPZ accumulated in the cells during the 14-day repeated exposure. Possible explanations for the accumulation of CPZ are a decrease in metabolism over time, inhibition of efflux transporters or binding to phospholipids. The biokinetics of CPZ differed between the three liver cell models and were influenced by specific cell properties as well as culture conditions. These results support the conclusion that in vitro biokinetics data are necessary to better interpret chemical-induced cytotoxicity data.

More than 30% of promising medications have failed in human clinical trials because they are determined to be toxic despite promising pre-clinical studies in animal models, and another 60% fail due to lack of efficacy. The challenge of accurately predicting drug toxicities and efficacies is in part due to inherent species differences in drug metabolizing enzyme activities and cell-type specific sensitivities to toxicants. to address this challenge in drug development and regulatory science, the NIH launched the Organs-on-Chips Program to develop alternative approaches that would enable early indications and potentially more reliable readouts of toxicity or efficacy, and provide suitable alternatives for animal testing. the goal of the program is to develop bio-engineered microdevices that represent functional units of the 10 major human organ systems: circulatory, respiratory, integumentary, reproductive, endocrine, gastrointestinal, nervous, urinary, musculoskeletal, and immune. The opportunities for significant advancements in the prediction of human drug toxicities require a multi-disciplinary approach that relies on an understanding of human physiology, stem cell biology, material sciences and bioengineering. this unique and novel in vitro platform could help ensure that safe and effective therapeutics are identified sooner, and ineffective or toxic ones are rejected early in the drug development process.

We recently found that rat hepatocyte transplantation was efficient (liver repopulation: 2.4%) in a sublethal nude mouse model (less than 33% mortality) of repeated liver injury generated using Jo2, a mouse-specific anti-Fas antibody, at sublethal dose of 250 μg/kg for 3 weeks. Genomic analysis of the livers revealed cell cycle blockade and an antiproliferative status of circadian genes, suggesting a selective advantage. By contrast, in the present study, freshly isolated human hepatocyte transplantation performed in the same mouse model resulted in implantation of less than 6,000 cells per liver (about 0.006% repopulation) in all animals. Genomic analysis of nude mouse livers revealed a lack of P21 upregulation, while a signature of stimulation of liver regeneration was observed, including upregulation of early response genes and upregulation of circadian genes. When we translated this sublethal model to a lethal model (65% mortality) by increasing the Jo2 repeated doses to 375 μg/kg, human hepatocyte engraftment was still very low; however, animal mortality was corrected by transplantation (only 20% mortality). Genomic findings in livers from the mice of the lethal Jo2 transplanted group were similar to those of the sublethal Jo2 transplanted group, that is, no selective advantage genomic signature and signature of mouse liver regeneration. In conclusion, transplanted human hepatocytes acted as if they modified nude mouse liver responses to Jo2 by stimulating liver regeneration, leading to an increased survival rate.

Drug-induced liver injury is the most frequent reason for market withdrawal of approved drugs, and is difficult to predict in animal models. Here, we analyzed transcriptomic data derived from short- and long-term cultured primary human hepatocytes (PHH) exposed to the well known human hepatotoxin chlorpromazine (CPZ). Samples were collected from five PHH cultures after short-term (1 and 3 days) and long-term (14 days) repeat daily treatment with 0.1 or 0.2 µM CPZ, corresponding to C(max). Two PHH cultures were additionally treated with 1 µM CPZ, and the three others with 0.02 µM CPZ. Differences in the total number of gene changes were seen between donors and throughout treatment. Specific transcriptomic hepatotoxicity signatures were created for CPZ and consisted of inflammation/hepatitis, cholestasis, and liver proliferation in all five donors, as well as fibrosis and steatosis, which were observed in four of five donors. Necrosis was present in three of five donors, and an indicative signature of cirrhosis was observed after long-term 14-day repeat treatment, also in three of five donors. The inter-donor variability in the inflammatory response to CPZ treatment was associated with variability in the strength of the response of the transcriptomic hepatotoxicity signatures, suggesting that features of inflammation could be related to the idiosyncratic hepatotoxic effects of CPZ in humans.

Rationale: The aim of the present study was to assess the stability of cryopreserved human hepatocytes over 5 years and to explore experimental condition-related variables such as seeding density, culture matrix and medium, start and duration of treatment that could potentially affect the quality of cultures and their response to cytochrome P450 (CYP) inducers. Results: 63/125 batches of cryopreserved human hepatocytes were plateable after thawing. Of those, 17 batches showed reproducible recovery, viability and plateability (less than 5% intra-batch variability) up to 5 years. When cultured in collagen home-coated 48-well plates at a seeding density allowing 70% confluence, cryopreserved human hepatocytes display activities equivalent to fresh counterparts. Their response to CYP inducers is maximal and equivalent to fresh counterpart for an incubation of 72 h starting at Day 2 or Day 3 after plating when cultured in modified Hepatocyte Maintenance Medium (HMM). The number of cryopreserved human hepatocytes can be further reduced by using a cocktail of CYP substrates for the assessment of their inducibility. Conclusions: Experimental condition-related variables, such as seeding density, culture matrix and medium, start and duration of treatment, affecting the response of plateable thawed cryopreserved human hepatocytes to cytochrome P450 inducers can be reduced by optimizing critical steps of the protocols.

As our knowledge of the species differences in drug metabolism and drug-induced hepatotoxicity has expanded significantly, the need for human-relevant in vitro hepatic model systems has become more apparent than ever before. Human hepatocytes have become the "gold standard" for evaluating hepatic metabolism and toxicity of drugs and other xenobiotics in vitro. In addition, they are becoming utilized more extensively for many kinds of biomedical research, including a variety of biological, pharmacological, and toxicological studies. This chapter describes methods for the isolation of primary human hepatocytes from liver tissue obtained from an encapsulated end wedge removed from patients undergoing resection for removal of liver tumors or from resected segments from whole livers obtained from multi-organ donors. In addition, methods are described for culturing primary hepatocytes under various matrix compositions and geometries, which reestablish intercellular contacts and normal cellular architecture for optimal phenotypic gene expression and response to drugs and other xenobiotics in vitro. Overall, improved isolation, cultivation, and preservation methods have expanded the number of applications for primary human hepatocytes in basic research, which has allowed for exciting advances in our understanding of the biochemical and molecular mechanisms of human liver toxicity and disease.

We have compared induction responses of human hepatocytes to known inducers of CYP1A2, CYP2B6, CYP2C and CYP3A4/5 to determine whether the culture format, treatment regimen and/or substrate incubation conditions affected the outcome. CYP induction responses to prototypical inducers were equivalent regardless of pre-culture time (24h or 48h), plate format (60mm or 24-well plates) used or whether CYP activities were measured in microsomes or whole cell monolayers. Fold-induction of CYP3A4/5 by 1000muM PB and 10microM RIF were equivalent. In contrast, the fold-induction of CYP2B6 by PB was 3-fold higher that by 10microM RIF. In addition to inducing CYP1A2, 50microM OME also induced CYP3A4/5 in 50% of the donors tested. CYP2B6 was induced in 14 out of 21 donors by BNF; however CYP3A4/5 was unaffected by BNF in these donors. In order to confirm that donor-to-donor variation was not due to inter-laboratory differences, the induction responses of 5 different batches of cryopreserved human hepatocytes were compared in two different laboratories. The induction of CYP1A2, CYP2B6 and CYP3A4 measured in our laboratory were equivalent to those obtained by the commercial companies, proving good between-laboratory reproducibility. In conclusion, there is some flexibility in the treatment and incubation protocols for classical CYP induction assays on human hepatocytes. Both RIF and PB are suitable positive control inducers of CYP3A4/5 but PB may be more appropriate for CYP2B6 induction. BNF may be more appropriate for CYP1A2 induction than OME since, in contrast to the latter, it does not induce CYP3A4. Induction responses using hepatocytes from the same donor but in different labs can be expected to be similar. The good reproducibility of induction responses between laboratories using cryopreserved hepatocytes underlines the usefulness of these cells for these types of studies.

The aim of the current work was to harmonise protocols between three laboratories by performing independent isolations and cultures of human hepatocytes and to assess their responses to prototypical cytochrome P450 (CYP) enzyme inducers, beta-naphthoflavone (BNF), rifampicin (RIF) or phenobarbital (PB). The magnitudes of the induction responses were CYP and donor-dependent but there was a good reproducibility between laboratories. CYP1A2 activity was evident in all cultures treated with BNF but not RIF or PB. Likewise, CYP3A4/5 activity was induced to the same extent by RIF and PB, while BNF did not affect this CYP in any of the cultures tested. All three compounds caused a concentration-dependent increase in CYP2B6 in cultures from 2 of the 3 laboratories and the response to PB was at least twice that of the other two inducers. In conclusion, the harmonised protocols used to study the response of primary cultures of human hepatocytes to prototypical inducers are transferable, reproducible within a given laboratory and between laboratories. The results obtained will support setting up a definitive validation study of the harmonised protocols.

Hepatocyte transplantation is a promising therapy for acute liver failure in humans. Recently, we succeeded in inducing various acute and chronic liver failures in nude mice. Engraftment of transplanted xenogeneic rat hepatocytes, visualized in the host liver by anti-MHC class I immunohistochemistry, revealed that liver repopulation was limited, and equivalent in nude mice with and without acute liver failure. In the present study, acute liver failure was induced in nude mice by a single injection of sublethal anti-Fas antibody Jo2, followed 24 h later by rat hepatocyte transplantation and than by a weekly repeated injection of Jo2. Rat hepatocyte engraftment into the recipient liver parenchyma 3 weeks following hepatocyte transplantation was about sevenfold increased when nude mice were subsequently subjected to weekly repeated Jo2 injection. Genomic analysis of these mice showed an overall transcriptome profile of upregulation of cellular cycle blocking transcripts, activation of liver injury inducing IFN-γ/STAT1 pathway, and circadian transcript signature of antiproliferative cell status compared to mice submitted to hepatocyte transplantation only. The findings of the present study suggest that the induction of cell proliferation blockade in recipient livers could promote sufficient engraftment of transplanted hepatocytes to allow transient or definitive treatment of liver failure in humans.

Curcumin (CUR) is a major component of a dietary spice derived from the roots of Curcuma longa. It has strong antioxidant activities and hepatoprotective properties. Primary human hepatocytes are clinically used in transplantation or in bioartificial liver devices for the treatment of patients with liver failure. Fresh and cryopreserved hepatocytes are also used in vitro for the study of drugs in pharmacotoxicology. We aimed to assess whether CUR could improve human liver cell viability and prevent oxidative damage responsible for large cell loss during cell preparation. Our study showed beneficial effects of CUR (25 microM) on freshly isolated human hepatocytes, increasing significantly metabolic activity of viable attached cells when seeded with CUR for 24 h. However CUR added during the cell isolation process did not have any significant impact on cell isolation outcomes or on cryopreservation outcomes. Conversely, CUR added during the thawing of frozen cells had a negative effect on the cell attachment capacity of hepatocytes that were cryopreserved in the presence or absence of CUR. In conclusion, although having positive effects on viability and challenge of oxidative stress on cultured human hepatocytes, CUR had no beneficial effect on cell isolation or cryopreservation outcomes.

Hydroxamic acid (HA)-based histone deacetylase (HDAC) inhibitors, with trichostatin A (TSA) as the reference compound, are potential antitumoral drugs and show promise in the creation of long-term primary cell cultures. However, their metabolic properties have barely been investigated. TSA is rapidly inactivated in rodents both in vitro and in vivo. We previously found that 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxyamide or 4-Me2N-BAVAH (compound 1) is metabolically more stable upon incubation with rat hepatocyte suspensions. In this study, we show that human hepatocytes also metabolize TSA more rapidly than compound 1 and that similar pathways are involved. Furthermore, structural analogs of compound 1 (compounds 2-9) are reported to have the same favorable metabolic properties. Removal of the dimethylamino substituent of compound 1 creates a very stable but 50% less potent inhibitor. Chain lengthening (4 to 5 carbon spacer) slightly improves both potency and metabolic stability, favoring HA reduction to hydrolysis. On the other hand, Calpha-unsaturation and spacer methylation not only reduce HDAC inhibition but also increase the rate of metabolic inactivation approximately 2-fold, mainly through HA reduction. However, in rat hepatocyte monolayer cultures, compound 1 is shown to be extensively metabolized by phase II conjugation. In conclusion, this study suggests that simple structural modifications of amide-linked TSA analogs can improve their phase I metabolic stability in both rat and human hepatocyte suspensions. Phase II glucuronidation, however, can compensate for their lower phase I metabolism in rat hepatocyte monolayers and could play a yet unidentified role in the determination of their in vivo clearance.