Investigation of genotoxic and antigenotoxic activities of chlorophylls and chlorophyllin in cultured V79 cells

Chlorophyll and its derivatives are examples of plant compounds (purified and/or extracted) which appear to protect DNA from damage caused by chemical or physical agents, although some studies have identified clastogenic activity of these compounds. This study was carried out to assess the genotoxic activity of chlorophyll-a (Chl-a), -b (Chl-b) and chlorophyllin (Chl) and their antigenotoxic activity against the DNA damage induced by methyl methanesulphonate (MMS) under conditions of simultaneous, pre-, post-treatment, and simultaneous treatment after pre-incubation of the chemical with MMS. The micronucleus (MN) test was used in binucleated cells (induced by cytochalasin-B) of a mammalian cell line (V79). The three concentrations of Chl-a, Chl-b or Chl (0.1375, 0.275, 0.55microM) were not genotoxic and the genotoxic action of MMS (400microM) decreased (74-117%) under all treatment conditions. The results showed that there was no significant difference among the treatment types, the concentration or the nature of chlorophyll used. The data obtained suggest that Chl-a, Chl-b and Chl when associated with the DNA damaging agent, MMS, may protect the DNA by desgenotoxic action and/or by bio-antigenotoxic mechanisms, with the similar efficiency.     

Coffee-mediated protective effects against directly acting genotoxins and gamma-radiation in mouse lymphoma cells

The cytokinesis-block micronucleus test was performed using L5178Y mouse lymphoma cells to ascertain whether or not standard (caffeinated) instant coffee, the commonly consumed polyphenolic beverage with antioxidant activity can protect against chromosomal damage induced by the directly acting agents N-methyl-N-nitro-N-nitrosoguanidine (MNNG), mitomycin C (MMC), methyl methanesulfonate (MMS) and gamma radiation. Our results demonstrated significant reductions in the in vitro genotoxic effects of MNNG, MMC, and MMS following co-treatment of mouse lymphoma cells with standard instant coffee. Subsequently, the comet assay was carried out to assess the effect of coffee co-treatment on the level of DNA damage induced by MMS in mouse lymphoma cells. The results demonstrated a significant reduction in MMS-induced DNA damage following co-treatment with standard instant coffee. Protective effects were observed in mouse lymphoma cells which were treated with coffee immediately after exposure to gamma radiation (1 and 2 Gy). Another experiment showed protection when the mammalian cells were irradiated (0.5 and 1 Gy) midway (at 2 h) during a 4 h coffee treatment. However, the protective effect against the lower dose (0.5 Gy) was not significant. In addition we assessed the modulatory effect of coffee on MNNG-induced apoptotic frequency by flow cytometry. The results revealed only a minor influence of coffee on the frequency of apoptotic cells induced by the test compounds, rendering an increase in sensitivity for apoptosis as a reason for the reduced genomic damage an unlikely or at least incomplete explanation.     

Anti-clastogenic activity of two structurally related pterocarpans purified from Bituminaria bituminosa in cultured human lymphocytes

Plant-derived isoflavones are currently receiving much attention because of their phyto-estrogenic, antioxidant, anti-mutagenic, and anti-tumor activities. In this study we have evaluated the clastogenic and anti-clastogenic activities in human lymphocytes of two structurally related pterocarpans, iso-flavonoid derivatives, termed erybraedin C and bitucarpin A, recently purified from Bituminaria bituminosa and chemically characterized. Mitomycin C (MMC) and the radio-mimetic bleomycin (BL) were used as reference clastogens. The end point studied was micronucleus formation. The results obtained in this study indicate that erybraedin C and bitucarpin A, when assayed alone, do not affect either the mitotic index or the cell-proliferation index of human lymphocytes. Interestingly, both compounds appear to be non-clastogenic in the range of concentrations used. In contrast, both substances seem to affect significantly the clastogenic effects induced by BL and MMC. A 1-h pre-exposition of the cell culture to erybraedin C was necessary to display its anti-clastogenic potential against BL, whereas bitucarpin A was inactive in this respect, with a structure-activity relationship. In contrast, the clastogenic activity of MMC was significantly reduced by both erybraedin C and bitucarpin A, using either a pre-incubation schedule or simultaneous treatment. These results suggest that the protective effects displayed by the two anti-clastogenic compounds against MMC could be due to the induction or inhibition of cellular reductive metabolic enzymes.     

Detection of aneuploidy in male germ cells of mice by means of a meiotic micronucleus assay.

The possibility of using a micronucleus (MN) assay in mouse germ cells for the identification of aneuploidogenic agents was evaluated by comparing the pattern of effects induced by 4 chemicals with different mechanisms of action (adriamycin, ADM; mitomycin C, MMC; chloral hydrate, CH; ethylenedinitrilotetraacetic acid, EDTA). The results obtained after treatment of spermatocytes at the premeiotic S-phase (preleptotene) indicated that only clastogenic agents (ADM and MMC) were able to induce MN at this cell stage. Previous data obtained with the same compounds demonstrated by contrast that the micronucleus spermatid assay may detect both clastogenic and aneuploidogenic effects after treatment of diakinesis/MI/MII cells. Analysis of MN size distributions, in the present and previous spermatid samples, revealed that the clastogens ADM and MMC produced relatively more small MN than CH and EDTA. These data are in agreement with the proposed mechanism of action of the chemicals tested.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. III. Induction of cell killing, chromosomal aberrations and sister-chromatid exchanges by bleomycin, mono- and bi-functional alkylating agents

Two X-ray-sensitive mutants of CHO-K1 cells, xrs 5 and xrs 6, were characterised with regard to their responses to genotoxic chemicals, namely bleomycin, MMS, EMS, MMC and DEB for induction of cell killing, chromosomal aberrations and SCEs at different stages of the cell cycle. In addition, induction of mutations at the HPRT and Na+/K+ ATPase (Oua) loci was evaluated after treatment with X-rays and MMS. Xrs 5 and xrs 6 cells were more sensitive than wild-type CHO-K1 to the cell killing effect of bleomycin (3 and 13 times respectively) and for induction of chromosomal aberrations (3 and 4.5 times). In these mutants a higher sensitivity for induction of chromosomal aberrations to MMS, EMS, MMC and DEB was observed (1.5-3.5 times). The mutants also showed increased sensitivity for cell killing effects of mono- and bi-functional alkylating agents (1.7-2.5 times). The high cell killing effect of X-rays in these mutants was accompanied by a slight increase in the frequency of HPRT mutation. The xrs mutants were also more sensitive to MMS for the increased frequency of TGr and Ouar mutants when compared to wild-type CHO-K1 cells. Though bleomycin is known to be a poor inducer of SCEs, an increase in the frequency of SCEs in xrs 6 cells (doubling at 1.2 micrograms/ml) was found in comparison to no significant increase in xrs 5 or CHO-K1 cells. The induced frequency of SCEs in all cell types increased in a similar way after the treatment with mono- or bi-functional alkylating agents. MMS treatment of G2-phase cells yielded a higher frequency of chromatid breaks in the mutants in a dose-dependent manner compared to no effect in wild-type CHO-K1 cells. Treatment of synchronised mutant cells at G1 stage with bleomycin resulted in both chromosome- and chromatid-type aberrations (similar to the response to X-ray treatment) in contrast to the induction of only chromosome-type aberrations in wild-type CHO-K1 cells. The frequency of chromosomal aberrations chromosome and chromatid types) also increased with MMC treatment in G1 cells of xrs mutants. DEB treatment of G1 cells induced mainly chromatid-type aberrations in all cell types. The possible reasons for the increased sensitivity of xrs mutants to the chemical mutagens studied are discussed and the results are compared to cells derived from radiosensitive ataxia telangiectasia patients.     

Detection of aneuploidy in male germ cells of mice by means of a meiotic micronucleus assay

The possibility of using a micronucleus (MN) assay in mouse germ cells for the identification of aneuploidogenic agents was evaluated by comparing the pattern of effects induced by 4 chemicals with different mechanisms of action (adriamycin, ADM; mitomycin C, MMC; chloral hydrate, CH; ethylenedinirrilotetraacetic acid, EDTA). The results obtained after treatment of spermatocytes at the premeiotic S-phase (preleptotene) indicated that only clastogenic agents (ADM and MMC) were able to induce MN at this cell stage. Previous data obtained with the saine compounds demonstrated by contrast that the micronucleus spermatid assay may detect both clastogenic and aneuploidogenic effects after treatment of diakinesis/MI/MII cells. Analysis of MN size distributions, in the present and previous spermatid samples, revealed that the clastogens ADM and MMC produced relatively more small MN than CH and EDTA. These data are in agreement with the proposed mechanism of action of the chemicals tested.     

Anticlastogenicity of chlorophyllin in the different cell cycle phases in cultured mammalian cells

Chlorophyllin (Chln), a sodium-copper salt derivative of chlorophyll, like chlorophyll-a and -b found in green plants, has been studied for its protective action against the carcinogenic effects of various physical and chemical agents and in relation to the mutagenic and clastogenic activities of genotoxic agents. The aim of the present study was to evaluate chlorophyllin in different phases of the cell cycle for clastogenicity and anticlastogenicity, the latter in reversing DNA damage induced by ethyl methane sulfonate (EMS). The test for chromosomal aberrations was performed in cultured mammalian cells (CHO-K1). The three Chln concentrations tested (6.25, 12.5 and 25 microg/ml) were not clastogenic and damage induced by EMS (1240 microg/ml) was reduced in cells treated with Chln as well during S (25-48%) and G2/S (70-80%). The results demonstrate a greater protective effectiveness of Chln against EMS during G2/S.     

Analysis of in vitro chemoprevention of genotoxic damage by phytochemicals, as single agents or as combinations

Cancer chemoprevention with low-dose combinations of bioactive phytochemicals instead of single agents has been suggested to induce less toxicity and improve efficacy. In this study, we selected four plant food-based phytochemicals, viz. chlorogenic acid (CLA), pelargonidin (PEL), resveratrol (RES) and epigallocatechin gallate (EGCG) to evaluate the in vitro chemoprevention of genotoxic damage in HL-60 cells. These agents were tested either individually or as a combination at two concentrations (with a 10-fold difference) against the genotoxins mitomycin C (MMC), diepoxybutane (DEB) and patulin (PAT). Our preliminary ferric reducing antioxidant power (FRAP) assay demonstrated additive effects when PEL, CLA, RES and EGCG were combined. Results of the cytokinesis-block micronucleus test showed significant protection against genotoxic damage induced by PAT, DEB and MMC when CLA, PEL, RES and EGCG were tested individually. This protective effect of the phytochemicals was not concentration-related. Both low- and high-concentration combinations of CLA, PEL, RES and EGCG showed significant reducing effects on the frequencies of micronuclei induced by PAT, DEB and MMC. However, the micronucleus test did not provide indications of additive or synergistic effects with this combination of phytochemicals. In conclusion, the chemo-preventive effects of PEL, CLA, RES and EGCG against genotoxic damage induced by MMC, DEB and PAT are indicative of a 'saturation effect' when higher concentrations and combinations of these phytochemicals are used.     

Antigenotoxic effect of lipoic acid against mitomycin-C in human lymphocyte cultures

Antitumor agents are used in therapy against many forms of human cancer. One of these is mitomycin-C (MMC). As with many agents, it can interact with biological molecules and can induce genetic hazards in non-tumor cells. One of the possible approaches to protect DNA from this damage is to supply antioxidants that can remove free radicals produced by antitumor agents. Lipoic acid (LA) is known as one of the most powerful antioxidants. The aim of this study was to investigate antigenotoxic effects of LA against MMC induced chromosomal aberrations (CA), sister chromatid exchanges (SCE) and micronucleus (MN) formation in human lymphocytes. Lymphocytes were treated with 0.2 μg MMC/heparinized mL for 48 h. Three different concentrations (0.5, 1, 2 μg/mL) of LA were used together with MMC in three different applications; 1 h pre-treatment, simultaneous treatment and 1 h post-treatment. A negative, a positive and a solvent control were also included. In all the cultures treated with MMC + LA, the frequency of abnormal cells and CA/cell significantly decreased compared to MMC. Statistically significant reduction was also observed in SCE/cell and MN frequencies in all treatments. These results demonstrated anticlastogenic and antimutagenic effects of LA against MMC induced genotoxicity. LA showed the most efficient effect during 1 h pretreatment. On the other hand, MMC + LA treatments induced significant reduction in mitotic index than that of MMC treatment alone. These results are encouraging that LA can be a possible chemopreventive agent in tumorigenesis in both cancer patients and in health care persons handling anti-cancer drugs.     

The micronucleus test with mouse peripheral blood on N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C

The micronucleus test using mouse peripheral blood was conducted with N-methyl-N'-nitro-N-nitro-soguanidine (MNNG) and mitomycin C (MMC) as part of the 5th collaborative study supported by the Environmental Mutagen Society of Japan (CSGMT/MMS · JEMS).Male CD-1 mice were intraperitoneally injected once with 12.5–100 mg/kg of MMC. Peripheral blood was drawn at different intervals after treatment, placed on slides previously coated with acridine orange and the numbers of reticulocytes with micronuclei (MNRETs) were scored.The experiments indicated that the maximum effect of both MNNG and MMC was found about 48 h after treatment, and that the micronucleus test using peripheral blood is useful for the screening of chemicals throughout the experimental period in a single animal.     

The micronucleus test with mouse peripheral blood on N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C.

The micronucleus test using mouse peripheral blood was conducted with N-methyl-N'-nitro-N-nitro-soguanidine (MNNG) and mitomycin C (MMC) as part of the 5th collaborative study supported by the Environmental Mutagen Society of Japan (CSGMT/MMS.JEMS). Male CD-1 mice were intraperitoneally injected once with 12.5-100 mg/kg of MMC. Peripheral blood was drawn at different intervals after treatment, placed on slides previously coated with acridine orange and the numbers of reticulocytes with micronuclei (MNRETs) were scored. The experiments indicated that the maximum effect of both MNNG and MMC was found about 48 h after treatment, and that the micronucleus test using peripheral blood is useful for the screening of chemicals throughout the experimental period in a single animal.     

Rates of micronuclei induction in different mouse strains

It has previously been shown that the inbred mouse strain MS/Ae was more sensitive in the micronucleus test to several mutagenic agents than outbred mice. To elucidate the possible influence of inbreeding, several inbred strains including MS/Ae, AKR, BALB/c, C57 BR were compared to the two OF1 and NMRI outbred strains. The 3 mutagenic agents MNNG, MMC and MMS all induced a significantly higher number of micronuclei in the MS/Ae strain than in any of the other mouse strains. AKR was especially resistant to the alkylating agents MMS and MNNG. Hence, except for the MS/Ae mouse strain, no inbred strain showed a systematically higher sensitivity than the outbred strains for all of the 3 mutagenic agents used.     

Short-term tests for transplacentally active carcinogens: I. Micronucleus formation in fetal and maternal mouse erythroblasts

A cell-kinetic model for the application of the micronucleus test to polychromatic erythrocytes in mouse fetal liver, fetal blood, and maternal bone marrow after exposure to clastogenic agents is described. The time of expression and dose-response relationships obtained with γ-radiation, methyl methanesulphonate, procarbazine, mitomycin C and benzo[a]pyrene are analysed in terms of this model. The numbers of target cells damaged per unit dose has been calculated and the dose equivalents obtained. Maternal and fetal cells show similar sensitivity to γ-radiation, but fetal cells are markedly more sensitive to MMS and procarbazine. This probably due to differences in tissue distribution and metabolism. Maternal and fetal erythroid tissues can show linear and exponential dose-response relationships, which may not coincide (e.g. with MMS). It is concluded that risks from fetal exposure to genotoxic agents cannot be reliably predicted from in vivo tests restricted to adult animals. However, the micronucleus technique appled to fetal erythroid cells proveds a rapid and reliable short-term test, appropriate to minimising risks of genome damage during prenatal development.     

Acetylsalicylic acid exhibits anticlastogenic effects on cultured human lymphocytes exposed to doxorubicin

Acetylsalicylic acid (ASA) is a non-steroidal anti-inflammatory drug (NSAID) with many pharmacological properties, such as anti-inflammatory, antipyretic and analgesic. Many studies have suggested the possible efficiency of ASA and other NSAIDs in preventing cancer. ASA could also have antimutagenic and antioxidant properties. The aim of this study was to investigate the possible clastogenic and anticlastogenic effects of different concentrations of ASA on doxorubicin-induced chromosomal aberrations in human lymphocytes. Human blood samples were obtained from six healthy, non-smoking volunteers; and the chromosomal aberration assay was carried out using conventional techniques. The parameters analyzed were mitotic index, total number of chromosomal aberrations and percentage of aberrant metaphases. The concentrations of ASA (25, 50 or 100 microg/mL) tested in combination with DXR (0.2 microg/mL) were established on the basis of the results of the mitotic index. The treatment with ASA alone was neither cytotoxic nor clastogenic (p>0.01). In lymphocyte cultures treated with different combinations of ASA and DXR, a significant decrease in the total number of chromosome aberrations was observed compared with DXR alone (p<0.01). This protective effect of ASA on DXR-induced chromosomal damage was obtained for all combinations, and it was most evident when ASA was at 25.0 microg/mL. In our experiments, ASA may have acted as an antioxidant and inhibited the chromosomal damage induced by the free radicals generated by DXR. The identification of compounds that could counteract the free radicals produced by doxorubicin could be of possible benefits against the potential harmful effects of anthracyclines. The results of this study show that there is a relevant need for more investigations in order to elucidate the mechanisms underlying the anticlastogenic effect of ASA.     

Effects of folic acid on the antiproliferative efficiency of doxorubicin,camptothecin and methyl methanesulfonate in MCF-7 cells by mRNA endpoints

There is a lack of consensus on whether the role of folate in cancer cells is protective or harmful. The use of folates in combination with cancer-targeting therapeutic regimens requires detailed information to ensure their safe and proper use. Therefore, we evaluated the effects of folic acid (FA) in combination with the chemotherapeutic compounds doxorubicin (DXR), camptothecin (CPT) and methyl methanesulfonate (MMS) on the growth of MCF-7 cells. The data generated from the RTCA assays demonstrated that FA did not affect proliferation in MCF-7 cells treated with DXR and CPT; however, FA reduced the efficacy of MMS treatment. RTCA data also confirmed that DXR and CPT exert their cytotoxic effects in a time-dependent manner and that CPT induced a significantly greater decrease in MCF-7 cell proliferation compared with DXR. The MTT assay failed to detect a reduction in cell proliferation in cells treated with MMS. We quantified the mRNA expression levels of genes associated with cellular stress response, cell cycle and apoptosis pathways using RT-qPCR. The addition of FA to DXR or CPT promoted a similar shift in the gene expression profile of MCF-7 cells compared with cells treated with DXR or CPT without FA; however, this shift did not alter the bioactivity of these drugs. Rather, it indicated that these drugs promoted cell death by alternative mechanisms. In contrast, the addition of FA to MMS reduced the efficacy of the drug without changing the gene expression profile. None of the genes encoding folate receptors that were analyzed were differentially expressed in cells treated with or without FA. In conclusion, supplementation with 450 μM FA was not cytotoxic to MCF-7 cells. However, the addition of FA to anti-cancer drugs must be performed cautiously as the properties of FA might lead to a reduction in drug efficacy.     

Use of HepG2 cell line for direct or indirect mutagens screening: comparative investigation between comet and micronucleus assays

In the present study, DNA-damage and clastogenic or aneugenic effects of genotoxic compounds were examined in a metabolically competent human cell line (HepG2 cells) using the micronucleus and the comet assays. Compounds with various action mechanisms were tested: direct mutagens such as 4-nitroquinoline-N-oxide (4-NQO) and methyl methanesulfonate (MMS) and indirect mutagens requiring biotransformation to be active such as N-nitrosodimethylamine (NDMA), benzo[a]pyrene (B[a]P) and 2-acetylaminofluorene (2-AAF). The compounds were first tested for cytotoxicity by measuring their effects on RNA synthesis inhibition in HepG2 cells. 4-NQO, B[a]P and 2-AAF were the most potent compounds; their IC(50) values were, respectively, 1.9 micro M (4h contact), 3.4 and 112 micro M after 20 h. MMS was mildly cytotoxic (IC(50)=0.9 mM) and NDMA had a weak effect (IC(50)=110 mM) after 4h contact. In the micronucleus and comet assays, concentrations required to obtain a significant genotoxic effect in HepG2 cells varied over a broad range, NDMA being active only at very high concentrations. To compare the sensitivity of the two assays, we measured the so-called FIC(2)-the concentration necessary to induce a 2-fold increase of the measured genotoxicity parameter. The data show that genotoxic effects were consistently observed at lower concentrations in the micronucleus test, except in the case of MMS. The measured FIC(2) values were 0.12 micro M (4-NQO), 0.17 micro M (2-AAF), 0.26 micro M (B[a]P) and 6.4mM (NDMA). MMS had such a weak effect in the HepG2 cells that we could not calculate its FIC(2) value. In the comet assay, FIC(2) values were observed, respectively, at 1.48 micro M (4-NQO), 3.67 micro M (B[a]P), 13.42 micro M (MMS) and 27 mM (NDMA). 2-AAF failed to induce DNA-damage in this assay. The present study shows that HepG2 cells could be a suitable tool for assessing the genotoxicity of direct and indirect mutagens and for establishing the lowest genotoxic concentration.     

Micronucleus evaluation in peripheral blood reticulocytes of mice treated with procarbazine hydrochloride or mitomycin C.

The usefulness of the acridine orange (AO) supravital staining technique for the mouse peripheral blood reticulocyte micronucleus test was investigated independently by three laboratories using the known clastogens procarbazine hydrochloride (PCZ) and mitomycin C (MMC). In all three laboratories the highest frequencies of micronucleated peripheral blood reticulocytes were observed 48 h after treatment of mice with a single dose of either MMC or PCZ. The animals responded to both chemicals in a dose-dependent manner. Although similar qualitative results were observed, mean micronucleus frequencies induced by a particular dose of a given test chemical did vary quantitatively among the three laboratories. This was most probably due to the use of slightly different scoring criteria by each examiner. This aspect needs special attention. To minimize inter-laboratory variability, therefore, we recommend establishing unequivocal criteria to distinguish the subclass of reticulocytes. These should then be used consistently by all investigators using this method. The most striking advantages of the AO supravital staining technique were the ease of slide preparation, the ease with which reticulocytes and mature erythrocytes could be distinguished by the examiners, and the occurrence of numerous scorable reticulocytes in each microscopic field, which greatly speeded up the manual counting process. The disadvantages of the staining technique were the limited scoring time due to the rapid fading of the fluorescence stain, the degradation of the cells with time, and the frequent need to search for adequate scoring areas within a microscopic field. Based on the data of this study the authors conclude that the AO supravital staining technique is highly suitable for the micronucleus assay in erythrocytic cells of mouse peripheral blood. In addition, we consider the mouse peripheral blood reticulocyte micronucleus test to be a useful tool with which to investigate the clastogenic potential of chemicals in vivo. As pretreatment of mice with Aroclor 1254 markedly increased the effect of PCZ on micronucleus induction, we suggest that the inclusion of inducers of drug metabolizing enzymes in the micronucleus test would be useful for the detection of the clastogenic potential of promutagenic chemicals.     

Digoxin reduces the mutagenic effects of Mitomycin C in human and rodent cell lines

Digoxin is a drug widely used to treat heart failure and studies have demonstrated its potential as anticancer agent. In addition, digoxin presents the potential to interact with a series of other compounds used in medicine. The aim of the present study was to evaluate in vitro the cytotoxicity, genotoxicity and mutagenicity of digoxin and its potential to interact with the mutagen Mitomycin C (MMC). The cytotoxicity of digoxin was assessed by employing the MTT method and the comet assay was performed to assess the genotoxicity of this medicine in CHO-K1 and HeLa cell lines. Besides, the cytokinesis-block micronucleus assay was performed to assess the mutagenicity and the antimutagenicity of this drug. The Ames assay was also performed with TA98 and TA100 strains of S. typhimurium. Results showed that digoxin was cytotoxic, genotoxic and mutagenic for HeLa and CHO-K1 cell lines at concentrations many times higher than those observed in human therapeutic conditions. Nevertheless, an antimutagenic effect against the mutagen MMC was observed on both cell lines in concentrations near those used therapeutically in humans. This chemoprotective effect observed is an interesting finding that should be better explored regarding its impact in anticancer chemotherapy.     

Radiation protection of human lymphocyte chromosomes in vitro by orientin and vicenin.

Orientin (Ot) and Vicenin (Vc), two water-soluble flavonoids isolated from the leaves of Indian holy basil Ocimum sanctum have shown significant protection against radiation lethality and chromosomal aberrations in vivo. In the present study the protective effect of Ot and Vc against radiation induced chromosome damage in cultured human peripheral lymphocytes was determined by micronucleus test. In order to select the most effective drug concentration, fresh whole blood was exposed to 4Gy of cobalt-60 gamma-radiation with or without a 30 min pre-treatment with 6.25, 12.5, 15.0, 17.5 or 20 microM of Ot/Vc. Micronucleus (MN) assay was done by cytochalasin induced cytokinesis block method. Radiation significantly increased the MN frequency (16 times normal). Pre-treatment with either Ot or Vc at all concentrations significantly (P<0.05-0.001) reduced the MN count in a concentration dependent manner, with the optimum effect at 17.5 microM. Therefore, fresh blood samples were incubated with/without 17.5 microM Ot/Vc for 30 min and then exposed to 0.5-4Gy of gamma-radiation. Radiation increased the MN frequency linearly (r(2)=0.99) with dose. Pre-treatment with Ot or Vc significantly (P<0.01-0.001) reduced the MN counts to 51-67% of RT alone values, giving DMFs of 2.62 (Ot) and 2.48 (Vc). Both the compounds showed significant antioxidant activity in vitro at the above concentrations, which was significantly higher than that of DMSO at equimolar concentrations. Thus, the results demonstrate that both the flavonoids give significant protection to the human lymphocytes against the clastogenic effect of radiation at low, non-toxic concentrations. The radioprotection seems to be associated with their antioxidant activity. The clinical potential of these protectors in cancer therapy needs to be investigated.     

Detection of micronuclei after exposure to mitomycin C, cyclophosphamide and diethylnitrosamine by the in vivo micronucleus test in mouse splenocytes.

A micronucleus detection test using mouse splenocytes has been adapted from a method previously carried out using human lymphocytes. An ex vivo protocol was chosen: male C57B16 mice were treated with various compounds. Splenocytes were then isolated and placed in culture for 48 h and stimulated with concanavalin A and conditioned medium. The cytokinesis-block method reported by Fenech and Morley was used to detect and score micronuclei in the proliferating lymphocytes (3 micrograms/ml of cytochalasin B for 16 h). Three mutagenic clastogens, mitomycin C (MMC), a direct alkylating agent (0.4, 0.8 and 1.6 mg/kg), cyclophosphamide (CP), an indirect alkylating agent (25, 50 and 100 mg/kg) and diethylnitrosamine (DEN), an indirect alkylating agent with labile metabolites (25, 50 and 100 mg/kg), were tested at four sampling times (2, 4, 8 and 15 days). All three compounds were detected from 48 h after treatment. This method was indeed able to detect clastogenic compounds normally detected by the mouse bone marrow micronucleus test (MMC, CP) as well as a compound with labile metabolites which is not usually detected by this test (DEN). Maximum micronucleus induction was observed after 4 days for MMC, 2 days for CP and 15 days for DEN. This method thus appears to offer a potentially useful toxicological test for assessing in vivo clastogenicity.