Collaborative Study of Thresholds for Mutagens: Adaptive Responses in the Micronucleus Test and Gene Induction by Mutagenic Treatments.

Background: We have been conducting a collaborative study on the thresholds of mutagens. In our previous examinations of cell activity and cell proliferation as endpoints, both displayed hormesis. This time, we conducted experiments to determine thresholds using the micronucleus test as an endpoint. Methods: The micronucleus test was conducted using Chinese hamster CHL/IU cells and mouse lymphoid L5178Y cells. Additionally, we conducted preliminary investigations into the gene expression using human TK6 cells. Results: When adhesive CHL/IU cells were treated with mitomycin C (MMC), and the hormetic response was examined, hormesis was not observed clearly. When L5178Y cells were treated with methyl methanesulfonate (EMS), AF-2, MMC, and colchicine, all of them exhibited an adaptive response. Additionally, cross-adaptive responses using AF-2 and MMC or EMS and MMC were conducted, both combinations showed a cross-adaptive response. When the gene expression patterns of six genes were investigated by RT-PCR after treatment with MMC, EMS, and H2O2 using TK6 cells, two genes, GADD45 A and P21, were induced in a dose- and time-dependent manner. Conclusion: Adaptive responses arise from preconditioning. As hormesis is inherently linked to preconditioning, adaptive responses observed in this study strongly suggest that hormesis was induced, hence existence of thresholds.

Bacterial mutagenicity test data: collection by the task force of the Japan pharmaceutical manufacturers association.

BACKGROUND: Ames test is used worldwide for detecting the bacterial mutagenicity of chemicals. In silico analyses of bacterial mutagenicity have recently gained acceptance by regulatory agencies; however, current in silico models for prediction remain to be improved. The Japan Pharmaceutical Manufacturers Association (JPMA) organized a task force in 2017 in which eight Japanese pharmaceutical companies had participated. The purpose of this task force was to disclose a piece of pharmaceutical companies' proprietary Ames test data. RESULTS: Ames test data for 99 chemicals of various chemical classes were collected for disclosure in this study. These chemicals are related to the manufacturing process of pharmaceutical drugs, including reagents, synthetic intermediates, and drug substances. The structure-activity (mutagenicity) relationships are discussed in relation to structural alerts for each chemical class. In addition, in silico analyses of these chemicals were conducted using a knowledge-based model of Derek Nexus (Derek) and a statistics-based model (GT1_BMUT module) of CASE Ultra. To calculate the effectiveness of these models, 89 chemicals for Derek and 54 chemicals for CASE Ultra were selected; major exclusions were the salt form of four chemicals that were tested both in the salt and free forms for both models, and 35 chemicals called "known" positives or negatives for CASE Ultra. For Derek, the sensitivity, specificity, and accuracy were 65% (15/23), 71% (47/66), and 70% (62/89), respectively. The sensitivity, specificity, and accuracy were 50% (6/12), 60% (25/42), and 57% (31/54) for CASE Ultra, respectively. The ratio of overall disagreement between the CASE Ultra "known" positives/negatives and the actual test results was 11% (4/35). In this study, 19 out of 28 mutagens (68%) were detected with TA100 and/or TA98, and 9 out of 28 mutagens (32%) were detected with either TA1535, TA1537, WP2uvrA, or their combination. CONCLUSION: The Ames test data presented here will help avoid duplicated Ames testing in some cases, support duplicate testing in other cases, improve in silico models, and enhance our understanding of the mechanisms of mutagenesis.

Collaborative Study of Thresholds for Mutagens: Hormetic Responses in Cell Proliferation Tests Using Human and Murine Lymphoid Cells.

BACKGROUND: We previously showed that hormetic responses can be established in cell activity tests using human and murine adherent cells. This time, we examined whether hormetic responses can be established in cell proliferation tests using suspended human and murine lymphoid cells. METHODS: Human lymphoblastoid cells (TK6) and mouse lymphoma cells (L5178Y) were cultured in multi-well culture plates and treated with mitomycin C, ethyl methansulfonate, hygromycin B, aclarubicin or colchicine at various dose levels and the number of cells was measured at varied times using a flow cytometer. RESULTS: When the ratio of the number of cells treated with a test chemical to those in the negative control was plotted, the dose-response relationship typically showed a reverse U-shaped curve, indicating the occurrence of hormesis and existence of thresholds in cell toxicity. The hormetic responses depended largely on the test chemical, dose level and exposure time. When examining responses over the course of time, a J-shaped or fallen S-shaped curve was also observed. CONCLUSIONS: The dose-response relationship showed a reverse U-shaped curve, a hallmark of hormesis, at least some time points for all chemicals tested here, indicating that chemical hormesis can be established in in vitro cell proliferation tests.

In Vitro and In Vivo Antibacterial Activities of a Novel Quinolone Compound, OPS-2071, against Clostridioides difficile.

OPS-2071 is a novel quinolone antibacterial agent characterized by low oral absorption that reduces the risk of adverse events typical of fluoroquinolone class antibiotics. The in vitro and in vivo antibacterial activities of OPS-2071 against Clostridioides difficile were evaluated in comparison to vancomycin and fidaxomicin. OPS-2071 exhibited potent antibacterial activity against 54 clinically isolated C. difficile strains with a MIC of 0.125 µg/ml (MIC50) and 0.5 µg/ml (MIC90), making it more active than vancomycin on a concentration basis (MIC50, 2 µg/ml; MIC90, 4 µg/ml) and comparable to fidaxomicin (MIC50, 0.063 µg/ml; MIC90, 8 µg/ml). OPS-2071 showed equally potent antibacterial activity against both hypervirulent and nonhypervirulent strains, while a significant difference in susceptibility to fidaxomicin was observed. Spontaneous resistance to OPS-2071 and vancomycin was not observed; however, resistance to fidaxomicin was observed at 4× MIC. The mutant prevention concentration of OPS-2071 was 16-fold lower than those of fidaxomicin and vancomycin, and the postantibiotic effect of OPS-2071 was longer than those of fidaxomicin and vancomycin. Also, OPS-2071 showed low systemic exposure, with OPS-2071 having 2.9% oral bioavailability at 1 mg/kg in rats. Furthermore, OPS-2071 showed significant in vivo efficacy at 0.0313 mg/kg/day (50% effective doses), 39.0-fold and 52.1-fold lower than those of vancomycin and fidaxomicin, respectively, in a hamster model of C. difficile infection. OPS-2071 has the potential to become a new therapeutic option for treating C. difficile infection.

Collaborative study of thresholds for mutagens: proposal of a typical protocol for detection of hormetic responses in cytotoxicity tests.

BACKGROUND: According to the linear no-threshold model (LNT), even the smallest amount of radiation is hazardous. Although the LNT is not based on solid data, this hypothesis has been applied to mutagens and carcinogens. As a result, it has been postulated that there are no thresholds for these chemicals. To demonstrate negativity by experiments is practically impossible, because negative data may leave behind the possibility that additional data might make the resolution power high enough to change negativity to positivity. Furthermore, additional data collection may be endless and we may be trapped in agnosticism. When hormesis is established, in which biological responses are higher at low-doses and lower at high-doses than the control, thresholds could be established between the low- and high-doses. Before examination of thresholds in chemical mutagenesis, hormetic responses in cytotoxicity were tested using cultured mammalian cells. METHOD: Human cells (HeLa S3 and TK6) or Chinese hamster cells (CHL/IU) were cultured in 96-well plates and treated with mitomycin C (MMC) or ethyl methanesulfonate (EMS) at various dose levels and optical density was measured after addition of a reagent to detect cellular activity. In hormetic responses, data might fluctuate to and fro; therefore, experimental conditions were examined from various aspects to eliminate confounding factors including cell numbers, detection time, the edge effect of 96-well plates, and measurement time after addition of the reagent for detection. RESULTS: The dose response relationship was never linear. Cellular activities after treatment with MMC or EMS were generally higher at lower doses levels and lower at higher doses than the control, showing hormesis and allowing the establishment of thresholds. Dose response curves sometimes showed two or three peaks, probably reflecting different cellular responses. CONCLUSION: Hormetic responses in cytotoxicity tests were observed and thresholds could be established. Based on the results of this investigation, we put forward a tentative protocol to detect chemical hormesis in cytotoxicity tests, i.e., inoculate 2000 cells per well, add various doses of a test chemical 48 h after inoculation, add a detection dye 10 h after treatment, and measure optical density 2 h after addition of the reagent for detection.

Nonclinical safety profile of tolvaptan.

PURPOSE: In the present study, the nonclinical safety profile of tolvaptan was evaluated. METHODS: A series of safety pharmacology and toxicology studies were performed in vitro and in mice, rats, dogs, rabbits and guinea pigs. RESULTS: In safety pharmacological studies, tolvaptan had no adverse effects on the central nervous, somatic nervous, autonomic nervous, smooth muscle, respiratory and cardiovascular, or digestive systems. In general toxicity studies, a single dose of tolvaptan up to 2,000 mg/kg was not lethal in rats and dogs. Tolvaptan did not cause any target organ toxicity in rats after treatment for 26 weeks or in dogs after treatment for 52 weeks at oral doses of up to 1,000 mg/kg/day. The toxicities observed in the present studies were generally attributable to the exaggerated pharmacological action of tolvaptan. In reproductive and developmental toxicity studies in rats, fertility was not affected. Suppressed viability or growth observed in the prenatal and postnatal progeny occurred at the maternally toxic dose of 1,000 mg/kg/day. In rabbits, tolvaptan showed teratogenicity at 1,000 mg/kg/day, a dose that was maternally toxic causing abortion. Tolvaptan was not genotoxic or carcinogenic, and did not induce phototoxicity, antigenicity or immunotoxicity. CONCLUSION: Nonclinical toxicity that precludes the safe administration of tolvaptan to humans was not observed. However, appropriate cautions should be taken in women of childbearing potential.