Toshihiko Kasahara

Test battery with the human cell line activation test, direct peptide reactivity assay and DEREK based on a 139 chemical data set for predicting skin sensitizing potential and potency of chemicals

To develop a testing strategy incorporating the human cell line activation test (h‐CLAT), direct peptide reactivity assay (DPRA) and DEREK, we created an expanded data set of 139 chemicals (102 sensitizers and 37 non‐sensitizers) by combining the existing data set of 101 chemicals through the collaborative projects of Japan Cosmetic Industry Association. Of the additional 38 chemicals, 15 chemicals with relatively low water solubility (log Kow > 3.5) were selected to clarify the limitation of testing strategies regarding the lipophilic chemicals. Predictivities of the h‐CLAT, DPRA and DEREK, and the combinations thereof were evaluated by comparison to results of the local lymph node assay. When evaluating 139 chemicals using combinations of three methods based on integrated testing strategy (ITS) concept (ITS‐based test battery) and a sequential testing strategy (STS) weighing the predictive performance of the h‐CLAT and DPRA, overall similar predictivities were found as before on the 101 chemical data set. An analysis of false negative chemicals suggested a major limitation of our strategies was the testing of low water‐soluble chemicals. When excluded the negative results for chemicals with log Kow > 3.5, the sensitivity and accuracy of ITS improved to 97% (91 of 94 chemicals) and 89% (114 of 128). Likewise, the sensitivity and accuracy of STS to 98% (92 of 94) and 85% (111 of 129). Moreover, the ITS and STS also showed good correlation with local lymph node assay on three potency classifications, yielding accuracies of 74% (ITS) and 73% (STS). Thus, the inclusion of log Kow in analysis could give both strategies a higher predictive performance. Copyright

Evaluation of combinations of in vitro sensitization test descriptors for the artificial neural network-based risk assessment model of skin sensitization

The skin sensitization potential of chemicals has been determined with the use of the murine local lymph node assay (LLNA). However, in recent years public concern about animal welfare has led to a requirement for non‐animal risk assessment systems for the prediction of skin sensitization potential, to replace LLNA. Selection of an appropriate in vitro test or in silico model descriptors is critical to obtain good predictive performance. Here, we investigated the utility of artificial neural network (ANN) prediction models using various combinations of descriptors from several in vitro sensitization tests. The dataset, collected from published data and from experiments carried out in collaboration with the Japan Cosmetic Industry Association (JCIA), consisted of values from the human cell line activation test (h‐CLAT), direct peptide reactivity assay (DPRA), SH test and antioxidant response element (ARE) assay for chemicals whose LLNA thresholds have been reported. After confirming the relationship between individual in vitro test descriptors and the LLNA threshold (e.g. EC3 value), we used the subsets of chemicals for which the requisite test values were available to evaluate the predictive performance of ANN models using combinations of h‐CLAT/DPRA (N = 139 chemicals), the DPRA/ARE assay (N = 69), the SH test/ARE assay (N = 73), the h‐CLAT/DPRA/ARE assay (N = 69) and the h‐CLAT/SH test/ARE assay (N = 73). The h‐CLAT/DPRA, h‐CLAT/DPRA/ARE assay and h‐CLAT/SH test/ARE assay combinations showed a better predictive performance than the DPRA/ARE assay and the SH test/ARE assay. Our data indicates that the descriptors evaluated in this study were all useful for predicting human skin sensitization potential, although combinations containing h‐CLAT (reflecting dendritic cell‐activating ability) were most effective for ANN‐based prediction. Copyright

The gene expression of hepatic proteins responsible for DNA repair and cell proliferation in tamoxifen-induced hepatocarcinogenesis.

Altered gene expression of the DNA repair‐ and cell proliferation‐associated proteins/enzymes was examined during the process of tamoxifen‐induced hepatocarcinogenesis in female Sprague‐Dawley rats. When rats were treated by gavage with a single dose of tamoxifen (20 mg/kg body weight) or with the same dose given at 24‐h intervals for 2, 12 or 52 weeks, no histopathological change was observed in the liver after 2 weeks. Pathologically altered cell foci and placental form of glutathione‐S‐transferase (GST‐P)‐positive foci were observed in the liver after 12 weeks of treatment. Treatment for 52 weeks resulted in the formation of liver hyperplastic nodules that strongly expressed GST‐P. During the process of carcinogenesis, changes in hepatic gene expression of DNA repair proteins/enzymes (XPA and XPC, xeroderma pigmentosum complementation groups A and C, respectively; APE, apurinic/apyrimidinic endonuclease) and of cell proliferation‐associated proteins (c‐myc; PCNA, proliferating cell nuclear antigen; cyclin D1, cyclin B, and p34cdc2) were examined by RT‐PCR. The gene expression of XPA and APE was increased by the tamoxifen treatment for 2 or 12 weeks, but no increase was observed after the 52‐week treatment. In addition, no significant change in XPC gene expression occurred at any period examined. The gene expression of c‐myc, PCNA, and cyclin D1 was increased in a time‐dependent fashion up to 12 weeks of treatment, and this increase was maintained up to 52 weeks of treatment. The gene expression of cyclin B and p34cdc2 was increased after the 1‐day treatment, reverted to the control level at 2 and 12 weeks of treatment, and was remarkably increased after the 52‐week treatment. In the present study, we demonstrate the altered gene expression of various proteins/enzymes involved in DNA repair, cell growth and the cell cycle during the process of tamoxifen‐induced hepatocarcinogenesis. We discuss the relationship between the altered gene expression and hepatocarcinogenesis.

Evaluation of high-throughput screening for in vitro micronucleus test using fluorescence-based cell imaging

The in vitro micronucleus (MN) test is an important component of genotoxicity screening and is used as an alternative to the in vitro chromosome aberration (CA) test. As the MN assay is more practical and simpler to use than the CA test, it is being applied as a high-throughput screening (HTS) assay. Therefore, we conducted a validation study of the MN test employing a confocal imaging plate reader, the IN Cell Analyzer 1000. We evaluated 30 chemicals, including clastogens and aneugens, using Chinese hamster lung cells (CHL/IU) seeded in 96-well microplates. The microplates were stained with Hoechst 33342 and CellMask Red for automated analysis, and MN were identified and counted automatically in fluorescence images. The MN test results for 30 chemicals obtained with this image analysis system, using the IN Cell Analyzer, were highly consistent with reference data for the in vitro MN test and CA test data obtained by microscopic analysis. In conclusion, this HTS assay for detecting MN offers high efficiency and accuracy in the early stages of chemical development.

Development of a prediction method for skin sensitization using novel cysteine and lysine derivatives.

INTRODUCTION The Direct Peptide Reactivity Assay (DPRA) was developed as an alternative simple and versatile method for predicting skin sensitization. Here, we describe a novel Amino acid Derivative Reactivity Assay (ADRA) involving 2 amino acid derivatives: N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC) and α-N-(2-(1-naphthyl)acetyl)-l-lysine (NAL), in which each amino-terminal residue is introduced into a naphthalene ring. ADRA measurements were conducted at 281nm, which improved baseline stability, and were less influenced by other substances in the reaction solutions than DPRA measurements that are conducted at 220nm. METHODS Chemically synthesized NAC and NAL were dissolved in phosphate buffers of pH9.5 and 12.0, respectively. Each solution, test chemical solution, and phosphate buffer, were mixed in 96-well microplates and incubated in the dark for 24h at 25°C. Following incubation, samples were diluted 10 times with a mixed solution of 25% acetonitrile/0.5% trifluoroacetic acid (TFA) in water, and NAC and NAL levels were quantified in each sample and control using a high-performance liquid chromatography (HPLC) system. The reactivity of NAC/NAL was calculated as the percent depletion based on the decrease in the non-reacted NAC/NAL concentration in the samples relative to the average concentration in the control; the average NAC/NAL reduction score was calculated. A 2-class classification model was developed using ADMEWORKS/ModelBuilder, and an optimal average score that could discriminate between sensitizers and non-sensitizers was determined. RESULTS A total of 82 test chemicals were applied to ADRA for comparison against DPRA. The prediction accuracy of ADRA was 88%, which was similar to that of DPRA. DISCUSSION ADRA was used to quantify NAC/NAL at 281nm, which showed high accuracy for the prediction of skin sensitization, similar to that of DPRA. Therefore, ADRA could be used to expand the range of chemicals tested in skin sensitization analyses.

A novel in chemico method to detect skin sensitizers in highly diluted reaction conditions.

The direct peptide reactivity assay (DPRA) is a simple and versatile alternative method for the evaluation of skin sensitization that involves the reaction of test chemicals with two peptides. However, this method requires concentrated solutions of test chemicals, and hydrophobic substances may not dissolve at the concentrations required. Furthermore, hydrophobic test chemicals may precipitate when added to the reaction solution. We previously established a high‐sensitivity method, the amino acid derivative reactivity assay (ADRA). This method uses novel cysteine (NAC) and novel lysine derivatives (NAL), which were synthesized by introducing a naphthalene ring to the amine group of cysteine and lysine residues. In this study, we modified the ADRA method by reducing the concentration of the test chemicals 100‐fold. We investigated the accuracy of skin sensitization predictions made using the modified method, which was designated the ADRA‐dilutional method (ADRA‐DM). The predictive accuracy of the ADRA‐DM for skin sensitization was 90% for 82 test chemicals which were also evaluated via the ADRA, and the predictive accuracy in the ADRA‐DM was higher than that in the ADRA and DPRA. Furthermore, no precipitation of test compounds was observed at the initiation of the ADRA‐DM reaction. These results show that the ADRA‐DM allowed the use of test chemicals at concentrations two orders of magnitude lower than that possible with the ADRA. In addition, ADRA‐DM does not have the restrictions on test compound solubility that were a major problem with the DPRA. Therefore, the ADRA‐DM is a versatile and useful method. Copyright

DNA damage and altered gene expression of enzymes for metabolism and DNA repair by tamoxifen and toremifene in the female rat liver

Effects of hepatocarcinogenic TAM and non‐hepatocarcinogenic TOR on the formation of hepatic DNA adducts and on the gene expression of hepatic drug‐metabolizing enzymes and DNA repair enzymes/proteins were comparatively examined in female Sprague‐Dawley rats treated with TAM (20 or 40 mg/kg/day, i.g.) or TOR (40 mg/kg/day, i.g.) for 1, 2 or 8 weeks. Hepatic TAM‐DNA adducts were formed even after 1 week of treatment with TAM at either dose, and the adduct levels increased in a dose‐ and treatment period‐dependent manner, whereas no DNA adducts were detected in any of the TOR‐treated rats. Conversely, TAM and TOR showed almost the same capacity for increasing the gene expression of drug‐metabolizing enzymes responsible for metabolic activation and detoxification, at least up to the 2‐week treatment mark. Accordingly, differences in DNA adduct formation between TAM‐ and TOR‐treated rats would not be primarily dependent on the capacity for inducing hepatic drug‐metabolizing enzymes. In addition, a drastic increase in the gene expression of cytochrome P4503A2 (CYP3A2), an activation enzyme of TAM, by the 8‐week treatment with TAM might have contributed to the increased formation of DNA adducts. Gene expressions of DNA repair enzymes/proteins responsible for a nucleotide excision repair system were not significantly changed in any of the rats treated with either drug. The present findings suggest that the difference between TAM and TOR in hepatocarcinogenic potency is dependent on the capacity to form DNA adducts rather than modulating the expression of drug‐metabolizing enzymes and DNA repair enzymes/proteins. (Cancer Sci 2006; 97: 468– 477)

An automated new technique for scoring the in vivo micronucleus assay with image analysis.

The mammalian erythrocyte micronucleus assay is frequently used to assess chemical-induced damage to the chromosomes or the mitotic apparatus of erythroblasts. Because quantitative analysis of micronuclei by microscopy is time consuming and laborious, several automatic scoring methodologies with image analysis have been reported. However, there have been cases in which it was difficult to examine the proportion of polychromatic erythrocytes (PCEs) among total erythrocytes as an index for bone marrow (BM) toxicity, and sample slide preparation has proven to be laborious with existing automatic methods. We developed an automatic scoring system with image analysis for the rodent erythrocyte micronucleus assay using 12-well plates employing high-content screening analyser. In our method, micronucleated PCEs (MNPCEs), PCEs and erythrocytes were identified from three kinds of images: bright field image, fluorescence image with Hoechst 33342, and fluorescence image with propidium iodide. The frequencies of MNPCEs and PCEs were subsequently calculated. A comparison of automatic and manual scoring was carried out using BM and peripheral blood (PB) obtained from mice treated with stepwise doses of mitomycin C. The scores obtained by automatic analysis corresponded to those obtained by manual scoring; the frequencies of MNPCEs in BM and PB obtained by automatic scoring were 132 and 113%, respectively, of those obtained by manual scoring, and the corresponding frequencies of PCEs were 95 and 120%, respectively. Furthermore, we performed five repeats of the examinations of mouse BM and PB treated with mitomycin C or vinblastine sulphate and showed that automatic scoring was equivalent to manual scoring in reproducibility. Meanwhile, the scoring data obtained by manual scoring tended to vary among observers. These results suggest that our automatic scoring system with image analysis is superior to manual microscopy scoring in terms of speed and objectivity, comparable in reproducibility and useful for the in vivo micronucleus assay.

Chemically induced strong cellular hypertrophy often reduces the accuracy of cytotoxicity measurements obtained using the ATP assay

The ATP assay is a highly sensitive and versatile method for measuring cytotoxicity. However, the correlation between the cell viability results obtained using the ATP assay and those obtained using direct cell counting has not been widely reported. Therefore, to evaluate the reliability and limitations of the ATP assay, we compared the results of ATP assay with those of automatic cell counter, which can measure the number and diameter of cells directly, by using 24 compounds and repeating individual experiments thrice. The correlation between the data was low for 7 of the 24 compounds (r2 < 0.8, at least 2 out of 3 experiments). These were the top 7 of the 11 compounds that induced cell hypertrophy. These 7 compounds were also observed to increase the area of mitochondria. However, the last 4 of the 11 compounds increased the cell size but did not increase the mitochondrial area. For the remaining 13 compounds, which had no effect on cell size, a good correlation was observed between the results of the two methods (r2 > 0.8, at least 2 out of 3 experiments), and the cell size was effectively the same as that of the controls. We concluded that the poor correlation between the two methods was attributable to an increase in the content of intracellular ATP because of the chemically induced cell and mitochondrial hypertrophy. We showed that the ATP assay is unsuitable for assessing the cytotoxicity of compounds that induce cell hypertrophy with increase in the mitochondrial area and ATP content.

High-content image analysis (HCIA) assay has the highest correlation with direct counting cell suspension compared to the ATP, WST-8 and Alamar blue assays for measurement of cytotoxicity

INTRODUCTION Various cytotoxicity assays measuring indicators such as enzyme activity, dye uptake, or cellular ATP content are often performed using 96-well microplates. However, recent reports show that cytotoxicity assays such as the ATP assay and MTS assay underestimate cytotoxicity when compounds such as anti-cancer drugs or mutagens induce cell hypertrophy whilst increasing intracellular ATP content. Therefore, we attempted to evaluate the reliability of a high-content image analysis (HCIA) assay to count cell number in a 96-well microplate automatically without using a cell-number indicator. METHODS We compared cytotoxicity results of 25 compounds obtained from ATP, WST-8, Alamar blue, and HCIA assays with those directly measured using an automatic cell counter, and repeating individual experiments thrice. RESULTS The number of compounds showing low correlation in cell viability measured using cytotoxicity assays compared to automatic cell counting (r2<0.8, at least 2 of 3 experiments) were follows: ATP assay; 7; WST-8 assay, 2; Alamar blue assay, 3; HCIA cytotoxicity assay, 0. Compounds for which correlation was poor in 3 assays, except the HCIA assay, induced an increase in nuclear and cell size. However, correlation between cell viability measured by automatic cell counter and the HCIA assay was strong regardless of nuclear and cell size. Additionally, correlation coefficients between IC50 values obtained from automatic cell counter and from cytotoxicity assays were as follows: ATP assay, 0.80; WST-8 assay, 0.84; Alamar blue assay, 0.84; and HCIA assay, 0.98. DISCUSSION From the above, we showed that the HCIA cytotoxicity assay produces similar data to the automatic cell counter and is highly accurate in measuring cytotoxicity.