Bone Marrow Micronucleus Assay Research Articles

Toxicity Profile Study of Antihypertensive Drug Prazosin in Pregnant Wistar Rats

The purpose of this research is to investigate and assess the cytotoxicity and genotoxicity of Prazosin HCL in pregnant rats. Prazosin (PZ) was administered to the animals intraperitoneally (IP) at dosages of 5, 15, and 25 mg/kg/body weight for single dose (14-day) toxicity tests. The following parameters have been examined for evaluating genotoxicity: bone marrow micronucleus assay, peripheral blood micronucleus assay, DNA damage is measured using the metaphase chromosomal analysis, DNA damage is measured using the DNA fragmentation test, and cytotoxicity is measured using a histological analysis. The results obtained clearly demonstrate that PZ induced hazardous responses at the higher dose in the hepatocytes, as evidenced by DNA damage, and increased DNA fragmentation in pregnant rats. Observing that PZ significantly increased DNA strand breakage and structural chromosomal aberrations in bone marrow cell lines is also fascinating. As a result, it is thought to be genotoxic to bone marrow and mouse hepatocyte cells. The current study showed that Prazosin had significant genotoxic effects in pregnant rats at the matching hepatotoxic dose level.

Monitoring hospital effluents through physico-chemical characterization and genotoxic testing.

Inefficient monitoring of the water resource recovery facility at various health care establishments may lead to detrimental effects on human population, especially when such water get mixed with potable municipal water supply. To ensure the efficiency and proper functioning of the water resource recovery facility and quality of water, before it being disposed of, the present study was conducted with the assessment of physico-chemical parameters of water and its genotoxic and cytogenetic effects in mice. The sample water was given ad libitum to animals for three different time durations, that is, 7, 15, and 30 days. Bone marrow chromosomal aberration and bone marrow micronucleus (MN) assay were performed to get the extent of genotoxicity and cytogenicity. The results showed presence of chromosomal aberrations like fragments breaks and ring formation in different groups. Besides this, a significant (p < 0.05*, p < 0.01**, p < 0.001***) decrease in the mitotic index was noted in the group which was given 100% concentrated sample water for 30 days. A significant (p < 0.05*, p < 0.01**, p < 0.001***) increase in MN induction and decreased ratio of polychromatic to normochromatic erythrocytes were observed in groups which were given samples at 10% and 100% concentrations for longer durations. The findings suggest that even the recovered sample water reflects a positive in vivo genotoxic potential when given for a longer duration of 30 days, indicating some lacuna in the treatment process.

Genotoxic and chemopreventive potentials of ethanol leaves extract of Annona muricata on N-Ethyl-N-Nitrosourea-induced pro-leukaemia carcinogen in mice model by bone marrow micronucleus assay

Background. Studies have proven the effect of several agents, including natural products, to induce, prevent and treat genotoxicity through experimental models and clinical trials. In this study, the genotoxic preventive potential of Annona muricata ethanol extract on N-Ethyl-N-Nitrosourea (ENU)-induced pro-leukaemia in mice models using micronuclei formation in bone marrow was assessed. Materials and methods. Forty-eight mice weighing 18-24g were randomly divided into six groups of eight mice. The mice were intravenously administered 20mg/kg of NEU 48 hourly 3 times, 80mg/kg of NEU 48 hourly 3 times. The negative control was fed with feed and water only. We introduced 0.2ml (0.1g/ml) ethanolic extract of Annona muricata for 3 weeks prior to NEU low dosage administration, 0.2ml (0.1g/ml) ethanolic extract of Annona muricata for 3 weeks prior to ENU high dosage and Annona muricata (ethanolic extract) administration, and gave commercial diet to the adverse/ toxicity group. The bone marrow was harvested, smeared and stained using MayGrumwald. The procedure enabled the determination of micronucleus polychromatic erythrocytes (MNPCEs) microscopically. Results. Groups exposed to various dosages of the ENU yielded significantly increased MNPCEs, with group B producing higher MNPCEs. The groups treated with the extract displayed a significant reduction in the MNPCEs despite prior exposure to concentrations of NEU. The adverse group displayed no difference in MNPCEs compared with the negative control. Conclusion. The ENU induced genotoxicity depending on its concentration. The extract displayed a profound capacity to prevent genotoxicity and alleviate leukaemia with good tolerance.

In vitro and in vivo assessments of the genotoxic potential of 3-chloroallyl alcohol.

3-Chloroallyl alcohol (3-CAA) can be found in the environment following the application of plant protection products. 3-CAA is formed in groundwater following the injection of 1,3-dichloropropene, a fumigant used to control nematodes. 3-CAA is also formed, in leafy crops, as a glycoside conjugate following application of the herbicide, clethodim. Human exposure may occur from groundwater used as drinking water or through dietary consumption. To characterize 3-CAA's potential to cause genotoxicity in mammals, in vitro and in vivo studies were conducted. 3-CAA was negative in an Ames test and positive in a mouse lymphoma forward mutation assay. 3-CAA was negative in an acute in vivo CD-1 mouse bone marrow micronucleus assay when administered up to a dose level of 125 mg/kg/day for two consecutive days. In a combined gene mutation assay and erythrocyte micronucleus assay, using transgenic Big Blue® Fischer 344 rats, 3-CAA was administered via drinking water at targeted dose levels of 0, 10, 30, and 100 mg/kg/day for 29 days. Peripheral blood samples, collected at the end of treatment, were analyzed for micronucleus induction in reticulocytes using flow cytometry. Liver and bone marrow samples, collected 2 days after the termination of the treatment, were analyzed for the induction of mutations at the cII locus. 3-CAA did not induce an increase in mutant frequency or micronuclei under the experimental conditions. In conclusion, the mutagenic response observed in the in vitro mouse lymphoma assay is not confirmed in the whole animal. 3-CAA is not considered to pose a mutagenic risk.

The effect of aging on the repeated-dose liver micronucleus assay using diethylnitrosamine

BackgroundThe repeated-dose liver micronucleus (RDLMN) assay has been well-developed and applied because of its simplicity and the ease of integration into general toxicity studies which is the preferred method from the 3R’s point of view. In this assay, we observed micronucleated hepatocytes which accumulated during a rather long-term dosing period. When considering integration into general toxicity studies, the effects of age of the animals used in the micronucleus assay becomes a major issue. The effect of age on the micronucleus induction rate has been reported in bone marrow micronucleus assays, and it is considered that the decrease in cell proliferation rate due to aging is the cause of the decrease in sensitivity. A decrease in sensitivity due to aging was also reported in a liver micronucleus assay using clofibrate and the cause is considered to be a decrease in hepatocyte proliferation activity due to aging. However, no actual decrease in hepatocyte proliferation rate due to aging has been reported. In addition, there are no reports, so far, on whether similar effects of aging appear when other substances were administered. To investigate the effects of aging in the RDLMN assay, this study focused on the effects of 14-day repeated administration of DEN, a well-known genotoxic hepatocarcinogen with the hepatocyte toxicity which should cause an elevation of cell proliferation rate as a reflective regeneration.ResultsThe liver micronuclei induced by DEN were equivalent between the two age groups (i.e., six and eight weeks of age at the start of dosing). In the histopathological examination for the liver, single cell necrosis, karyomegaly, and increased mitosis were observed in the hepatocytes, and the frequency and severity were increased dose-dependently. Ki-67 immunohistochemical analysis which can detect all cells in the cell cycle other than those in the G0 phase revealed dose-dependent increase of cell proliferation activity, and the difference between ages was not observed.ConclusionThe effect of aging on the RDLMN assay could not be recognized when DEN was administered for 14 days in rats. Meanwhile, it was supported by the histopathological examination and Ki-67 immunohistochemical analysis that such an effect of aging was masked by the compensatory hepatocyte proliferation which was induced by the hepatocyte toxicity of DEN.

Search for the optimal genotoxicity assay for routine testing of chemicals: Sensitivity and specificity of conventional and new test systems

Many conventional in vitro tests that are currently widely used for routine screening of chemicals have a sensitivity/specificity in the range between 60 % and 80 % for the detection of carcinogens. Most procedures were developed 30–40 years ago. In the last decades several assays became available which are based on the use of metabolically competent cell lines, improvement of the cultivation conditions and development of new endpoints. Validation studies indicate that some of these models may be more reliable for the detection of genotoxicants (i.e. many of them have sensitivity and specificity values between 80 % and 95 %). Therefore, they could replace conventional tests in the future. The bone marrow micronucleus (MN) assay with rodents is at present the most widely used in vivo test. The majority of studies indicate that it detects only 5–6 out of 10 carcinogens while experiments with transgenic rodents and comet assays seem to have a higher predictive value and detect genotoxic carcinogens that are negative in MN experiments. Alternatives to rodent experiments could be MN experiments with hen eggs or their replacement by combinations of new in vitro tests. Examples for promising candidates are ToxTracker, TGx-DDI, multiplex flow cytometry, γH2AX experiments, measurement of p53 activation and MN experiments with metabolically competent human derived liver cells. However, the realization of multicentric collaborative validation studies is mandatory to identify the most reliable tests.

Efficacy of Carob Leaves and Pods in Reducing Cyclophosphamide-Induced Toxicity on Bone Marrow and Peripheral Blood Leukocytes

Cyclophosphamide is one of the chemotherapeutics for cancer, however this drug has severe side effects in multiple organs and is a well-known mutagenic agent. In this work the protective activity of antioxidant-rich Carob (Ceratonia siliqua L.) pods and leaves extracts against cyclophosphamide induced genotoxicity was studied. Safety of these extracts was evaluated in acute and subacute toxicity tests at a dose of 1500 mg/kg/day in albino rats. Both extracts exhibited no mortality or significant changes in rats' general behavior. Moreover, organ weights and blood chemistry revealed no toxic effects of these extracts (p&gt;0.05).The anti-mutagenic potential of carob pods and leaves extracts were evaluated using in vivo bone marrow chromosomal aberration and micronucleus assays and in vitro comet assay for peripheral blood white cells. Results showed that rats that were treated with cyclophosphamide along with carob pods had a significant decrease in chromosomal aberrations when compared with cyclophosphamide-treated rats (p&lt;0.05). Pods extract also showed significant reduction in cyclophosphamide-induced DNA migration (p&lt;0.05). There were also highly statistically significant differences in the means of micronuclei (MNs) in immature red blood cells between cyclophosphamide group and carob leaves group (p&lt;0.01).It could be concluded that Carob extracts had protective effect against cyclophosphamide- induced genotoxicity and may be considered as anti-mutagenic agent after conducting more studies.

Abstract 1804: IND-ready clinical candidate for HPK1 developed with excellent efficacy and safety profile

Abstract Background Hematopoietic progenitor kinase 1 (HPK1) is a member of the mitogen-activated protein kinase (MAP4K) family of protein serine/threonine kinases1,2 and is a negative regulator of T and B cell receptor signaling3. Inhibition of HPK1 is an attractive therapeutic strategy for immuno-oncology based treatment of solid tumors3. We present in vitro, in vivo, pharmacokinetic (PK) and early safety profiles of a novel and differentiated HPK1 inhibitor GRC 54276. Methods GRC 54276 is our clinical candidate, designed and developed using intuitive medicinal chemistry design and supported by computational approaches. SAR studies included a battery of biochemical assays, functional read-outs and primary human in vitro T-cell activation assays. In vivo efficacy was demonstrated in syngeneic mouse tumor models, both as a single agent and combination with immune check-point blockers (ICB), mouse anti-CTLA4 antibody or Atezolizumab (human anti-PD-L1 antibody). ADME-PK properties was evaluated cross-species. GLP and non-GLP safety tolerability studies were conducted in mice and monkeys. Results GRC 54276 demonstrated sub-nanomolar HPK1 potency, strong target engagement of pSLP76 inhibition, anti-tumor cytokines (IL-2 and IFN-γ) induction, reversal of immunosuppression by prostaglandin E2 (PGE2) or adenosine in both human and mouse systems. GRC 54276 demonstrated very strong tumor growth inhibition (TGI) efficacy as single agent and significantly enhanced TGI in combination with ICB antibodies anti-CTLA4 (CT26 model) or Atezolizumab (MC38-hPD-L1 model). The in vivo TGI efficacy mechanistically correlated with increased immune responses of cytokine induction, infiltration of cytotoxic T cells, tumor rejections accompanied by immune memory T cells induction. Pharmacokinetic profile of GRC 54276 included cross-species oral bioavailability (30 to 100%), predominant clearance by CYP3A4 with no significant inhibition of major CYP isoforms, negative activation potency in human PXR assay at several-fold over EDmax exposures. Safety profile demonstrated that GRC 54276 is non-genotoxic in the bone marrow micronucleus assay in mice and has no hERG liability. The no observed adverse effect levels in the 14-day and 17-day exploratory studies in mice and monkeys were 50 and 15 mg/kg/day, respectively. Conclusions GRC 54276, our clinical candidate is potent, selective, orally bioavailable HPK1 inhibitor demonstrating strong single-agent and combination efficacy, low DDI liability accompanied by acceptable early safety profile in mice and monkeys. GRC 54276 is undergoing IND enabling studies to advance to Phase 1 clinical trial. Acknowledgements We thank Vidya Kattige, Pooja Sawant, Shital More, Rahul B. Bhadane, Ajit Jagadale, Sanjay Gaikwad, Pramod Sagar for their contributions to the project

Abstract 1171: Genotoxic preventive potential of ethanol leaves extract of Annona muricata on N-Nitroso-N-ethylurea pro-leukaemia carcinogen in mice by bone marrow micronucleus assay

Abstract Background: Genotoxicity which is an important indicator of DNA damage, repair and cell rejuvenation has been a predisposing factor to cancer development evidenced by bone marrow suppression in leukemia and studies have proven the effect of several agents inclusive of natural products to induce, prevent and treat genotoxicity through experimental models and clinical trials. Aim:The genotoxic preventive potential of Annona muricata ethanol leaves extract on N-nitroso-N-ethylurea (NEU) pro-leukemia carcinogen in mice model using micronuclei formation in bone marrow was assessed. Materials and Methods: 48 mice weighing 18-24g were randomly divided into six groups of eight mice each comprising of the; mice intravenously administered 20mg/kg of NEU 48 hourly 3 times (A); 80mg/kg of NEU 48 hourly 3 times (B); negative control fed with feed and water only (C); 0.2ml (0.1g/ml) ethanolic leaves extract of Annona muricata for 3 weeks prior to administration of low dose of NEU (D); 0.2ml (0.1g/ml) ethanolic extract of Annona muricata for 3 weeks prior to administration of high dose of NEU (E); and Annona muricata (ethanolic leaves extract) and commercial diet for adverse/toxicity group (F). After six weeks of last exposure, animals were ethically sacrificed, bone marrow harvested, smeared, stained using MayGrumwald and number of micronucleus polychromatic erythrocyte (MNPCEs) were determined microscopically.Result: Groups exposed to various dosage of the NEU yielded significantly increased MNPCEs with group B producing higher MNPCEs than A. The groups treated with extract displayed a significant reduction in the MNPCEs despite the prior exposure concentrations of NEU. The adverse group displayed no difference in MNPCEs when compared with the negative control (Table 1). Conclusion: The extract displayed profound capacity to prevent genotoxicity thereby alleviating leukemia condition and it is well tolerated. Week Number Groups (Mean±Standard deviation of MNPCE) A B C D E F 2 14.13±0.56 50.45±2.31 4.52±0.34 6.15±0.49 17.11±1.11 4.53±0.45 3 15.01±0.18 53.77±1.95 4.61±0.67 7.85±0.98 20.34±2.34 4.62±0.23 4 16.11±0.22 56.95±2.01 4.91±0.88 8.01±1.01 25.11±1.67 5.63±0.45 5 19.64±1.68 68.23±1.56 4.82±0.75 10.07±0.67 30.54±2.34 5.92±0.67 Citation Format: Olufemi Emmanuel Akanni, Eunice Oluwaseyi Bamisaye, Sunday Anthony Fashina, Fatai Adesina Abdulraheem, Solomon Olufemi Fadiora. Genotoxic preventive potential of ethanol leaves extract of Annona muricata on N-Nitroso-N-ethylurea pro-leukaemia carcinogen in mice by bone marrow micronucleus assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1171.

Evaluation of the Genotoxic Potential of the Selective COX-2 Inhibitor Enflicoxib in a Battery of in vitro and in vivo Genotoxicity Assays.

Aim Enflicoxib, a selective COX-2 inhibitor approved for the treatment of pain and inflammation associated with osteoarthritis in dogs (Daxocox ® [Ecuphar/Animalcare Group]) was assessed for its genotoxic potential in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse mutation assay (Ames test), an in vitro human lymphocyte chromosome aberration assay and an in vivo mouse bone marrow micronucleus assay. Methods Relevant vehicle and positive control cultures and animals were included in all assays. In the Ames test, enflicoxib was tested at concentrations of up to 5000 μg/plate. Signs of cytotoxicity were observed at the highest tested concentrations for several of the bacterial strains, both in absence and presence of S9. In human lymphocytes, enflicoxib was assessed for the induction of chromosomal aberrations when exposed at concentrations of up to 62.5 (3 hours) and 29.6 µg/mL (20 hours) in the absence of S9, and up to 66.7 µg/mL (3 hours) in presence of S9. Signs of cell toxicity, evidenced as a decrease in the mitotic index, were observed at these concentrations. In the mouse micronucleus assay, enflicoxib dose levels of up to 2000 mg/kg were administered (single dose) to male and female animals, and bone marrow samples were taken 24 and 48 hours (high-dose animals only) after administration. Results Enflicoxib was shown to lack genotoxic activity in the conducted assays. Conclusions The administration of enflicoxib as a therapeutic analgesic agent would not pose a genotoxic risk to animals or humans.

Genotoxicity and mutagenicity evaluation of isoquercitrin-γ-cyclodextrin molecular inclusion complex using Ames test and a combined micronucleus and comet assay in rats.

Flavonoids such as quercetin and its glucosides, especially isoquercitrin are well known as anti-inflammatory, anti-allergic, and anti-carcinogenic, etc. The safety of isoquercitrin formulations needs to be established prior to their use in functional food applications. The mutagenicity and genotoxicity of the IQC-γCD inclusion complex were assessed with three standard assays of the bacterial reverse mutation assay (Ames test) and using a combined in-vivo micronucleus and comet assay under the Organisation for Economic Co-operation and Development (OECD) guidelines. In combined rat bone marrow micronucleus and rat liver comet assay performed in male Sprague Dawley (SD) rats, the various doses of IQC-γCD inclusion complex (max. 2000 mg/kg bw) and positive controls ethyl methanesulfonate (EMS) and mitomycin C (MMC), respectively, and negative control (vehicle) were administrated. The results of the Salmonella typhimurium mutagenicity assay (strains TA100, TA1535, WP2uvrA, TA98, and TA1537) after exposure to the IQC-γCD inclusion complex with the absence and presence of the metabolic activation system (S9 fraction from rat liver) revealed a weakly positive response but with no biologically relevant mutagenicity at the conditions examined according to recommended regulatory guidelines. The combined micronucleus and comet assay results reveal that the IQC-γCD inclusion complex did not induce in-vivo genotoxic potential or indication of any oxidative DNA damage in rat liver tissues. Altogether, considering the results of the study, it is unlikely that the consumption of IQC-γCD inclusion complex as food or supplement would present any concern for humans regarding the mutagenicity and genotoxicity.

Toxicological safety of VOHO Hemp Oil; a supercritical fluid extract from the aerial parts of hemp.

VOHO Hemp Oil (Verdant Nature LLC (in collaboration with HempFusion)) is an extract of the aerial parts of hemp (Cannabis sativa L) manufactured using a supercritical CO2 extraction process. The results of four safety studies are reported here including a bacterial reverse mutation assay, an in vivo mammalian micronucleus study, a maximum tolerated dose study in rats and a 90-day repeat dose subchronic toxicity study in rats. VOHO Hemp oil can contain up to 30% phytocannabinoids and less than 0.2% is tetrahydrocannabinol (THC). VOHO Hemp Oil was found to be non-mutagenic in the bacterial reverse mutation assay and was negative for inducing micronuclei in the rat bone marrow micronucleus assay. The maximum tolerated dose in male and female Wistar rats was 2250 mg/kg bw/day. A 90-day repeat dose study was conducted in male and female Wistar rats according to OECD Guideline 408 and included a 21-day recovery period. The doses used in the study were 0, 25, 90 and 324 mg/kg bw per day in the main study, and in the recovery phase a control and 324 mg/kg bw/day group were included. One mortality was reported during the study, a high dose female, and test substance-related adverse clinical signs were reported in the high dose group. Other test substance-related changes noted in the high dose group included changes in body weights, activated partial thromboplastin time (APTT) values, and in absolute and relative organ weights. Based on the results of the study, the no observed adverse effect level (NOAEL) for VOHO Hemp Oil was determined to be 90 mg/kg bw/day in both male and female Wistar rats.

Decrease of the DXR-induced genotoxicity and nongenotoxic effects of Theobroma cacao revealed by micronucleus assay.

This study evaluated the genotoxicity of lyophilized glycolic extract of Theobroma cacao Linné seeds (TCL), using the micronucleus assay in bone marrow of mice. The interaction between TCL and doxorubicin (DXR) was also analyzed. Experimental groups were evaluated 24-48 h after treatment with N-Nitroso-N-ethylurea (NEU: 50 mg/kg), DXR (5 mg/kg), NaCl (145 mM), TCL (0.5-2 g/kg), and TCL (2 g/kg) in combination with DXR (antigenotoxic assays). Analysis of micronucleated polychromatic erythrocytes (MNPCEs) showed no significant differences between all the treatment doses of TCL and NaCl control. Mice experimentally treated with DXR and NEU significantly induced MNPCEs. However, a significant reduction of MNPCEs was also observed when TCL was administered in combination with the chemotherapeutic agent DXR. The analysis of the PCE/NCE ratio revealed no significant differences between the NaCl control, all doses of TCL, and DXR. However, there were significant differences in the PCE/NCE ratio between positive NEU control and all other treatments. The PCE/NCE ratio observed after treatment with TCL and DXR showed significant differences and intermediate values to controls (NaCl and NEU). This study suggests absence of genotoxicity and cytotoxicity of TCL, regardless of dose, sex, and time. TCL reduced genotoxic effects induced by DXR, suggesting potential antigenotoxic effects.

Genotoxicity Evaluation of Propyl-Propane-Thiosulfinate (PTS) from Allium genus Essential Oils by a Combination of Micronucleus and Comet Assays in Rats.

Propyl-propanethiosulfinate (PTS) is a component of Allium essential oils. This organosulfur molecule can be used as a feed additive to decrease the appearance of bacterial resistances caused by the residues of antibiotics. In previous in vitro genotoxicity studies, contradictory results were reported for PTS. In this work, the in vivo genotoxicity of PTS in male and female rats was assessed for the first time, following OECD (Organisation for Economic Co-operation and Development) guidelines. After oral administration (doses: 5.5, 17.4, and 55.0 mg/kg PTS body weight), a combination of the micronucleus (MN) assay (OECD 474) in bone marrow and the standard and enzyme-modified comet assay (OECD 489) was performed. After necropsy, histopathological studies were also carried out. The results did not show the in vivo genotoxicity of PTS at any doses assayed, revealed by the absence of increased MN, and DNA strand breaks or oxidative DNA damage in the standard and enzyme-modified comet assays. The histopathological study revealed that only the highest dose tested (55.0 mg/kg) in the liver and all dose groups in the stomach presented minimal pathological lesions in the organs studied. Consequently, the present work confirms that PTS is not genotoxic at the doses assayed, and it is a promising natural alternative to synthetic preservatives and antibiotics in animal feed.

Genotoxicity evaluation of self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG), a novel siRNA nanoparticle for the treatment of fibrotic disease

The self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG) is a novel small-interfering RNA (siRNA) nanoparticle that is used for treatment of pulmonary fibrosis. We investigated the potential genotoxicity of SAMiRNA-AREG based on the guidelines published by the Organization for Economic Cooperation and Development. In the bacterial reverse mutation assay (Ames test), SAMiRNA-AREG did not induce mutations in Salmonella typhimurium TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2uvrA at concentrations of up to 3000 μg/plate with or without metabolic activation. The SAMiRNA-AREG (concentrations up to 500 μg/mL) did not induce chromosomal aberrations in cultured Chinese hamster lung cells with or without metabolic activation. In the in vivo mouse bone marrow micronucleus assay, the SAMiRNA-AREG (concentrations up to 300 mg/kg body weight) did not affect the proportions of polychromatic erythrocytes and total erythrocytes, nor did it increase the number of micronucleated polychromatic erythrocytes in ICR mice. Collectively, these results suggest that SAMiRNA-AREG is safe with regard to genotoxicity such as mutagenesis or clastogenesis under the present experimental conditions. These results might support the safety of SAMiRNA-AREG as a potential therapeutic agent for pharmaceutical development.

In Vivo Genotoxicity Evaluation of a Stilbene Extract Prior to Its Use as a Natural Additive: A Combination of the Micronucleus Test and the Comet Assay.

Genotoxic data of substances that could be used as food additives are required by the European Food Safety Authority. In this sense, the use of an extract from grapevine shoots containing a stilbene richness of 99% (ST-99), due to its antioxidant and antibacterial activities, has been proposed as an alternative to sulfur dioxide in wine. The aim of this work was to study, for the first time, the in vivo genotoxic effects produced in rats orally exposed to 90, 180, or 360 mg ST-99/kg body weight at 0, 24, and 45 h. The combination of micronucleus assay in bone marrow (OECD 474) and standard (OECD 489) and enzyme-modified comet assay was used to determine the genotoxicity on cells isolated from stomach, liver, and blood of exposed animals. The ST-99 revealed no in vivo genotoxicity. These results were corroborated by analytical studies that confirm the presence of stilbenes and their metabolites in plasma and tissues. Moreover, to complete these findings, a histopathological study was performed under light microscopy in liver and stomach showing only slight modifications in both organs at the highest concentration used. The present work confirms that this extract is not genotoxic presenting a good profile for its potential application as a preservative in the wine industry.

Mutagenic and teratogenic toxicity evaluation of Forsythia suspensa leaves aqueous extract

Forsythia suspensa leaves (FSL), rich in phillyrin, forsythiaside A, phillygenin, rutin, and other compounds, is a known traditional Chinese medicine (TCM). It has been effective in heat retreat and detoxification. In this study, we performed the mutagenic and teratogenic toxicity evaluation of FSL aqueous extract (FSLAE) using the bacterial reverse mutation assay (Ames test), mouse bone marrow micronucleus assay, spermatocyte chromosomal aberration assay in mice. Kunming mice and SD rats were used were for the mutagenic and the teratogenic studies, respectively. We found that FSLAE was not mutagenic and did not induce unfavorable chromosomal events. Additionally, the Ames test revealed FSLAE was not genotoxic and showed no mutagenic activity in histidine dependent strains of Salmonella typhimurium at concentrations up to 5000 μg/plate. Likewise, in vivo test revealed no induced micronucleus of mouse bone marrow or chromosome aberration in spermatocytes up to the dose of 10.00 g/kg BW. For the teratogenic evaluations, pregnant rats were treated with 1.04, 2.08, and 4.17 g/kg FSL, and fetuses were examined on the 6–15 day of pregnancy. We observed no maternal toxicity and embryotoxicity related to the treatment. Based on these in vitro and in vivo studies, we concluded the genotoxic and teratogenic safety of FSL.

Assessment of the Genotoxic Potential of Cizolirtine a Substance-P and Calcitonin Gene-Related Peptide Release Modulator.

The analysis of the genotoxic potential of cizolirtine, a compound being developed as a drug for analgesia and for urinary incontinence, was carried out using a battery of in vitro and in vivo assays as recommended in the guidelines for medicinal products. Negative results were obtained in an Ames test (up to 5000 µg/plate), in a Mouse Lymphoma assay (up to 2000 µg/ml) and in a single dose mouse bone marrow micronucleus assay (up to 300 mg/kg). In a human lymphocyte chromosome aberration assay, a slight statistical increase in the frequency of cells with chromosome aberrations including gaps was reported for the concentrations of 200 and 1600 μg/ml at the 24-h sampling time. This minor increase in chromosome aberrations was considered of questionable biological relevance since it was moderate, was within the laboratory historical control values, did no show a dose-dependent effect and was not observed at similar concentrations in a repeat assay. Taking into considerations the results obtained in the different in vitro and in vivo assays and a weight-of-evidence analysis, it suggests that cizolirtine would not pose a genotoxic risk when administered to humans.

Safety evaluation of Eucommia ulmoides extract

Eucommia ulmoides Oliver is native to China and frequently used in traditional Chinese medicine formulations. However, studies show that Eucommia ulmoides extract (EUE) are potentially genotoxic and nephrotoxic. To evaluate its safety, the Ames test, bone marrow micronucleus assay and chromosomal aberration assay, along with acute (24 h) and sub-chronic (13 weeks) toxicity were conducted. EUE was non-genotoxic within the dose ranges of 0.0352–22 mg/plate (raw plant equivalent as below), 22–88 g/kg body weight and 2–20 mg/mL. The maximum tolerated dose of EUE was not less than 168 g/kg, which is 1260 times that of clinical doses in human adults. Long-term (13 weeks) administration led to dose-dependent increase in nephrotoxicity-related indices, and pathological changes in renal tissues. These changes were alleviated 5 weeks after ceasing the low dosage of 11.2 g/kg but persisted at the high dosage of 56 g/kg. Conclusively, EUE is non-genotoxic, and do not result in acute toxicity. However, long-term and high-dose administration can lead to partly reversible nephrotoxicity.

Gallic acid: A promising genoprotective and hepatoprotective bioactive compound against cyclophosphamide induced toxicity in mice.

Cyclophosphamide (CP) is a potent chemotherapeutic agent and is also known to interact with specific biological molecules and produce numerous side effects such as genotoxicity, neurotoxicity, reproductive toxicity and nephrotoxicity. CP induces genotoxicity by generating oxygen/nitrogen derived free radicals during chemotherapy and causes DNA damage. Hence, to overcome such side effects of chemotherapeutic agents antioxidants are recommended. Gallic acid (GA) a phenolic compound is commonly exists in variety of fruits and in many plants. In the present study, genoprotecive effects of GA against CP induced genotoxicity in Swiss albino mice were evaluated by using comet assay, bone marrow, and peripheral micronucleus (MN) assays. CP produced oxidative stress induced hepatic damage was assessed by estimating the activity of liver superoxide dismutase (SOD), reduced glutathione content (GSH), and also by examining the histological alterations induced by CP in hepatic tissue of mice. It was noticed that, GA pretreatment significantly reduced the frequency of MN and DNA strand breaks induced by CP. GA also protected the hepatic tissue against CP induced reactive oxygen species (ROS) mediated damage and was evident by increased levels of liver SOD and GSH. GA ameliorated the histological changes induced by CP in hepatic tissue. These findings suggest that, GA is a versatile antioxidant with promising protection against CP induced genotoxicity and hepatic damage in Swiss albino mice.