Annual Meeting of the Japanese Society of Toxicology

We performed a 52-week toxicity study in minipigs by daily oral gavage dosing. The female reproductive system was identified in earlier rodent studies as a potential target for toxicity of the drug. In this study, we incorporated a number of observations and measurements to assess the reproductive system in the females in order to identify potential signals for toxicity as early as possible. The purpose of these measurements was to avoid the need to sacrifice groups of animals periodically during the study to enable assessment of the relevant organs macroscopically and microscopically. Evaluations undertaken were; evaluation of the estrus cycle, regular blood sampling for progesterone, periodic (every 3 months) ultrasound scanning of the ovaries and post-mortem evaluation (macroscopic, organ weight and microscopic changes). Through these investigations, we gained useful information concerning measurement and observation of the reproductive behaviour of the animals. Normal cycling (estrus cycle length of ca 19 to 20 days) was seen in all but 1 animal which was found, both by scanning and by post-mortem examination, to have ovarian cysts. Ultrasound scanning was useful to monitor the normal size and morphology of the ovaries in-life, and could identify those animals that were shown to have abnormalities post-mortem. We conclude that the battery of tests incorporated into this study is useful to detect possible changes in the female reproductive system in minipigs during the course of a study. A significant benefit was avoiding use of interim kills to detect the timing of potential changes in ovarian morphology.

View full abstract

By current estimates around 50% of preclinical in vivo rodent toxicity screens failing to predict subsequent human toxicity, representing a significant challenge in drug development. As a result, regulatory agencies have led the call for improved in vitro screening assays to better predict human toxicity. Here we demonstrate the use of aProximate™ human proximal tubule cell (hPTC) monolayers as an in vitro tool to investigate nephrotoxicity using the clinically relevant biomarkers NGAL, KIM-1, and Clusterin. Freshly isolated hPTCs were seeded onto Transwell filters, and grown to confluence over 3 days before addition of nephrotoxicants. Biomarker generation was assessed by ELISA using samples removed from the apical chamber at various time points. In addition, measurements of trans-epithelial electrical resistance (TEER) were taken to determine monolayer integrity. Exposure of the hPTCs to gentamycin (200 µg/ml), cyclosporine A (10 µM), cisplatin (10 µM), or methotrexate (10 µM) for periods of up to 120 hours resulted in significant (P < 0.001) increases in biomarker production. Furthermore, we observed that for hPTC exposed to cyclosporine A, cisplatin, or methotrexate, the elevation in biomarkers at 120 hours was also accompanied by a significant decrease in TEER, suggesting that the monolayer integrity was compromised. In contrast, TEER values were unchanged upon exposure to gentamycin. In summary, these data suggest that aProximate™ hPTC monolayers express clinically relevant biomarkers of nephrotoxicity, and so have excellent potential for in vitro predictive human toxicology screening during drug development.

View full abstract

New tobacco products with the potential to reduce individual risk and population harm in comparison to smoking cigarettes are under development and require a careful safety assessment strategy. In line with the 21st century toxicology paradigm and with the use of human in vitro organotypic models as substitute of animal testing, we exposed human nasal and bronchial epithelial tissue culture to an aerosol generated by a candidate Reduced Risk Product (RRP), Tobacco Heating System (THS) 2.2, versus air (sham control) or cigarette smoke (CS) with the same nicotine content as THS2.2. Different endpoints (cytotoxicity, cilia beating frequency, CYP1A1/1B1 enzyme activity, inflammatory markers release, morphological and transcriptomic changes) were analyzed at 4, 24, 48 and 72h after exposure to identify and compare the dose- and time-dependent effect of the different test item used.
By using systems toxicology-based risk assessment approaches combining computable biological network models and gene expression changes, we compared the molecular perturbations in both conventional cigarette and THS2.2 exposure conditions. A significant impact was quantified over different CS post-exposure time points in the networks representing cell death, inflammation, proliferation and cellular stress; instead, the impact of THS2.2 was closer to sham controls and detectable only shortly after exposure (4h). These results indicate a reduced toxicity of THS2.2 acute exposure on both nasal and bronchial epithelial tissue culture when compared to CS.

Reduced Risk Products (“RRPs”) is the term we use to refer to products with the potential to reduce individual risk and population harm in comparison to smoking cigarettes.

View full abstract

Show abstractHide abstract

[Purpose] Drug-induced liver injury (DILI) is a major cause for termination of drug development and withdrawal of approved drugs from the market. Although toxic potential of compounds is often evaluated in in vitro, current in vitro systems generally have a low concordance with human hepatotoxicity. In the past years, human hepatoma HepaRG line has been shown to be a valuable tool to study the mechanism of DILI. However, high level of glutathione in the cell is considered to lessen the cytotoxicity by drugs. In this study, we investigated utility of a glutathione-depleted HepaRG cells in detecting toxicity caused by reactive metabolites.
[Methods] HepaRG and HepG2 cells were pre-incubated with 400 µM_L-buthionine-S,R-sulfoximine (BSO) for 3 h, and then the cells were treated with 30 test compounds classified as withdrawn, boxed-warning, warning and safe at 1.6-, 6.4-, 25-, and 100-fold of the therapeutic maximum plasma concentration for 24 h. Cytotoxicity was evaluated by LDH assay. Sensitivity and specificity were calculated (cut-off = 70%) to evaluate the predictability of the assay.
[Results and Discussion] Cytotoxicity was strongly enhanced in glutathione-depleted HepaRG cells but not obviously in glutathione-depleted HepG2 cells. For example, co-treatment of boxed-warning drug flutamide with BSO decreased HepaRG cell viability from 82% to 54%, but did not affect HepG2 cell viability. Among 30 test compounds, BSO-treated HepaRG cells exhibited the highest sensitivity of 43%, while non-treated HepaRG cells was only 21%, both of them exhibited 100% specificity. BSO-treated HepG2 cells exhibited the sensitivity of 36% and 100% specificity, while non-treated HepG2 cells was only 21% and 92% specificity. These results indicate that glutathione-depleted HepaRG cell model is useful in predicting potential DILI risks caused by reactive metabolites.

View full abstract

Download PDF (662K)

Register with J-STAGE for free!