Frequency Of Micronucleus Formation Research Articles

Abstract 3927: Radium-223 α-particle radiation: Characterization of the in vitro effects on cancer cells in monotherapy and in combination with DNA repair inhibitors

Abstract Purpose: Targeted alpha therapy (TAT) represents an emerging treatment approach attempting to deliver systemic radiation selectively to cancer cells and bypassing mechanisms of acquired resistance while minimizing systemic toxic effects. Radium-223 dichloride (Ra-223) is a first-in-class TAT shown to prolong survival in patients with metastatic castration-resistant prostate cancer and bone metastases. Yet, the understanding of the biological effects of α-particle irradiation is still limited. Here, we used a novel in vitro assay designed to assess alpha irradiation specific effects to investigate the biological effects of Ra-223 in various cancer cell lines. Furthermore, the combination of α-particle irradiation with DNA repair inhibitors was tested. Methods: The following cancer cell lines were used: lung (H460), ovarian (OVCAR-3, COV362, COV644, ES2) and prostate (22Rv1, LNCaP). The effects of different radiation doses and exposure times of Ra-223 on various biological parameters were analyzed using a Transwell® system where cells grow on a 10 µm membrane located on top of an underlying Ra-223 coating. Results: After 4 hours of exposure to α-particle irradiation (5, 10 and 20 kBq/3.8 cm2), a dose-dependent induction of DNA double strand breaks (DSB) as assessed by 53BP1 positivity was observed. Exposure to α-particle irradiation (10 and 20 kBq/3.8 cm2) for 1 hour dose-dependently induced DNA damage in a comet assay. Exposure to α-particle irradiation (5 kBq/3.8 cm2) for 1, 4 and 8 hours time-dependently reduced the surviving fraction in the subsequent colony formation assay. The frequency of micronuclei formation was increased in an activity- and time-dependent manner after α-particle exposure at 5, 10, 20, 40 kBq/3.8 cm2 for 1, 4 and 8 hours. In the same assay, the number of dead cells was significantly increased after an 8-hour exposure in all dose groups tested. Pre-incubation with the ATR inhibitor BAY 1895344 (5 nM for 2 hours prior to radiation) synergistically enhanced micronuclei formation (< 5% vs 12% vs 42% in ATR inhibitor only vs α-particle irradiation only vs ATR inhibitor + α-particle irradiation, respectively). Conclusion: Using several cancer cell lines, we demonstrated that short term exposure to α-particle irradiation in the range of 1 to 8 hours is sufficient to induce DNA damage including DNA DSB which leads to cellular damage and cell death. Synergistic effects were observed when Ra-223 was combined with the ATR inhibitor BAY 1895344 . Citation Format: Kristina Bannik, Sabine Zitzmann-Kolbe, Arne Scholz, Sabrina Jarke, Marco Jarzombek, Andreas Sutter, Gerhard Siemeister, Dominik Mumberg. Radium-223 α-particle radiation: Characterization of the in vitro effects on cancer cells in monotherapy and in combination with DNA repair inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3927.

Antioxidant and genoprotective properties of the antidiabetic drug metformin under X-ray irradiation

The antioxidant and genoprotective properties of the antidiabetic drug metformin have been investigated. The research was performed both in vitro and in vivo under X-ray irradiation. It was shown that metformin reduced the formation of hydroxyl radicals and hydrogen peroxide in aqueous solutions irradiated with X-rays. The particular features of its influence on the formation of H2O2 were revealed. It has been established that metformin reduced the formation of 8-oxoguanine, a key biomarker of oxidative damage to DNA, in vitro. The effect of metformin on the frequency of micronucleus formation in polychromatic erythrocytes of bone marrow of irradiated mice was investigated. Its genoprotective and radiomimitatory properties at different concentrations and times of application are shown.

Doramectin induced cytotoxic and genotoxic effects on bovine peripheral lymphocytes and cumulus cells in vitro

The effect of doramectin (DOR) was tested on two experimental somatic bovine cells in vitro: peripheral lymphocytes (PL) and cumulus cells (CC). The cytotoxicity and genotoxicity of DOR were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, single cell gel electrophoresis assay (SCGE) and cytokinesis-block micronucleus cytome (CBMN Cyt) assay. Both cells were treated with three concentrations of DOR (20, 40, 60 ng mL–1) for 24 h. The results obtained from PL demonstrated that DOR was able to induce cytotoxic effect and DNA damage with all concentrations tested. Additionally, DOR increased micronuclei (MNi) frequency and nuclear buds (NBuds) with 20, 40, 60 ng mL–1, and nucleoplasmic bridges (NPBs) only with 40 ng mL–1. On the other hand, the three concentrations of DOR were not able to induce cytotoxic effect and DNA damage using SCGE in the bovine CC. Nevertheless, the two higher concentrations of DOR (20, 40 µg mL–1) significantly increased the frequency of micronucleus formation in bovine CC. These results represent the first experimental evidence of genotoxic and cytotoxic effects exerted by DOR on bovine PL and CC.

Effect of aqueous extract of Rosemary officinalis on cytotoxicity of CCL4 induced albino male mice

This study focused the line on the effect of aqueous extract of Rosemary officinalis, as well as, effect of toxic compound CCL4, on micronucleus formation and mitotic index assay in albino male mice. This work started at September 2017 at Biotechnology Research center \Al-Nahrain University, by using 20 albino male mice. The result indicated that aqueous extract of rosemary caused significant increased in mitotic index and decrease micronucleus formation for two doses tested 50,100 mg/kg in comparison with negative and positive controls, also the results revealed that CCL4 showed significant mutagenic action on biological system of treated mice by increased frequency of micronucleus formation and decreased the percentage of mitotic index in bone marrow cells. Pre-and post –treatment between aqueous extract and CCL4 were also made. The results of pre and post treatment with rosemary extract were also caused a significant decreased in micronucleus formation and increase the percentage of mitotic index for two doses 50,100 mg/kg in comparison with its corresponding controls which caused increased in the frequencies of micronucleus formation and decrease the percentage of mitotic index in bone marrow cells. Conclusions: Rosemary officinalis enhanced immunity, reduced mutagenic effects against cytotoxicity of CCL4.

Genotoxic evaluation of common commercial pesticides in human peripheral blood lymphocytes.

This study aims to evaluate the genotoxic potential of four commercial pesticides with diverse health categorizations by different world associations currently in use. We tested the fungicide mancozeb and the insecticides pirimicarb, monocrotophos and permethrin. The research was done with in vitro human peripheral blood lymphocytes using the DNA single gel electrophoresis assay and the cytokinesis-block micronucleus (MN) test, where we analysed common parameters such as the tail moment and the frequency of MN formation. We also measured other parameters like frequency of nucleoplasmic bridges, nuclear buds, apoptosis and necrosis with the MN test. Each pesticide induced significant differences in all of these parameters when compared with the negative control and showed different behaviours in the concentration-dependent response. This could be attributed to their genotoxic potential where mancozeb and monocrotophos induced the highest genetic damage, permethrin caused mainly cell death and pirimicarb had the least impact upon cells. This research provides valuable data about the harmful effects of these pesticides on human cells and may be an important contribution in the construction of a unique international classification of health and to reinforce the use of genotoxic analyses to regulate the use of pesticides.

Limited ability of DNA polymerase kappa to suppress benzo[a]pyrene-induced genotoxicity in vivo.

DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock-in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP-induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven-week-old inactivated Polk KI and wild-type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP-induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644-653, 2017. © 2017 Wiley Periodicals, Inc.

Immunological, Cytogenetic and Hepatoprotective Effect of Viola odorata Methanolic Extract on Methotrexate Induced Albino Male Mice

Pants used in traditional medicine contain a vast array of substances that can be used to treat chronic and infectious diseases. One of these medical plant Viola odorata which a popularly known as “Banafshah” and sweet violet in Asia and Europe respectively. It is found in high altitudes of Himalyas, Europe and throughout North America. The study was designed to assess immunological (total and differential count of white blood cells), cytogenetic (micronucleus formation) and hepatoprotective activity of methanolic extract of Viola odorata by using two doses 100 and 200mg/kg. In addition, these effects were assessed after an interaction between the two doses of the plant and methtrexte drug. Results:The results of this study indicated that Viola odorata had the ability to enhance immunity and reduced the frequency of micronucleus formation in bone marrow cells in addition to its hepatoprotective activity. Alternatively, the plant was able to counteract the damage induced by methotrexate 40 mg\kg.

An oxovanadium(IV) complex protects murine bone marrow cells against cisplatin-induced myelotoxicity and DNA damage

Cisplatin (CDDP) is one of the first-line anticancer drugs that has gained widespread use against various forms of human malignancies. But, the therapeutic outcome of CDDP therapy is limited due to its adverse effects including myelotoxicity and DNA damage which may lead to the subsequent risk of developing secondary cancer. Hence, in search of a suitable cytoprotectant, this study investigated the probable protective efficacy of an oxovanadium(IV) complex, namely oxovanadium(IV)-L-cysteine methyl ester complex (VC-IV) against CDDP-induced myelosuppression and genotoxic damage in the bone marrow cells of Swiss albino mice. CDDP was administered intraperitoneally (5 mg/kg b.w.) and VC-IV was administered orally (1 mg/kg b.w.) in concomitant and 7 d pretreatment schedule. Treatment with VC-IV in CDDP-treated mice significantly (p < 0.01) enhanced bone marrow cell proliferation and inhibited cell death in the bone marrow niche indicating improvement of CDDP-induced myelotoxicity. The organovanadium compound also significantly (p < 0.01) reduced the percentage of chromosomal aberrations, the frequency of micronuclei formation, and the extent of DNA damage. The observed chemoprotective effect of VC-IV was attributed to its anti-oxidant efficacy which significantly (p < 0.01) attenuated CDDP-induced generation of free radicals, and restored (p < 0.01) the levels of oxidized and reduced glutathione. Hence, VC-IV may serve as a promising candidate for future development to decrease the deleterious effects of CDDP in the bone marrow cells of cancer patients and associated secondary complications.

The effect of particle size on the genotoxicity of gold nanoparticles.

Despite the increasing biomedical applications of gold nanoparticles (AuNPs), their toxicological effects need to be thoroughly understood. In the present study, the genotoxic potential of commercially available AuNPs with varying size (5, 20, and 50 nm) were assessed using a battery of in vitro and in vivo genotoxicity assays. In the comet assay, 20 and 50 nm AuNPs did not induce obvious DNA damage in HepG2 cells at the tested concentrations, whereas 5 nm NPs induced a dose-dependent increment in DNA damage after 24-h exposure. Furthermore, 5 nm AuNPs induced cell cycle arrest in G1 phase in response to DNA damage, and promoted the production of reactive oxygen species (ROS). In the chromosomal aberration test, AuNPs exposure did not increase in the frequency of chromosomal aberrations in Chinese hamster lung (CHL) cells. In the standard in vivo micronucleus test, no obvious increase in the frequency of micronucleus formation was found in mice after 4 day exposure of AuNPs. However, when the exposure period was extended to 14 days, 5 nm AuNPs presented significant clastogenic damage, with a dose-dependent increase of micronuclei frequencies. This finding suggests that particle size plays an important role in determining the genotoxicity of AuNPs. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 710-719, 2017.

Protective Effect of Curcumin Against Imidacloprid-Induced Genotoxicity in Rats

Aim : To study the effect of curcumin in imidacloprid (IMI)-induced genotoxicity by comet and micronucleus assays. Materials and Methods : Experiments were conducted on adult rats that were randomly divided into six groups of six rats as follows: Group 1: Sham; Group 2: Curcumin (50 mg/kg po); Group 3: IMI (20 mg/kg po); Group 4: IMI (40 mg/kg po); Group 5: IMI (20 mg/kg po) + curcumin (50 mg/kg po); and Group 6: IMI (40 mg/kg po) + curcumin (50 mg/kg po). Micronucleus and comet assays were used to study the DNA damage in all the groups. Results : There was a significant (P &lt; 0.05) increase in the comet tail length (μm) and the frequency of micronuclei formation in the Group 4 treated with 40 mg/kg IMI when compared to other groups. Curcumin treatment in Group 6 showed a significant (P &lt; 0.05) decrease in the comet tail length, and micronuclei as compared to Group 4 though the decrease was significantly (P &lt; 0.05) low as compared to other groups. Conclusions : This study revealed that IMI has a tendency to induce the genotoxicity on exposure to 40 mg/kg b.wt. Curcumin, due to its anti-oxidant and anti-mutagenic property, alleviated the genotoxicity induced by IMI.

Abstract 3031: Replication stress and DNA damage promote genomic instability in near-tetraploid colorectal cancer cells

Abstract Aneuploidy represents a hallmark of most solid tumours, and potentially has a causal role in carcinogenesis. Also, cancer cells exhibit high rates of chromosome missegregation, which leads to chromosomal instability (CIN). However, a large amount of tumours present near-triploid karyotypes likely to go through a tetraploid transient stage. Here, by using DLD-1 isogenic diploid and tetraploid cell lines generated by single cell FACS-sorting, we aimed at exploring how polyploidization affects cellular functions and whether tetraploid per se generates CIN in a genomically stable background. First, gene expression and subsequent gene set enrichment analysis revealed that genes involved in the machinery of the DNA synthesis and replication, such as MCM2 and RRM2, and genes involved in cell cycle, were significantly upregulated in near-tetraploid cells compared to their diploid counterparts. Functionally, polyploidy cells exhibited replication stress, as indicated by higher levels of pCHK1, RPA and 53BP1 foci, which resulted in increased DNA damage in S- and M-phase. In addition, tetraploid cells displayed impaired proliferative capabilities as a consequence of a cell cycle delay confirmed by BrdU pulse and flow cytometry. Furthermore, near-tetraploid clones showed a higher amount of intracellular karyotypic heterogeneity due to the higher frequency of micronuclei formation compared to their diploid counterparts. In fact, polyploid cells displayed an increased tendency of abnormal anaphase events, including lagging chromosomes and acentric fragments. Interestingly, these heterogeneous cellular populations showed strengthen migratory capabilities and preliminary experiments suggested an increment in tumor invasiveness too. Taken together, our data suggest that near-tetraploid cells systematically undergo replication stress, which can be responsible for the increased levels of genomic instability. Citation Format: Isabel Quintanilla, Darawalee Wangsa, Markus Brown, Amaia Ercilla, Greg Klus, Maria Vila, Juan José Lozano, Zoltan Szallsi, Neus Agell, Antoni Castells, Thomas Ried, Jordi Camps. Replication stress and DNA damage promote genomic instability in near-tetraploid colorectal cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3031. doi:10.1158/1538-7445.AM2015-3031

Genetic instability in plants associated with vehicular traffic and climatic variables

To characterize the effect of vehicular traffic on air quality, the micronuclei of Tradescantia pallida tetrads were counted. Young inflorescences of T. pallida (Rose) D.R. Hunt var. purpurea were collected in 2010, 2011, and 2012, from three sites subjected to different intensities of vehicular traffic. The sites were located in the municipality of Dourados, in the State of Mato Grosso do Sul, Brazil. A standardized methodology was used to analyze the Tradescantia micronuclei, in order to evaluate the mutagenic potential of the local air pollutants. Statistical analyses using the Pearson's linear correlation were employed to determine the relationship between relative humidity and temperature, and the average number of micronuclei. In this study, an increase in the average number of passing vehicles was correlated with an increase in the frequency of micronucleus formation. Climatic factors also influenced micronucleus formation, although vehicular traffic remained the most important factor. Thus, the Tradescantia micronuclei assay may be a useful method of assessing air quality.

Prevention of myelosuppression and genotoxicity induced by cisplatin in murine bone marrow cells: effect of an organovanadium compound vanadium(III)-l-cysteine.

Cisplatin (CDDP) is one of the first-line anticancer drugs indicated for use against various form of human malignancies; but, the therapeutic outcome of CDDP chemotherapy is limited due to the development of myelosuppression and genotoxicity which may lead to secondary cancer. Induction of oxidative stress in normal host cells is thought to be responsible for these adverse effects. Therefore, in search of a potential chemoprotectant, an oraganovanadium compound, viz., vanadium(III)-l-cysteine (VC-III) was evaluated against CDDP-induced clastogenicity and cytotoxicity in bone marrow cells of Swiss albino mice. CDDP was administered intraperitoneally (5mg/kg body weight [b.w.]) and VC-III was given by oral gavage (1mg/kg b.w.) in concomitant and pretreatment schedule. The results showed that VC-III administration significantly (P < 0.001) enhanced cell proliferation and inhibited apoptosis in the bone marrow niche indicating recovery of CDDP-induced myelosuppression. VC-III also significantly (P < 0.001) decreased the percentage of chromosomal aberrations, the frequency of micronuclei formation and the extent of DNA damage. The observed antigenotoxic and cytoprotective effect of VC-III was attributed to its attenuation of free radicals status and restoration of oxidised and reduced glutathione levels. These results suggest that VC-III is a potential candidate for future development as a chemoprotective agent against chemotherapy-associated primary and secondary complications.

A role for reactive oxygen species in the resolution of persistent genomic instability after exposure to radiation

Reactive oxygen species (ROS) are involved in amplifying the radiation-induced damage to macromolecules as well as in downstream early responses leading to DNA repair or cell death and in delayed responses leading to tissue damage and/or tumorigenesis. We found in immortalized normal human bronchial epithelial cells (HBEC-3KT) following exposure to 1 Gy low (X-rays) or high (Fe ions) LET radiation that ROS levels persist elevated for up to eight population doublings (2 weeks) in the progeny of cells that repaired the radiation-induced damage and survived. To evaluate the function of persistently elevated ROS levels, we interrogated cells over the same period for genomic instability, proliferation and senescence biomarkers and evaluated whether ROS are functionally related to any of these phenotypes. We found that increased ROS production overlapped temporally with the persistence of reporters for genomic instability, increased micronucleus frequency and the presence of γH2AX-53BP1 positive ionizing radiation-induced foci. Although low LET radiation at high doses and high LET radiation induced a senescence-like phenotype dependent on ATM and p38 MAPK activity, this pathway was not a causative factor in ROS generation and did not affect cell proliferation rates at day 7 post-irradiation. On the contrary, when this pathway was inhibited, ROS levels increased further and micronucleus formation frequency was reduced. Because these results suggest a positive effect for increased ROS levels in the resolution of persistent micronucleus formation, their role was further confirmed by exposure to exogenous hydrogen peroxide, which resulted in a reduced micronucleus frequency. These results implicate ROS as an effector in the resolution of genomic instability and suggest that the senescence phenotype induced by high LET radiation or high low LET radiation dose promotes genomic instability by interfering with the cellular mechanism regulating ROS levels or the DNA repair machinery.

Validation of DNA Damage Progression with Days after Single Exposure of Sublethal Dosage of Electron Beam Radiation

Article history: The aim of this study was to asses the DNA damage progression with days after single exposure of 6Gy electron beam radiation (EBR). Molecular DNA damage in lymphocytes of mice was assessed using alkaline comet assay and in bone marrow cells by micronucleus assay. In comet assay %DNA in tail, Comet length, Olive Tail Moment (OTM) served as a measure of DNA damage and in micronucleus test, frequency of micronucleus formation in bone marrow cells was measured to evaluate the DNA damage. The experiment was performed by taking 24 healthy Swiss albino mice with body weight 30+5g. The animals were grouped into four. Group1 served as control, Group 2, 3 and 4 were irradiated by 6Gy EBR. Animals of Group 2, 3 and 4 were sacrificed on 5 th , 10 th and 15 th day post irradiation respectively. The comet assay procedure was carried out for all these groups by using lymphocytes separated from EDTA treated blood. The micronucleus test was performed in bone marrow cells (PCE-Poly chromatic erythrocytes, NCE-normochromatic erythrocytes). The slides prepared for this were scored for the measure of DNA damage. The results showed all comet parameter were significantly (P<0.05) affected by prolonging the post irradiation days from zero (control) to 5, 10 and 15. There was an alteration found in the PCE/PCE+NCE ratio in irradiated mice. A linear increase in the micronucleus formation was observed in post irradiated days. These results conclude a progression in DNA damage with days, post irradiation.

Assessing the Anti-mutagenic Potentials of Sage Salvia officinalis L. Leaf Aqueous Extract in Cultured Blood Cells of Acute Lymphocytic Leukaemia Patients Using the Micronucleus Formation Assay

three concentrations (250, 500, 1000) µg/ml of sage Salvia officinalis L. leaf aqueous extract were assessed for their anti-mutagenic potentials in cultured blood cells of 10 acute lymphocytic leukaemia (ALL) patients and a similar number of apparently healthy controls. The parameter of assessment was micronucleus (MN) formation, which was either spontaneous or induced by a treatment with the mutagen cytostar 125 µg/ml, and accordingly, eight cultures were set-up. Blood cells in culture I was negative control (untreated cells), while in culture II, the cells were treated with the mutagen cytostar (cytarabine) at a concentration 125 µg/ml. Cultures III, IV and V were treated with the three concentrations of sage extract respectively, while in cultures VI, VII, VIII, interactions between the extract (the three concentrations respectively) and cytosar were carried out. The results demonstrated that ALL patients showed a significant increased frequency of MN formation in the eight cultures as compared to the corresponding cultures in healthy controls. With respect to the spontaneous formation of MN formation (cultures III, IV, V), the three concentrations of sage extract demonstrated a gradual significant reduction of MN frequency in patients (0.0144, 0.0098, 0.0062) MN/cell, respectively and control subjects (0.0104, 0.0076, 0.0038) MN/cell, respectively. In the induction cultures (VI, VII, VIII), a similar reduction was observed and the dose 1000 µg/ml exerted the highest reduction in both patients and controls (0.0108, 0.0086) MN/cell, respectively. These results demonstrate the anti-mutagenic importance of sage leaf aqueous extract.

Implementation of the Three Rs in the Human Hazard Assessment of Brazilian Medicinal Plants: An Evaluation of the Cytotoxic and Genotoxic Potentials of Dipteryx alata Vogel

In Brazil, medicinal plants are widely used by the indigenous people, which leads to a constant requirement for toxicity tests to be performed on the plant extracts. Although the current Brazilian Directive 90/2004 on the preclinical toxicity testing of phytotherapeutics recommends only in vivo tests, some Brazilian researchers would like to change this situation by implementing the Three Rs in the toxicological testing of medicinal plants. The present study evaluated the cytotoxic and genotoxic potentials of bark extracts from Dipteryx alata Vogel, a medicinal plant of the Brazilian cerrado, by using CHO-K1 (Chinese hamster ovary) cells. An IC50 value was obtained, which corresponded to 0.16mg/ml of plant extract, and from this the equivalent LD50 was determined as 705mg/kg. In order to determine the genotoxic potential of the sample, the frequency of micronucleus formation was assessed. CHO-K1 cells were exposed, during targeted mitosis, to different concentrations of plant extract and cytochalasin B, in the presence and absence of an appropriate metabolic activation system (an S9 mix). The results obtained indicated that it might be possible to implement the Three Rs in assessing the potential human hazard of medicinal plants. The publication of such data can increase awareness of the Three Rs by showing how to optimise the management of animal use, if in vivo toxicological experiments are required.

Abstract 2480: Low dose radiation- and thiol-induced effects on micronuclei formation following fractionated irradiation

Abstract Exposure of cells to low doses of ionizing radiation (≤ 10 cGy) or amifostine induces pro-survival pathways that result in protection against the damaging effects of high dose ionizing radiation exposure. We have identified one such signaling pathway whose molecular mechanism involves the elevation of active manganese superoxide dismutase (SOD2) levels in the cell. SOD2 is a mitochondrial matrix protein that serves as the primary mitochondrial defense against superoxide formation. Treatment of RKO36 human colon carcinoma cells with 40 μM WR1065 or 10 cGy x-rays causes a significant increase in SOD2 activity that peaks 24 h after exposure. This increase in SOD2 activity is completely eliminated in cells subsequently exposed to 2 Gy. Following the dismutation of O2−, SOD2 is rapidly converted back into active enzyme in the case where enzyme and substrate concentrations are approximately equal. However, when substrate concentration is much greater than enzyme concentration, as is the case following exposure to 2 Gy radiation, the conversion from inactive to active enzyme occurs with much slower kinetics, taking minutes rather than seconds to occur. The protective effect of the thiol- and radiation-induced elevation in active SOD2 was examined using the endpoint of micronuclei formation as a measure of chromosomal damage. Exposure of RKO36 cells to 4 Gy results in a significant increase in the mean number of micronuclei observed. The frequency of micronuclei formation is significantly reduced when cells are treated with 40 μM WR1065 or exposed to 10 cGy 24 h prior to being irradiated with 4 Gy. No protective response is observed in cells exposed to 40 μM WR1065 30 min, 1 h or 3 h prior to two 2 Gy fractionated radiation doses separated by 24 h, or in cells exposed to 10 cGy 30 min, 3 h or 6 h before the first of two 2 Gy fractions. Treatment of cells with 40 μM WR1065 30 min, 1 h or 3 h after the first of the two 2 Gy fractions, or with 10 cGy 30 min, 3 h or 6 h after the first 2 Gy fraction also did not protect cells against the formation of micronuclei relative to cells exposed to the 2 Gy fractions alone. This work was supported by NIH/NCI grant R01 CA132998 and DOE Low Dose Program/Project Grant DE-SC0001271 (D.J.G). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2480. doi:10.1158/1538-7445.AM2011-2480

Genotoxicity Evaluation of Nanomaterials: DNA Damage, Micronuclei, and 8-Hydroxy-2-deoxyguanosine Induced by Magnetic Doped CdSe Quantum Dots in Male Mice

Quantum dots (QDs) are a novel class of inorganic fluorophores which are gaining widespread recognition as a result of their exceptional photophysical properties and their applications as a biomarker and in molecular biomedical imaging. The aim of this study was to evaluate the in vivo genotoxicity in mice exposed to CdSe quantum dots of average size 5.0 ± 0.2 nm and CdSe doped with 1% cobalt ions of similar size. The quantum dots are surface modified using mercaptoacetic acid (MAA) in order to be biocompatible and water-soluble. The MAA-QDs were given to the mice orally at doses of 500, 1000, and 2000 mg/kg by weight of MAA-QDs. Bone marrow and liver samples were collected after two and seven days of treatment. The results indicated that after two days of treatment, the high dose of doped MAA-QDs was significantly able to induce DNA damage, formation of micronuclei (MNs), and generation of DNA adduct (8-hydroxy-2-deoxyguanosine, 8-OHdG). However, increasing DNA damage and the frequency of MNs formation as well as the generation of DNA adducts were observed with both the undoped MAA-QDs (2000 mg/kg) and doped MAA-QDs (1000 and 2000 mg/kg) after seven days of treatment. The results of our study indicate that exposure to high doses of pure MAA-QDs or MAA-QDs doped with cobalt has the potential to cause indirect in vivo genetic damage, which may be attributed to free radical-induced oxidative stress in mice.

Curcumin protects against cypermethrin-induced genotoxicity in rats

Cypermethrin is a synthetic pyrethroid insecticide used worldwide in agriculture, home pest control, protection of foodstuff and disease vector control. The aim of the present study was to investigate the protective effect of curcumin on cypermethrin-induced genotoxicity in rats. Administration of cypermethrin (25 mg/kg, p.o.) for 28 days resulted in significant increase in the frequency of micronuclei formation in bone marrow cells and DNA damage in blood cells. Curcumin (100 mg/kg, p.o.) administration caused significant reduction in micronuclei formation and, marked reduction in DNA damage. The present study revealed that presence of curcumin could diminish cypermethrin-induced genotoxicity in rats.