Integration of liver and blood micronucleus and Pig-a gene mutation endpoints into rat 28-day repeat-treatment studies: Proof-of-principle with diethylnitrosamine

Khanal, Sumee; Singh, Priyanka; Avlasevich, Svetlana L.; Torous, Dorothea K.; Bemis, Jeffrey C.; Dertinger, Stephen D.

doi:10.1016/j.mrgentox.2018.02.005

Cited by 12 publications

(5 citation statements)

References 28 publications

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“…Our findings revealed that CGDCM treatment decreased the levels of the liver proliferative marker Ki-67 in DEN-induced HCC rats, implying anti-proliferative activity against liver cancer. DEN has been reported to enhance the proliferation and initiation of hepatocyte tumors by activating the G1/S phase of the cell cycle 96 , 119 , 120 . The enhanced proliferation of liver cells after DEN treatment supports compensatory responses for the development of primary HCC following DNA damage and necrotic cell death in the liver 3 , 88 , 121 .…”

Section: Discussionmentioning

confidence: 99%

Calotropis gigantea stem bark extracts inhibit liver cancer induced by diethylnitrosamine

Sawong

Pekthong

Suknoppakit

et al. 2022

Sci Rep

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Several fractions of Calotropis gigantea extracts have been proposed to have potential anticancer activity in many cancer models. The present study evaluated the anticancer activity of C. gigantea stem bark extracts in liver cancer HepG2 cells and diethylnitrosamine (DEN)-induced primary liver cancer in rats. The carcinogenesis model induced by DEN administration has been widely used to study pathophysiological features and responses in rats that are comparable to those seen in cancer patients. The dichloromethane (CGDCM), ethyl acetate, and water fractions obtained from partitioning crude ethanolic extract were quantitatively analyzed for several groups of secondary metabolites and calactin contents. A combination of C. gigantea stem bark extracts with doxorubicin (DOX) was assessed in this study to demonstrate the enhanced cytotoxic effect to cancer compared to the single administration. The combination of DOX and CGDCM, which had the most potential cytotoxic effect in HepG2 cells when compared to the other three fractions, significantly increased cytotoxicity through the apoptotic effect with increased caspase-3 expression. This combination treatment also reduced ATP levels, implying a correlation between ATP and apoptosis induction. In a rat model of DEN-induced liver cancer, treatment with DOX, C. gigantea at low (CGDCM-L) and high (CGDCM-H) doses, and DOX + CGDCM-H for 4 weeks decreased the progression of liver cancer by lowering the liver weight/body weight ratio and the occurrence of liver hyperplastic nodules, fibrosis, and proliferative cells. The therapeutic applications lowered TNF-α, IL-6, TGF-β, and α-SMA inflammatory cytokines in a similar way, implying that CGDCM had a curative effect against the inflammation-induced liver carcinogenesis produced by DEN exposure. Furthermore, CGDCM and DOX therapy decreased ATP and fatty acid synthesis in rat liver cancer, which was correlated with apoptosis inhibition. CGDCM reduced cleaved caspase-3 expression in liver cancer rats when used alone or in combination with DOX, implying that apoptosis-inducing hepatic carcinogenesis was suppressed. Our results also verified the low toxicity of CGDCM injection on the internal organs of rats. Thus, this research clearly demonstrated a promising, novel anticancer approach that could be applied in future clinical studies of CGDCM and combination therapy.

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Section: Discussionmentioning

confidence: 99%

Calotropis gigantea stem bark extracts inhibit liver cancer induced by diethylnitrosamine

Sawong

Pekthong

Suknoppakit

et al. 2022

Sci Rep

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“…The studies described herein employed previously established flow cytometric methods for scoring the frequency of rat blood micronucleated reticulocytes (MN-RET) and Pig-a gene mutant phenotype reticulocytes (RET CD59− ) and erythrocytes (RBC CD59− ) (Dertinger et al 2004(Dertinger et al , 2011. The incidence of rat liver micronuclei (MNHEP) was scored using a recently reported flow cytometric method Khanal et al 2018).…”

Section: Introductionmentioning

confidence: 99%

3Rs friendly study designs facilitate rat liver and blood micronucleus assays and Pig‐a gene mutation assessments: Proof‐of‐concept with 13 reference chemicals

Dertinger

Avlasevich

Torous

et al. 2019

Environ and Mol Mutagen

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Regulatory guidance documents stress the value of assessing the most appropriate endpoints in multiple tissues when evaluating the in vivo genotoxic potential of chemicals. However, conducting several independent studies to evaluate multiple endpoints and/or tissue compartments is resource intensive. Furthermore, when dependent on visual detection, conventional approaches for scoring genotoxicity endpoints can be slow, tedious, and less objective than the ideal. To address these issues with current practices we attempted to (1) devise resource sparing treatment and harvest schedules that are compatible with liver and blood micronucleus endpoints, as well as the Pig‐a gene mutation assay, and (2) utilize flow cytometry‐based methods to score each of these genotoxicity biomarkers. Proof‐of‐principle experiments were performed with 4‐week‐old male and female Crl:CD(SD) rats exposed to aristolochic acids I/II, benzo[a]pyrene, cisplatin, cyclophosphamide, diethylnitrosamine, 1,2‐dimethylhydrazine, dimethylnitrosamine, 2,6‐dinitrotoluene, hydroxyurea, melphalan, temozolomide, quinoline, or vinblastine. These 13 chemicals were each tested in two treatment regimens: one 3‐day exposure cycle, and three 3‐day exposure cycles. Each exposure, blood collection, and liver harvest was accomplished during a standard Monday–Friday workweek. Key findings are that even these well‐studied, relatively potent genotoxicants were not active in both tissues and all assays (indeed only cisplatin was clearly positive in all three assays); and whereas the sensitivity of the Pig‐a assay clearly benefitted from three versus one treatment cycle, micronucleus assays yielded qualitatively similar results across both study designs. Collectively, these results suggest it is possible to significantly reduce animal and other resource requirements while improving assessments of in vivo genotoxicity potential by simultaneously evaluating three endpoints and two important tissue compartments using fit‐for‐purpose study designs in conjunction with flow cytometric scoring approaches. Environ. Mol. Mutagen., 60:704–739, 2019. © 2019 Wiley Periodicals, Inc.

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“…This has contributed to an increase in the assessment of multiple genotoxic endpoints from the same animals . As an example, it is common that data from assays which combine a subacute toxicology study in the rodent, with micronucleus assessment in the blood, as well as comet assay in the liver [10] science will be a substantial part of it. However, the most dramatic change in thinking, will be that the test substance and not the list of tests (the strategy) should be the main consideration.…”

Section: Discussionmentioning

confidence: 99%

Strategies in genotoxicology: Acceptance of innovative scientific methods in a regulatory context and from an industrial perspective

Steiblen

Benthem

Johnson

2020

Mutation Research/Genetic Toxicology and Environmental Mutagene

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The tests used and the general principles behind test strategies are now often over 30 years old. It may be time by now, given that our knowledge of genetic toxicology has improved and that we also technically are better able to investigate DNA damage making use of modern molecular biological techniques, to start thinking on a new test strategy. In the present paper, it is demonstrated that the time is there to consider a new approach for genotoxicity assessment of substances. A fit for all test strategy was discussed making use of the most recent technological methods and techniques. It was also indicated that in silico tools should be more accepted by regulatory institutes/bodies as supporting information to better conclude which tests should be required for each separate substance to demonstrate its genotoxic potency. Next to that there should be a good rationale for performing in vivo studies. Finally, the need for germ cell genotoxicity testing, essential when classification and labeling of substances is mandatory, was discussed. It was suggested to change the GHS for genotoxicity classification and labelling from in vivo tests in germ cells into in vivo tests in somatic cells. Quantitative genotoxicology was also discussed. It appeared that we are currently at a transition, where the science developing to justify carrying out human health risk assessments based on genetic toxicology data sets supported by mechanistic data and exposure data. However, implementation will take time, and acceptance will be supported through the development of numerous case studies. Major remaining questions are: is genetic damage a relevant endpoint in itself, or should the risk assessment be carried out on the apical endpoint of cancer and which genotoxic endpoint should be used to derive the point of departure (PoD) for the human exposure limit?

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Integration of liver and blood micronucleus and Pig-a gene mutation endpoints into rat 28-day repeat-treatment studies: Proof-of-principle with diethylnitrosamine

Cited by 12 publications

References 28 publications

Calotropis gigantea stem bark extracts inhibit liver cancer induced by diethylnitrosamine

Calotropis gigantea stem bark extracts inhibit liver cancer induced by diethylnitrosamine

3Rs friendly study designs facilitate rat liver and blood micronucleus assays and Pig‐a gene mutation assessments: Proof‐of‐concept with 13 reference chemicals

Strategies in genotoxicology: Acceptance of innovative scientific methods in a regulatory context and from an industrial perspective

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