Clastogenicity Of Cyclophosphamide Research Articles

Flow cytometric analysis of micronuclei in rat peripheral blood: An interlaboratory reproducibility study

In anticipation of proposed OECD guideline changes that may include increasing the number of reticulocytes scored for micronuclei, an inter-laboratory reproducibility study of the rat peripheral blood micronucleus assay was performed using flow cytometry. In this experiment, male Sprague–Dawley (SD) rats were treated with the model clastogen cyclophosphamide (CP: 5, 10 or 15mg/kg) by a single oral administration. As controls, rats were treated with physiological saline (solvent) in the same manner as for the model clastogen. Peripheral blood was collected from each rat 48h after the treatment. The blood samples were prepared at the Public Interest Incorporated Foundation, BioSafety Research Center (BSRC) in duplicate using the rat MicroFlowPLUS Kit. After fixation, one replicate set of samples was shipped to Litron Laboratories, and each sample was analyzed by flow cytometry at the two laboratories. In addition, the frequency of micronucleated reticulocytes (MNRETs) was determined at the BSRC by microscopic analysis using supravital acridine orange (AO) staining. The reproducibility of micronucleated reticulocyte frequencies analyzed by microscopy and flow cytometry showed good correlation (r2=0.84). The frequencies of micronucleated reticulocytes analyzed by flow cytometry at the two independent laboratories showed good concordance (r2=0.97). The data indicate that the flow cytometric micronucleus analysis method is a good alternative to manual microscopic analysis. Flow cytometry allows groups to readily score 5000 or even 20,000 RETs in a matter of minutes compared to manual analysis. This results in increased reliability of the assay by achieving better statistical power.

Flow cytometry peripheral blood micronucleus test in vivo: Determination of potential thresholds for aneuploidy induced by spindle poisons

Non-DNA binding genotoxins (e.g., aneugens), unlike DNA-binding genotoxins, are theoretically expected to show thresholded concentration-effect response curves. This is a major issue in genetic toxicology testing because the identification of thresholds in vivo can provide a safety margin for exposure to a particular compound. In the current study we measured micronucleus induction by flow cytometry to determine the dose-response curves for tubulin interacting agents, a specific class of aneugens. All experiments with aneugens, which include colchicine, vinblastine, vincristine, as well as the clastogen cyclophosphamide (CP) were performed in mice to avoid the splenic elimination of micronucleated reticulocytes, which has been described in rats. Flow cytometry analysis revealed a non-linear dose-dependent increase in micronuclei frequencies for all tested aneugens, and a linear dose response curve for the clastogen, CP. To determine whether micronucleus induction at higher doses was due to chromosome loss (aneuploidy) or chromosome breakage (clastogenicity), flow sorting of the micronucleated reticulocytes and fluorescent in situ hybridization (FISH) with a mouse pan centromeric probe were performed for vinblastine, vincristine, and colchicine. Statistical evaluation of the flow cytometry and FISH data was performed to determine the threshold levels for chromosome loss in vivo. The threshold concentrations for vinblastine, vincristine, and colchicine were found at 0.35, 0.017, and 0.49 mg kg(-1), respectively.

Evaluation of the micronucleus assay in bone marrow and peripheral blood of rats for the determination of cigarette mainstream-smoke activity

The mammalian in vivo micronucleus assay is widely used as part of the genotoxicity testing battery required during the development of new drugs. As such, the in vivo micronucleus assay has been used in a battery of assays for the assessment of cigarette ingredients or design modifications to help ensure that there is no increase in risk or any new risk introduced by these additions or modifications. The present series of studies was conducted to optimize and evaluate this assay for the assessment of the effects of mainstream smoke on the micronucleus frequency in the bone marrow and peripheral blood of rats. In a first experiment, the optimal conditions for performing the micronucleus assay in these tissues were determined. This was done by use of two compounds known for their micronucleus-inducing activity, i.e., the clastogen cyclophosphamide and the aneugen colchicine. In a second experiment, the effects of tube restraint on untreated control rats were investigated. In a third experiment, the optimal conditions were used to assess the clastogenic/aneugenic activity of cigarette smoke in Sprague-Dawley rats. The rat micronucleus assay in both bone marrow and peripheral blood is able to detect clastogenic and aneugenic activity. The flow cytometric determination of micronucleated cells in rat blood is at least as sensitive as determinations in bone marrow. No statistically significant differences were observed in micronucleus frequencies between rats with and without the additional stress of tube restraint; however, the cautious approach would be to use a fresh-air-exposed group (with tube restraint) as the negative control in inhalation experiments. Using the conditions identified as optimal in the above-mentioned experiments, the micronucleus assay was not able to detect effects induced by smoke from conventional cigarettes. Nevertheless, the micronucleus assay will remain a valuable tool as part of a testing battery used to investigate possible adverse effects related to product modifications.

Aquatic ecotoxicology approaches in Western Mexico

A series of bioindicator organisms for aquatic ecosystems are being maintained under laboratory conditions in order to analyze effects of pollution on aquatic wildlife and potential effects on human health. Growth kinetics of algae Pseudokirchneriella subcapitata was used to evaluate effects of the surfactant nonilphenol (NP). Brachionus calyciflorus was used to set up a model of endocrine disruption using the fungicide vinclozolin (Vc). We exposed salamanders from the genus Ambystoma sp., to different concentrations of both the aneugen colchicine (COL) and the clastogen cyclophosphamide (CP) and we determined the frequency of micronucleated cells (MNC) in their shed skin. The presence of spontaneous micronuclei in peripheral blood erythrocytes from 10 fish species in Lake “La Alberca,” Michoacan (Mexico), was evaluated as a possible biological indicator of genotoxic agents. Results confirm the sensivity of Pseudokirchneriella subcapitata to growth kinetics: the range of concentration of NP (20, 200 and 2000 μ g L− 1) shows an inverted U shape in its maximum growth rate; Dimethyl sulfoxide (DMSO) used as a positive control and to solvate NP induced an inverse stimulatory effect on growth rate in the range of concentrations analyzed (0.0023, 0.023 and 0.23% v v− 1). In the use of the rotifer Brachionus calyciflorus, the range of Vc from 0.185 mg L− 1 to 3 mg L− 1 clearly showed an inverted U shape characteristic of endocrine disruptions. We were able to use shed skin from Ambystoma sp., to measure MNC frequencies induced either by an aneugenic or a clastogenic compound. The MNC frequency was increased significantly by all doses of COL and CP, administered either as single or repeated exposures. The presence of MNC in the shed skin and the speed of sloughing lead us to propose that the sheds of Ambystoma sp., or other amphibians that slough their skin, as suitable alternative models for detecting genotoxic exposures relevant to aquatic environments. In the survey to determine potential biological fish indicators for genotoxic agents, the frequency of spontaneous micronucleated erythrocytes (MNE) found in the goodeid Xenotoca melanosoma (3.7 ± 1.6 MNC) and the cichlid Oreochromis aureus (2.0 ± 1.0 MNC) suggests that these species can be considered as potential biological indicators.

Induction of micronucleated cells in the shed skin of salamanders (Ambystoma sp.) treated with colchicine or cyclophosphamide

The micronucleus (MN) assay can be used to detect the genotoxic effects of chemical agents in virtually any cell that divides frequently. Salamanders (Ambystoma sp.) are amphibians that can be easily maintained and bred in the laboratory and spontaneously shed their skin every 2.5-4 days. In this present study, we have evaluated the usefulness of this shed skin for the MN assay. We exposed salamanders to different concentrations of both the aneugen colchicine (COL) and the clastogen cyclophosphamide (CP) and we determined the frequency of micronucleated cells (MNCs) in their sheds. Fragments of shed skin were placed on clean slides, fixed, stained, observed with a light microscope, and the number of MNCs was counted. The MNC frequency was increased significantly by all doses of COL and CP tested, administered either as single or repeated exposures. The presence of MNCs in the shed skin and the speed of sloughing lead us to propose that the sheds of Ambystoma sp., or other amphibians that slough their skin, are suitable alternative models for detecting genotoxic exposures relevant to aquatic environments.

A comparison between mouse and fish micronucleus test using cyclophosphamide, mitomycin C and various pesticides

A comparative analysis between mouse and fish erythrocyte micronuclei (MN) assays was carried out using cyclophosphamide, mitomycin C and various pesticides such as alliete, brestanid, decis 25 CE (deltamethrin), kelthane 480 CE (dicofol), roundup (glyphosate), imazapyr and thiram. The aim of this study was to evaluate the fish species Tilapia rendalli as a suitable organism for the detection of genotoxicants in water. The clastogens cyclophosphamide and mitomycin C induced MN in both test-systems. Insecticides: decis 25 CE increased T. rendalli MN frequencies at doses of 1.0 and 5.0 mg/kg, but not at the highest dose, and in mice there was no MN induction. Kelthane 480 CE also induced a significant MN frequency in T. rendalli, but not in mice. Fungicides: alliete and brestanid induced MN only in T. rendalli, while thiram was negative in both assays. Herbicides: imazapyr induced MN in T. rendalli at the maximum tolerated dose only, while roundup induced MN at three dosed levels. In mice both herbicides presented negative results. This study revealed that fish MN assay can be used as a genotoxicological test-system since some methodological particularities were observed.

Transplacental inhibitory effect of carrot juice on the clastogenicity of cyclophosphamide in mice

Genetic damage during the prenatal period can provoke important neoplastic alterations and other diseases in postnatal life. Beta-carotene (ßC) is considered to be one of the most important anticarcinogens in the diet and can protect mammalian cells against genotoxic events. As carrots are important dietary source of ßC, we decided to test the effect of fresh carrot juice (CaJ) on cyclophosphamide (CP)-induced genotoxicity in maternal and fetal erythropoietic tissues. The treatment with CaJ started on the 7th day of the pregnancy of BALB/c female mice. We observed, on the 16th gestational day, that this treatment did not modify the spontaneous frequency of micronucleated polychromatic erythrocytes (mPCE) in the bone marrow of the females nor in the livers of their fetuses. The mPCE frequency observed 24 h after an intraperitoneal injection of CP (40 mg/kg) on the 15th day was significantly lower in CaJ-pretreated pregnant female bone marrow and in the liver of their fetuses than those observed in the group treated with CP only. These results demonstrate the presence of natural anticlastogens in carrots.

Inhibition of clastogenic effect of cyclophosphamide and mitomycin C by Neem leaf-extract in mice

Azadirachta indica commonly known as ‘Neem’ is well known for its medicinal properties in the indigenous Indian system of medicine. Almost every part of the tree has some beneficial use. The anticlastogenic activity of ‘Neem’ against cyclophosphamide (CP) and mitomycin C (MMC) was studied in vivo in bone marrow cells of mice. Aqueous leaf-extracts of A. indica were injected intraperitoneally at doses 3, 6, 12 and 24 mg/kg body weight. Simultaneously, two known clastogens CP (10 mg/kg) and MMC (1.5 mg/kg) were administered individually to animals treated with 6 and 12 mg/kg of the leaf-extract. The end-points screened were chromosomal aberrations and damaged (aberrant) cells. Neem leaf-extract per se was found to be a weak clastogen; 6 and 12 mg/kg of the leaf-extract inhibited the clastogenicity of CP and MMC. The extent of inhibition was different for the two clastogens. An ANOVA test showed that the reduction in the frequency of chromosomal aberrations was significantly less when the leaf-extract was given in combination with CP. MMC co-administered with the leaf-extract showed a trend that was not statistically significant. The difference may be attributed to the degree of modulation of bioactivation of cytochrome P-450 enzymes, or the repair of damaged DNA or a difference in detoxification of the reactive species of the two genotoxicants. © 1998 John Wiley & Sons, Ltd.

Studies on the anticlastogenic effect of turmeric and curcumin on cyclophosphamide and mitomycin C In Vivo

Turmeric and its main constituent curcumin were assessed in vivo for their anticlastogenic potential. In one experimental set, Swiss albino male mice were given turmeric (8, 12 and 16 mg/kg body weight) or curcumin (2, 4 and 8 mg/kg body weight) as a single intraperitoneal injection. In another set, the mice were given 8 mg/kg body weight of turmeric or one of three concentrations of curcumin (2, 4 and 8 mg/kg body weight) as a dietary supplement by gavage for 7 consecutive days. 30 min after the last dose the mice were administered a single acute dose of two known clastogens, cyclophosphamide (CP) (20 mg/kg body weight) or mitomycin C (MMC) (1.5 mg/kg body weight). After 18 hr, chromosome preparations were made from bone marrow cells. The endpoints studied were chromosome aberrations and damaged cells. Clastogenicity of the chemicals was compared using turmeric- or curcuminprimed and non-primed animals. As single agents turmeric and curcumin were not clastogenic even after 7 days of priming. Turmeric/curcumin could not inhibit CP- or MMC-induced clastogenicity. Although curcumin is reported to be the active chemopreventive principle in turmeric effective against a number of potential carcinogens in several experimental systems, it was virtually ineffective against the clastogenicity of CP or MMC at the doses tested.

Phenethyl isotiocyanate modulates clastogenicity of mitomycin C and cyclophosphamide in vivo

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, is an effective chemopreventive agent against N-nitrosamine-induced carcinogenesis. We have investigated the extent to which PEITC modulates the clastogenicity of standard genotoxicants, mitomycin C and cyclophosphamide, using bone marrow cells of Swiss albino mice. PEITC, 1 μmol/kg body weight in corn oil was administered by gavage for 7 consecutive days to prime the animals. 24 h later, mice received a single dose of cyclophosphamide (10 or 20 mg/kg body weight)or mitomycin C (1 or 2 mg/kg body weight) intraperitoneally. Clastogenicity of the chemicals was compared using PEITC-primed and non-primed animals 24 h after clastogen treatment. As a single agent, PEITC is not clastogenic even after 7 days of priming. Oral priming with PEITC decreased the aberrations per cell values by 22–67% in all cases. PEITC could only alleviate the clastogenicity of I mg/kg body weight mitomycin C to near-control values ( p ≤ 0.05). Although PEITC is reported to be effective against N-nitrosamine-induced tumorigenesis by preventing metabolic activation and by blocking the reactive species formed, it is virtually ineffective against the clastogenicity of cyclophosphamide. The results of inhibition by PIETC of the clastogenicity of mitomycin C suggest that the modulation of mitomycin C bio-activation contributes to, but may not be sufficient for, PIETC chemoprevention of clastogenicity by mitomycin C.

An assessment of the in vitro hepatocyte micronucleus assay

The in vitro hepatocyte micronucleus assay was tested for its practicability and its usefulness in detecting mutagens. The assay protocol developed by Alati et al. (1989) was shown to give reproducible levels of proliferating hepatocytes and the formation of micronuclei could be readily assessed by fluorescence microscopy. Epidermal growth factor and insulin were used as mitogens, yielding mitotic indices of 2.4 ± 0.74% after 72 h of culture. The high number of 8.0 ± 3.33% micronucleated hepatocytes in control cultures at that time, typically for in vitro stimulated hepatocytes, is probably due to disordered mitoses frequently leading to chromosome loss. The direct acting mutagen N-methyl- N′-nitro- N-nitrosoguanidine and the clastogens cyclophosphamide and retrorsine, which require metabolic activation, induced dose dependent increases in the frequencies of micronucleated hepatocytes. The carcinogen 2-AAF also yielded significantly enhanced rates of micronuclei. The non-mutagen KCl as well as the peroxisome proliferator clofibrate, which is considered to be a non-genotoxic hepatocarcinogen, yielded consistently negative results. Problems occurred when chemicals exerting strong cytotoxic effects were tested in this assay. The mutagen and hepatocarcinogen aflatoxin B1 did not enhance the number of micronucleated hepatocytes. Rather a reduction of micronuclei and of mitoses was observed at AFB1 concentrations considered positive in other genotoxicity assays. Hepatocyte proliferation seems to be highly susceptible to the cytotoxic action of chemicals. A decrease in the proliferating activity of hepatocytes can obviously prevent the detection of mutagenic effects. Further studies on the in vitro hepatocyte micronucleus assay are necessary to clarify its role in mutagenicity testing.

Beta-carotene as a modulator of chromosomal aberrations induced in mouse bone marrow cells.

The inhibitory effects of beta-carotene on cyclophosphamide (CPA)-induced chromosomal aberrations in mouse bone marrow cells were investigated. Male Balb C mice, 8-10 weeks old, were treated with beta-carotene (0.5, 1.0, 2.0, 5.0, 10, 25, 50, 100, and 200 mg/kg) or with corn oil (0.05 ml/10 g b.w.) by gavage for 5 consecutive days. Four hours after the last treatment with or without beta-carotene, the animals were intraperitoneally injected with CPA and killed 24 hr later for cytological preparations and analysis. The results obtained show that beta-carotene provides significant protection against the clastogenicity of CPA. The maximum reduction in the frequency of aberrant metaphases (26.9%) and in total number of chromosomal aberrations were observed when beta-carotene was used at 50 mg/kg. Nevertheless, no direct dose-response relationship was detected, suggesting that beta-carotene might act through different mechanisms at different doses. The results obtained in animals studies have served as part of the basis for the human intervention studies now underway to determine if beta-carotene does indeed function as a chemopreventive agent in human nutrition.

Clastogenicity of cyclophosphamide in Fanconi's anemia lymphocytes without exogenous metabolic activation

The indirect mutagen cyclosphosphamide (CP) is known to induce chromosomal aberrations in human lymphocyte cultures only after exogenous metabolic activation. We document here that lymphocytes from five patients with Fanconi's anemia (FA) exhibited extensive chromosomal breakage when cultured in the continuous presence of 1 mM CP without deliberate activation of the drug, whereas, cultures from healthy subjects were not significantly susceptible to breakage under these conditions. CP exposure also enhanced the frequency of sister chromatid exchanges (SCE), but to similar extents in control and FA subjects, suggesting that there is a similar low level of CP metabolism in FA and normal lymphocytes. The direct clastogenic effect of nonactivated CP as demonstrated here in FA lymphocyte cultures may be of value in screening for possible hypersensitivity in patients considered for CP therapy.

Qualitative and quantitative differences between micronuclei morphology induced with the clastogen cyclophosphamide and the spindle poison vincrlstine in mouse reticulocytes

Qualitative and quantitative differences between micronuclei morphology induced with the clastogen cyclophosphamide and the spindle poison vincrlstine in mouse reticulocytes

Rapid detection of mutagen induced micronucleated erythrocytes by flow cytometry

The micronucleus test is a cytogenetic method for the screening of mutagens and carcinogens which exhibit clastogenic mechanisms of action. After application of clastogenic agents chromosomal fragments or even whole chromosomes can remain as conspicuous structures (micronuclei) in a small fraction of anucleated polychromatic erythrocytes which can be visually scored using a microscope following staining with May-Grünwald-Giemsa solutions. These time-consuming, painstaking, and tedious manual evaluations are often sources of unreliability and uncertainty. Here, a fluorescence technique is presented which applies DNA and protein fluorochromes to discriminate normal anucleated erythrocytes from micronucleated erythrocytes using a fluorescence microscope. This method is particularly tailored to be applied to flow cytometric instrumentation. Data obtained manually and automatically in flow show a strong linear correlation with high significance (r = 0.96) as far as the percentage of micronucleated erythrocytes as an indicator for the mutagenicity of a given drug is concerned. These results have been obtained by means of the established clastogens cyclophosphamide and mitomycin C.