The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 μl of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.     

The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats.

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 microliters of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.     

The genotoxic and cytotoxic effects of ribavirin in rat bone marrow

The genotoxic and cytotoxic effects of the antiviral drug, ribavirin, was studied in rat bone marrow by employing the micronucleus assay. Ribavirin in doses of 10, 15, 20, 30, 50, 75, 100 and 200 mg/kg, and cyclophosphamide (CP) 40 mg/kg (only for sex-difference study) were injected intraperitoneally. Bone marrow was collected at 24 h and 48 h following the injection. To evaluate the recovery, the bone marrow was also sampled at 72 h from 20, 100 and 200 mg/kg treated rats. The micronucleus assay was conducted according to the standard procedure. Ribavirin elevated the incidence of micronuclei (except 10 mg/kg) in erythrocytes (P<0.01). The micronucleated polychromatic erythrocytes showed the initial steep increase at 15 and 20 mg/kg dose level, then with the gradual increase, possibly due to the limited metabolism and action of higher doses. The incidence of micronucleated normochromatic erythrocytes was not dose dependent. The effect was more at 48 h than 24 h due to prolonged toxicity of the drug or its metabolites, and by 72 h, recovery was observed eventhough the genotoxicity was significant. The PCE% decreased as the dose was increased up to 75 mg/kg, then without much difference between two higher doses. Only 100 mg/kg ribavirin and CP showed more toxicity on male rats. Cytotoxicity was seen due to hindered erythropoiesis or cell destruction. Our findings suggest that ribavirin is genotoxic and cytotoxic agent for rat bone marrow.     

Comparison of the effects of 1,2-dimethylhydrazine and cyclophosphamide on micronucleus incidence in bone marrow and colon

An in vivo micronucleus assay has been developed that utilizes colonic epithelial cells. The genotoxic effects of 1,2-dimethylhydrazine (54-07-3), a colon carcinogen, and of the nitrogen mustard, cyclophosphamide (50-18-0), on the bone-marrow polychromatic erythrocytes and on colonic epithelium from mice were compared using micronucleus induction in each organ as the end point. In the bone marrow, cyclophosphamide was a potent inducer of micronuclei, while 1,2-dimethylhydrazine administration had little effect on the micronucleus incidence. In the colon, 1,2-diemthylhydrazine was an effective inducer of micronuclei. Thus, the colonic micronucleus assay appears to be a potentially useful test for the detection of colon carcinogens.     

Use of the cytokinesis-block micronucleus method in mouse splenocytes

Mouse splenocytes have been used in the cytokinesis-block method for the evaluation of micronuclei induced by mutagenic agents in vitro as well as in vivo. Stimulation with concanavalin A for 48 h followed by 16-24-h treatment with 5 micrograms/ml cytochalasin B was found to be an optimum condition to obtain micronuclei in the binucleated splenocytes after the cells were cultured in vitro. Under the above conditions splenocytes from mice pretreated with a single i.p. injection of cyclophosphamide gave a significant increase in micronucleus production. This increase was dependent on the dose of cyclophosphamide (r = 0.99). A dose of 50 mg/kg resulted in 22% of the binucleated cells producing micronuclei, more than 20 times the level in the untreated control. The increase was also dependent on the time of cyclophosphamide injection before removal of the spleen. A duration of 4-8 h after cyclophosphamide injection gave rather sharp optimum values for the production of micronuclei. When splenocytes from non-treated mice were treated with mitomycin C together with cytochalasin B in the above in vitro condition, there was a significant increase in micronucleus production in the binucleated cells. It was also dependent on the dose of mitomycin C (r = 0.975) and a dose of 0.5 micrograms/ml resulted in a more than 20-fold increase over the untreated control. Thus, the use of mouse splenocytes in the cytokinesis-block micronucleus assay was shown to be sensitive enough for testing mutagenic agents in vivo as well as in vitro.     

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.     

The micronucleus assay as a test for the detection of aneugenic activity

The aim of this work was to determine the usefulness of the micronucleus assay for the detection of aneugenic potential. Chemicals affecting microtubule assembly, i.e., colchicine, vinblastine sulfate and tubulazole, and chemicals affecting targets other than microtubuli, i.e., mitomycin C, cyclophosphamide and miconazole, and the clastogens azathioprine and procarbazine were administered once orally or intraperitoneally to male and female mice. Bone marrow preparations were made at 24, 48 and 72 h after dosing. All the clastogens and aneugens, except miconazole, yielded positive results in the micronucleus test. Measurements of the area of the micronuclei and their distribution clearly showed that the chemicals affecting microtubule assembly produced larger micronuclei than did the clastogens. The pattern of area distribution of the micronuclei found with cyclophosphamide and mitomycin C was between those found for the tubulin inhibitors and the clastogens. These findings indicate that the micronucleus test not only detects chemicals affecting microtubule assembly, but also can discriminate them from clastogens by measurements of the area of the micronuclei.     

Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound

AMP397 is a novel antiepileptic agent and the first competitive AMPA antagonist with high receptor affinity, good in vivo potency, and oral activity. AMP397 has a structural alert (aromatic nitro group) and was mutagenic in Salmonella typhimurium strains TA97a, TA98 and TA100 without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. The amino derivative of AMP397 was negative in wild-type strains TA98 and TA100. AMP397 was negative in a mouse lymphoma tk assay, which included a 24h treatment without S9. A weak micronucleus induction in vitro was found at the highest concentrations tested in V79 cells with S9. AMP397 was negative in the following in vivo studies, which included the maximum tolerated doses of 320mg/kg in mice and 2000mg/kg in rats: MutaMouse assay in colon and liver (5x320mg/kg) at three sampling times (3, 7 and 31 days after the last administration); DNA binding study in the liver of mice and rats after a single treatment with [14C]-AMP397; comet assay (1x2000mg/kg) in jejunum and liver of rats, sampling times 3 and 24h after administration; micronucleus test (2x320mg/kg) in the bone marrow of mice, sampling 24h after the second administration. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. In particular, no genotoxic metabolite is formed in mammalian cells, and, if formed by intestinal bacteria, is unable to exert any genotoxic activity in the adjacent intestinal tissue. These data were considered to provide sufficient safety to initiate clinical development of the compound.     

Use of the cytokinesis-block method for the analysis of micronuclei in V79 Chinese hamster lung cells: results with mitomycin C and cyclophosphamide

The cytochalasin B (CYB)-blocked binucleated cell assay has been explored to analyze micronuclei and cell cycle kinetics using 2 known mutagenic carcinogens in V79 Chinese hamster lung cells. To determine the optimum time to obtain the maximum number of binucleated cells for micronucleus analysis, duplicate cultures of exponentially growing cells were treated with 3 micrograms/ml CYB for varying durations (8-48 h). A peak appearance of binucleated cells at 16 h in the presence of CYB suggested this as an optimum time for micronucleus analysis in binucleated V79 cells. To evaluate the capacity for induction of micronuclei in V79 cells, 2 mutagenic carcinogens, mitomycin C (0.125-1.0 micrograms/ml) and cyclophosphamide (2-12 micrograms/ml) were tested in duplicate cultures. Mitomycin C, a direct-acting alkylating agent, caused approximately an 18-fold increase in micronucleus frequency over controls at the highest concentration tested (1.0 micrograms/ml), and this increase occurred in a dose-related manner (r = 0.92). The concentrations of mitomycin C tested also caused a significant dose-related cell cycle delay, thus suggesting cytotoxicity to V79 cells. Cyclophosphamide, an indirect-acting alkylating agent, requiring the presence of S9 mix, caused approximately a 17-fold increase in micronucleus frequency over controls at the highest tested concentration (12 micrograms/ml), with a clear dose response (r = 0.99). The various concentrations of cyclophosphamide also caused cytotoxicity in a dose-related fashion. Thus, this study demonstrates the usefulness of the cytokinesis-block method in V79 cells as a possible screen to analyze micronucleus induction and cytotoxicity. Because this approach is much less labor intensive than conducting a structural chromosomal analysis, this assay has great potential both as an initial screen for clastogenic activity and as a tool for investigating the underlying mechanisms for clastogenicity.     

Malaria infection modulates effects of genotoxic chemicals in the mouse bone-marrow micronucleus test

Malaria has been reported to modulate the activity of cytochrome-P450 enzymes (CYP). Since CYPs are involved both in the activation and detoxication of xenobiotics, we investigated whether malaria would modify the effects of chemical carcinogens in the bone-marrow micronucleus assay. Female C57BL6 mice were infected with Plasmodium berghei (ANKA) and treated (ip route) with cyclophosphamide (CPA, 25 mg/kg body weight), 7,12-dimethylbenz[a]anthracene (DMBA, 50mg/kg body weight) or ethyl methanesulfonate (EMS, 150 mg/kg body weight), on post-infection days 9-12 when parasitemia was > or =9% of RBC. Controls were age-paired non-infected mice. Bone marrows were sampled at 24 and 48 h (CPA), 24 h (EMS) or 48 h (DMBA) after treatment. The background incidence of polychromatic erythrocytes with micronuclei (MN-PCE) in malaria-infected mice was approximately twofold the background incidence in non-infected controls. Effects of indirect clastogens (CPA and DMBA) in the micronucleus assay were attenuated while the effect of EMS, a direct clastogen, was enhanced by infection. In a separate experiment, malaria was shown to decrease activities of ethoxy-(EROD, a marker for CYP1A) and benzyloxy-(BROD, CYP2B) resorufin-O-dealkylases in liver microsomes. The foregoing findings are consistent with the hypothesis that malaria-caused attenuation of genotoxicity arose from a down modulation of CYP isoforms that convert CPA (CYP2B) and DMBA (CYP1A) into their active metabolites.     

Development and validation of an in vitro micronucleus assay platform in TK6 cells

The Organization for Economic Co-operation and Development (OECD) has recently adopted Test Guideline 487 (TG487) for conducting the in vitro micronucleus (MNvit) assay. The purpose of this study is to evaluate and validate treatment conditions for the use of p53 competent TK6 human lymphoblastoid cells in a TG487 compliant MNvit assay. The ten reference compounds suggested in TG487 (mitomycin C, cytosine arabinoside, cyclophosphamide, benzo-a-pyrene, vinblastine sulphate, colchicine, sodium chloride, nalidixic acid and di(2-ethylhexyl)phthalate and pyrene) and noscapine hydrochloride were chosen for this study. In order to optimize the micronucleus response after treatment with some positive substances, we extended the recovery time after pulse treatment from 2 cell cycles recommended in TG487 to 3 cell cycles for untreated cells (40h). Each compound was tested in at least one of four exposure conditions: a 4h exposure followed by a 40h recovery, a 4h exposure followed by a 24h recovery, a 4h exposure in the presence of an exogenous metabolic activation system followed by a 40h recovery period, and a 27h continuous direct treatment. Results show that the direct acting clastogens, clastogens requiring metabolic activation and aneugens caused a robust increase in micronuclei in at least one test condition whereas the negative compounds did not induce micronuclei. The negative control cultures exhibited reproducibly low and consistent micronucleus frequencies ranging from 0.4 to 1.8% (0.8±0.3% average and standard deviation). Furthermore, extending the recovery period from 24h to 40h produced a 2-fold higher micronucleus frequency after a 4h pulse treatment with mitomycin C. In summary, the protocol described in this study in TK6 cells produced the expected result with model compounds and should be suitable for performing the MNvit assay in accordance with guideline TG487.     

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.     

Assessment of the genotoxicity of propineb in mice bone marrow cells using micronucleus assay

Propineb, a dithiocarbamate fungicide, is commonly used for the control of disease in a wide range of crops in agriculture. The genotoxic effects of commercial formulation of propineb in bone marrow cells of mice was investigated in vivo by micronucleus (MN) assay. The three different concentrations of propineb (12.5, 25 and 50 μg/mL; 0.01 mL per gram) were injected intraperitoneally (i.p.) to mice for 24 and 48 h. The results of the MN assay indicated that propineb induced a significant increase in frequency of micronucleated polychromatic erythrocytes (MNPCE) at 25 and 50 μg/mL concentrations for 24 h and at the highest (50 μg/mL) concentration for 48 h when compared with negative control. Also significant reduction for the polychromatic erythrocyte/normochromatic erythrocyte (PCE/NCE) ratio which is indicative for bone marrow cytotoxicity was observed at the same concentrations for 24 and 48 h. These results lead us to the conclusion that propineb may have genotoxic and cytotoxic potential due to induction in the frequency of MN and a reduction in PCE/NCE ratio in the bone marrow cells of mice.     

Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining: The summary report of the 5th collaborative study by CSGMT/JEMS · MMS

The main goal of the Collaborative Study Group for the Micronucleus Test (CSGMT) was to validate a new method for the micronucleus test, recently introduced by Hayashi et al. (1990), using mouse peripheral blood cells stained supravitally with acridine orange (AO). The micronucleus tests were performed on CD-1 mice using 23 chemicals with various modes of action. As a rule, one chemical was studied by two participants. Peripheral blood sampled from the same animal was examined 0, 24, 48, and 72 h (or longer) after treatment. The frequencies of micronucleated peripheral reticulocytes (MNRETs) were recorded based on observation of 1000 reticulocytes per mouse.All chemicals induced MNRETs dose-dependently. Interlaboratory differences in the induction of MNRETs were in an acceptable range for most chemicals tested. Although differences were observed with some chemicals, there were no discrepancies in qualitative judgment. Most chemicals gave the greatest response 48 h after treatment, which was less variable than in the bone marrow assay (greatest response, 24–48 h). These results suggest that the peripheral blood assay using the AO supravital staining technique generates reproducible and reliable data to evaluate the clastogenicity of chemicals. This makes the peripheral blood micronucleus assay an attractive alternative to the conventional bone marrow assay.     

Genotoxic and cytotoxic effects in the bone marrow of rats exposed to a low dose of paraquat via the dermal route

The genotoxic effect of the herbicide paraquat was studied in rat bone-marrow by means of the micronucleus assay. Paraquat at dose levels of 6, 15 and 30 mg/kg body weight was given to rats in a single application via the dermal route. Marrow was collected at 24, 48 and 72 h after the application. The micronucleus assay was done as recommended by standard procedures. Paraquat gave rise to an increase in the number of micronuclei in a dose-dependent manner. The number of micronucleated polychromatic erythrocytes showed a maximum at 48 h and the toxicity was further prolonged, as there was no complete recovery at 72 h. These findings suggest a genotoxic effect of paraquat even after exposure via dermal application.     

Validation of the mouse peripheral blood micronucleus assay using acridine orange supravital staining with urethane.

The mouse peripheral blood micronucleus assay using acridine orange supravital staining was compared with the standard bone marrow assay using urethane (ethyl carbamate)-treated mice. Urethane was intraperitoneally injected to CD-1 and BDF1 mice at doses ranging from 62 to 1000 and 62 to 250 mg/kg, respectively. Peripheral blood was collected from the tail 0, 24, 48, and 72 h and bone marrow cells were smeared at 24 and 42 h after the treatment. Although the response of micronucleus induction in peripheral reticulocytes was delayed by about 24 h compared to that in bone marrow polychromatic erythrocytes, the maximum frequencies of micronucleated young erythrocytes were comparable. Therefore, the peripheral blood micronucleus assay using the acridine orange supravital staining method may provide a good alternative to the conventional bone marrow assay.     

The cytonucleus test in the rat: a combined metaphase and micronucleus assay

The suitability of the rat as a species choice for the micronucleus assay and the possibility of combining both metaphase and micronucleus analysis using one set of animals were investigated. Cyclophosphamide, trenimon, vinblastine sulphate and dimethyl benzanthracene were used to optimise the study design and experimental procedures. The sample times ranged from 12 to 72 h after a single dose of the compound. A maximal response in both micronucleus and chromosome aberration induction was observed 24 h after dosing. This sample time is recommended as the single sample time for the screening of all classes of compound. Using the optimal conditions, a number of different mutagens/carcinogens were then analysed for the induction of both micronuclei and chromosome aberrations in the same animal. The compounds selected were mitomycin C, methotrexate, 5-fluorouracil, hexamethyl phosphoramide, benzo[a]pyrene, benzidine and diaminoterphenyl. The results show that the rat is a responsive test species and that it is possible to combine both metaphase and micronucleus analysis in the same animal.     

Validation of the mouse peripheral blood micronucleus assay using acridine orange supravital staining with urethane

The mouse peripheral blood micronucleus assay using acridine orange supravital staining was compared with the standard bone marrow assay using urethane (ethyl carbamate)-treated mice. Urethane was intraperitoneally injected to CD-1 and BDF₁ mice at doses ranging from 62 to 1000 and 62 to 250 mg/kg, respectively. Peripheral blood was collected from the tail 0, 24, 48, and 72 h and bone marrow cells were smeared at 24 and 42 h after the treatment. Although the response of micronucleus induction in peripheral reticulocytes was delayed by about 24 h compared to that in bone marrow polychromatic erythrocytes, the maximum frequencies of micronucleated young erythrocytes were comparable. Therefore, the peripheral blood micronucleus assay using the acridine orange supravital staining method may provide a good alternative to the conventional bone marrow assay.     

Induction of micronuclei and other nuclear abnormalities in European minnow Phoxinus phoxinus and mollie Poecilia latipinna: an assessment of the fish micronucleus test

In this work, we have measured both micronuclei and other nuclear abnormalities in renal erythrocytes from European minnow Phoxinus phoxinus and mollie Poecilia latipinna, with the aim to contribute to the standardisation of the micronucleus test for fish species. Intraperitoneal injections of colchicine (10 mg/kg), cyclophosphamide (40 mg/kg), or mitomycin C (20 mg/kg) for 24 h induced diverse nuclear abnormalities in minnow erythrocytes, therefore nuclear abnormalities should be added to micronuclei as genotoxicity indicators in fish micronucleus tests. The adequacy of administration protocols based on intraperitoneal injections has been evaluated by injecting saline solution to both species: single or double injections have not induced neither micronuclei nor other nuclear abnormalities in any case. Finally, the differential sensitivity of both species to toxic heavy metals was evaluated by exposing individuals of both species to different doses (0.17, 1.7, 2x1.7, and 3.4 mg/kg) of cadmium and mercury for 24 h; we concluded that the mollie is sensitive to both metals whereas the minnow is not sensitive to mercury.     

Mitigation by vitamin C of the genotoxic effects of nicotine in mice, assessed by the comet assay and micronucleus induction

Nicotine has been reported to cause acute toxicity and to present long-term risks, such as chromosomal damage and genetic instability. The genotoxicity of nicotine may be mediated partly by an oxidative mechanism. We have evaluated the effects of the antioxidant vitamin C on nicotine-induced genotoxicity in mice. The comet assay and the micronucleus test were used to assess the effects of nicotine (15mg/kg) at different exposure times (2, 4, and 24h in the comet assay; 24h in the micronucleus test). Pretreatment with vitamin C 24h before nicotine exposure strongly protected mice against nicotine-induced DNA damage.