The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides.

Ultra-vital staining with acridine orange (AO) is introduced into the micronucleus assay with mouse peripheral blood cells. Peripheral blood was stained vitally by dropping whole blood on an AO-coated slide and covering the sample with a coverslip. With this method, reticulocytes are identified easily by their red fluorescing reticulum structure. The distinction between young and mature erythrocytes was clearer and less subjective than the distinction between polychromatic and normochromatic erythrocytes by Giemsa staining or by conventional AO fluorescent staining. Although the induction of micronucleated peripheral reticulocytes (MNRETs) was delayed by about 12 h compared to that of micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow, the frequencies of MNRETs and MNPCEs were almost identical at each optimal sampling time. It is concluded that bone marrow cells can be replaced by peripheral blood as material for the micronucleus assay.     

Micronucleus induction in mouse peripheral reticulocytes by 7,12-dimethylbenz[a]anthracene.

Micronucleus assays using mouse peripheral blood stained vitally on acridine orange (AO)-coated slides were evaluated at two laboratories with 7,12-dimethylbenz[a]anthracene (DMBA) and compared with the standard bone marrow assay. DMBA was administered by single intraperitoneal injection to CD-1 mice at doses ranging from 5 to 80 mg/kg, then 5 microliters of peripheral blood was sampled from a tail vein at 24, 48, 72, 96, and 120 h after treatment. Similar incidences of micronucleated young erythrocytes were observed in peripheral blood reticulocytes and bone marrow polychromatic erythrocytes. The dose response of micronucleated reticulocytes was delayed compared to that of micronucleated polychromatic erythrocytes. The dose-response curves after treatment with DMBA differed depending on the sampling times, which revealed the difficulty of obtaining accurate dose-response relations in the micronucleus assay. The present result demonstrated that the simple and rapid AO supravital staining method is a valuable and easier method for obtaining dose- and time-response data for quantification of micronucleus induction by chemicals.     

Relative distribution of mature erythrocytes, polychromatic erythrocytes (PE) and micronucleated PE on mouse bone-marrow smears: control observations

Maps are presented showing the distribution of normal erythrocytes (NE), polychromatic erythrocytes (PE) and micronucleated PE (MPE) on 3 mouse bone-marrow smears. Adjacent areas of each slide were assessed along their whole length, and it was found that both the incidence of MPE and the ratio of PE:NE varied between different regions of the slide. These two variables were essentially independent of each other. The maps give the impression of clusters or islands of MPE on the slide, but these become less significant when the number of erythrocytes in those areas is taken into account. The present data confirm our previous conclusion that the resolving power of the bone-marrow micronucleus assay is related to the number of PE assessed for MPE per slide. Our data also illustrate that in instances where small departures from control values are observed, clarification/confirmation of the result can be obtained by extending the assessment of slides for MPE. It is proposed that the present recognition of the inherent sensitivity contained within this assay necessitates careful examination of the criteria for a positive response. The selection of PE suitable for assessment is discussed, and our recent 24-h oral corn oil control data are analysed.     

Gamma ray induced genetic changes in different organs of chick embryo using peripheral blood micronucleus test and comet assay

Ionizing radiation is known to produce a variety of cellular and sub cellular damage in both prokaryotic and eukaryotic cells. Present studies were undertaken to assess gamma ray induced DNA damage in different organs of the chick embryo using alkaline comet assay and peripheral blood micronucleus test. Further the suitability of chick embryo, as an alternative model for genotoxicity evaluation of environmental agents was assessed. Fertilized eggs of Rhode island red strain were exposed to 0.5, 1 and 2 Gy of gamma rays delivered at a dose rate of 0.316 Gy/min using a ⁶⁰Co teletherapy machine. Peripheral blood smears were prepared from 8- to 11-day-old chick embryos for micronucleus test. Alkaline comet assay was performed on 11-day-old chick embryos in different organs such as the heart, liver, lung, blood, bone marrow, brain and kidney.Analysis of the data revealed a significant increase in the frequency of micronucleated polychromatic erythrocytes, micronucleated normochromatic erythrocytes and total micronucleated erythrocytes in the peripheral blood of gamma irradiated chick embryos at all the doses tested as compared to the respective controls. The polychromatic to normochromatic erythrocytes ratio which is an indicator of proliferation rate of hematopoetic tissue, decreased in the irradiated groups as compared to the controls. Data obtained from comet assay, clearly demonstrated a significant increase in DNA strand breaks in all the organs of irradiated chick embryos as compared to the respective controls. However, maximum damage was observed in the heart tissue on all the doses tested, followed by kidney, brain, lung, blood and liver. The lowest damage was observed in the bone marrow tissue. Both micronucleus test and comet assay were found to be suitable biomarkers for the evaluation of genotoxicity of gamma radiation in the chick embryo.     

An in vivo mouse micronucleus assay on musk ketone

Musk ketone (3,5-dinitro-2,6-dimethyl-4-tert-butyl-acetophenone) was evaluated in an in vivo mouse micronucleus assay. Male and female mice were dosed with 250, 500 or 1000 mg musk ketone/kg body weight by a single intraperitoneal injection in corn oil. Results of the assay showed that under the conditions of this test evaluated at 24, 48 and 72 h after dosing, musk ketone did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice at any dose or any time period. Musk ketone was considered to be negative in the mouse in vivo micronucleus test as well as in a battery of previously published in vitro genotoxicity tests. Based on the total weight of evidence available, it was concluded that musk ketone does not have significant potential to act as a genotoxic carcinogen.     

Determination of micronucleus frequency by acridine orange fluorescent staining in peripheral blood reticulocytes of mice treated topically with different lubricant oils and cyclophosphamide

To ascertain whether used and re-refined lubricant oil absorbed through the skin can produce a genotoxic effect or cytotoxicity in mouse bone marrow cells, we examined the induction of micronucleated erythrocytes of peripheral blood after cutaneous application. Both re-refined and used lubricant oils showed a weak but significant induction of micronucleated polychromatic erythrocytes compared with control, while virgin oil did not show micronucleus induction. Cyclophosphamide (CP) was used not only as positive control but also to compare the sensitivity between intraperitoneal and dermal routes of administration of the test compounds in mice. The efficacy of intraperitoneal injection of CP is well known. On the other hand, dermal exposure is not so common and when CP was diluted in glycerin statistically significant values (P = 0.0036) of micronuclei were also found. Topically applied lubricant oils (virgin, re-refined and used) have the capacity to interfere with mouse bone marrow hematopoiesis evidenced by a statistically significant decrease in the proportion of polychromatic erythrocytes in the peripheral blood. Physical and chemical analysis revealed that used oil is more viscous than other lubricants, suggesting the presence of insoluble compounds, oxidized products and water as well as aromatic hydrocarbons. Used oil differs from other lubricant oils in metal and polyaromatic hydrocarbon content. Re-refined oil revealed a neutral value typical of pure mineral oil. This assay is an important tool to evaluate environmental pollutants that cause genotoxicity and/or cytotoxicity through skin exposure.     

Slide preparation and sampling as a major source of variability in the mouse micronucleus assay

This paper describes the results of a study in which mouse bone marrow micronucleus assay slides were assessed for homogeneity of micronucleated polychromatic erythrocytes (MPE) among polychromatic erythrocytes (PE). The slides were prepared by 3 distinct methods and several methods of slide reading were assessed. Observations made using our slides were confirmed by re-analysis of slides from 3 independent laboratories. It is concluded that the method of slide preparation and assessment can significantly influence the variability of data obtained from a study. The extent of this variability casts doubt upon the validity of certain assumptions concerning this assay--such as sex differences in MPE incidence, responder variability, etc. Results are discussed within the context of the very recent literature for this assay. Some laboratories appear to have adequate methods of slide preparation and data accumulation, while others do not. Methods to improve the sensitivity of this assay are suggested within the context of the recommendations made by the Gene-Tox review group. In particular, it is suggested that individual investigators present evidence of the adequacy of their data accumulation techniques in order to enhance the value of future studies.     

In vivo micronucleus assays on 2,4-dichlorophenoxyacetic acid and its derivatives.

The potential for 2,4-D and seven of its salts and esters to induce cytogenetic abnormalities in mammalian cells in vivo was investigated in the mouse bone marrow micronucleus test. All the test materials were administered to male and female mice by oral gavage and the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in the bone marrow were determined at intervals of 24, 48 and 72 h following dosing. There were no significant increases in the incidence of MN-PCE in the treated mice at any of the bone marrow sampling times. These results are consistent with the reported lack of in vitro genetic toxicity for these materials in various in vitro genotoxicity assays as well as the absence of carcinogenic potential for 2,4-D in both mice and rats.     

Studies on three structurally related phenylenediamines with the mouse micronucleus assay system.

Three structurally related compounds, 4-chloro-o-phenylenediamine (COP), 4-nitro-o-phenylenediamine (NOP) and p-phenylenediamine dihydrochloride (PPD), are used in fur dyes, inks and hair coloring formulations. COP has been reported to be carcinogenic in both rats and mice. NOP and PPD are non-carcinogens, but have consistently tested positive in short-term in vitro genotoxicity assays. Studies were undertaken to evaluate their genotoxicity with the in vivo mouse bone-marrow micronucleus assay. Five CD-1 male mice per dose were injected i.p. with the compounds and sacrificed at intervals of 24, 48 and 72 h. 2000 cells were scored per animal to determine the frequency of micronucleated-polychromatic erythrocytes (MPCE). COP induced significant dose-related increases in MPCE over the 3 doses tested at each of the sampling intervals. The peak response occurred at 24 h. No response was observed in animals treated with PPD or NOP.     

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.     

Evaluation of the genotoxic potential of three phenyltetrahydropyridinyl butylazole-derived sigma-receptor ligand drug candidates

Three structurally related phenyltetrahydropyridinyl butylazole (PTHPB)-derived drug candidates with sigma receptor-binding properties were evaluated for genotoxic potential in the ICH standard battery of genetic toxicology assays. These comprised an Ames test, a mouse-lymphoma assay, and a mouse bone-marrow micronucleus test. The maximum test concentrations in the in vitro assays were determined by the solubility and/or the cytotoxicity of the compounds. In the mouse micronucleus assay, the compounds were administered orally at three levels up to the maximum tolerated dose (MTD). Negative results were obtained for all three drug candidates in the Ames test and in the mouse-lymphoma assay, both in the absence or presence of metabolic activation. In the mouse micronucleus test, there was no effect on the frequency of micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after oral administration of any of the three test compounds, at any dose level or sampling time (24 and 48h). Administration of all three compounds at the MTD induced a clear decrease in mouse body-temperature of 3.1-4.8 degrees C below normal; the temperature returned to normal within 8h of dose administration. The produced mild hypothermia and absence of micronucleus induction was in contrast to the induction of MNPCE secondary to marked hypothermia reported for a structurally similar PTHPB-derived sigma-receptor ligand, the antipsychotic compound E-5842. The results obtained in the current series of studies suggest that exposure to the three tested PTHPB-derived drug candidates would not pose a genotoxic risk under clinical conditions.     

Activity of 1,1,1- and 1,1,3-trichloroacetones in a chromosomal aberration assay in CHO cells and the micronucleus and spermhead abnormality assays in mice

1,1,1- and 1,1,3-trichloroacetones (TCA) result from the disinfection of municipal water supplies with chlorine, and are direct-acting mutagens in the Ames/Salmonella assay. The objective of this study was to further investigate the genotoxicity of these compounds in mammalian cells using an in vitro chromosomal aberration assay in Chinese hamster ovary (CHO) cells and the micronucleus and spermhead abnormality assays in mice. Both compounds induced significant increases in structural chromosomal aberrations in CHO cells in the presence and in the absence of rat S9 metabolic activation (MA). 1,1,3-TCA was more cytotoxic to CHO cells but 1,1,1-TCA resulted in a higher proportion of cells with aberrations. The clastogenic activities of both compounds were reduced in assays conducted with MA. Neither compound resulted in the induction of a significant increase in micronucleated polychromatic erythrocytes from bone marrow of Swiss-Webster mice when administered by oral gavage; nor were effects seen on the incidence of sperm with head-shape abnormalities, testis weight, or epididymal sperm concentration in B6C3F1 mice 21 or 35 days after treatment. These data indicate that the drinking water contaminants 1,1,1- and 1,1,3-TCA are clastogenic in vitro, but are not clastogenic to bone marrow cells in vivo, and do not adversely affect several indicators of testicular function in mice.     

Evaluation of genotoxicity using the micronucleus assay and nuclear abnormalities in the tropical sea fish Bathygobius soporator (Valenciennes, 1837) (Teleostei, Gobiidae)

The micronucleus and nuclear abnormalities assays have been used increasingly to evaluate genotoxicity of many compounds in polluted aquatic ecossystems. The aim of this study is to verify the efficiency of the micronucleus assay and nuclear abnormality assay in field and laboratory work, when using erythrocytes of the tropical marine fish Bathygobius soporator as genotoxicity biomarkers. Gill peripheral blood samples were obtained from specimens of Bathygobius soporator. In order to investigate the frequencies of micronuclei and to assess the sensitivity of species, the results were compared with samples taken at the reference site and maintained in the laboratory, and fish treated with cyclophosphamide. The micronucleus assay was efficient in demonstrating field pollution and reproducing results in the labotatory. There were significant higher frequencies of micronuclei in two sites subject to discharge of urban and industrial effluents. The nuclear abnormality assay did not appear to be an efficient tool for genotoxicity evaluation when compared with field samples taken at a reference site in laboratory, with a positive control.     

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.     

Evaluation of the potential in vivo genotoxicity of quercetin

Quercetin, a naturally occurring flavonol commonly detected in apples, cranberries, blueberries, and onions, has been reported to possess antioxidant, anti-carcinogenic, anti-inflammatory, and cardioprotective properties. While positive results have been consistently reported in numerous in vitro mutagenicity and genotoxicity assays of quercetin, tested in vivo, quercetin has generally produced negative results in such studies. Furthermore, no evidence of carcinogenicity related to the oral administration of quercetin was observed in chronic rodent assays. In order to further define the in vivo genotoxic potential of quercetin, a bone marrow micronucleus assay and an unscheduled DNA synthesis (UDS) assay were conducted in Wistar rats. Administered orally to male rats at dose levels of up to 2000 mg/kg body weight, quercetin did not increase the number of micronucleated polychromatic erythrocytes (MN-PCE) 24 or 48 h following dosing in the micronucleus assay. Likewise, orally administered quercetin (up to 2000 mg/kg body weight) did not induce UDS in hepatocytes of male or female rats. While measurable levels of metabolized quercetin were observed in rat plasma samples for up to 48 h after dosing, peaking at 1h following treatment administration, the unmetabolized aglycone was not identified in either plasma or bone marrow. With the exception of only a few rats, the aglycone was also not detected in liver tissue. These results demonstrate that quercetin is not genotoxic under the conditions of these assays and further support the negative results of previously conducted in vivo assays.     

Evaluation of the potential in vivo genotoxicity of quercetin

Quercetin, a naturally occurring flavonol commonly detected in apples, cranberries, blueberries, and onions, has been reported to possess antioxidant, anti-carcinogenic, anti-inflammatory, and cardioprotective properties. While positive results have been consistently reported in numerous in vitro mutagenicity and genotoxicity assays of quercetin, tested in vivo, quercetin has generally produced negative results in such studies. Furthermore, no evidence of carcinogenicity related to the oral administration of quercetin was observed in chronic rodent assays. In order to further define the in vivo genotoxic potential of quercetin, a bone marrow micronucleus assay and an unscheduled DNA synthesis (UDS) assay were conducted in Wistar rats. Administered orally to male rats at dose levels of up to 2000 mg/kg body weight, quercetin did not increase the number of micronucleated polychromatic erythrocytes (MN-PCE) 24 or 48 h following dosing in the micronucleus assay. Likewise, orally administered quercetin (up to 2000 mg/kg body weight) did not induce UDS in hepatocytes of male or female rats. While measurable levels of metabolized quercetin were observed in rat plasma samples for up to 48 h after dosing, peaking at 1 h following treatment administration, the unmetabolized aglycone was not identified in either plasma or bone marrow. With the exception of only a few rats, the aglycone was also not detected in liver tissue. These results demonstrate that quercetin is not genotoxic under the conditions of these assays and further support the negative results of previously conducted in vivo assays.     

Genotoxic and mutagenic effects of diesel oil water soluble fraction on a neotropical fish species

Numerous spills and leakages involving petroleum and its derivatives have recently occurred in Brazilian rivers. Considering the lack of information regarding the genotoxic response of neotropical fish to these events and the predominance of information regarding saltwater fish, which offers no genuine comparisons, the present work aimed to evaluate the genotoxicity and mutagenicity of the diesel water soluble fraction (DWSF) on the neotropical fish Prochilodus lineatus under acute (6, 24 and 96h) and subchronic (15 days) exposures, using the comet (SCGE) and micronucleus assays. The results indicated genotoxic and mutagenic damage in erythrocytes of P. lineatus exposed to DWSF. Comet scores for fish exposed to DWSF in all experimental periods were significantly higher than the respective negative control groups (fish exposed to clean water for the same period). The relative frequencies of micronucleated erythrocytes for P. lineatus exposed to DWSF under acute and subchronic treatment were also significantly higher than their respective negative controls. Taken together these results showed that acute and subchronic exposures to DWSF produce mutagenic and genotoxic effects on the blood cells of P. lineatus and that the combination of comet and micronucleus assays proved to be both suitable and useful in the evaluation of the genotoxicity of diesel oil due to their complementary action.     

Induction of micronuclei by Zearalenone in Vero monkey kidney cells and in bone marrow cells of mice: protective effect of Vitamin E

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin mainly produced by Fusarium graminaerum, found as a world-wide contaminant mainly of corn and wheat. Previous studies have demonstrated that among several other effects on animals and humans, ZEN also displays hepatotoxicity, immunotoxicity and nephrotoxicity. ZEN is mainly known as a hormonal disrupter due to its estrogenic activities and consequent toxicity for reproduction. Furthermore, mutagenic and genotoxic proprieties of ZEN were disclosed recently, the molecular mechanisms of which are not yet well understood. In the present study, the genotoxic potential of ZEN was evaluated using genotoxicity tests: the 'cytokinesis block micronucleus assay' in Vero monkey kidney cells and the 'in vivo mouse bone marrow micronucleus assay'. In cultured cells treated with 5, 10 and 20 microM ZEN, the frequency of binucleated micronucleated cells (BNMN) was assessed in 1000 binucleated cells and in mice given oral doses of 10, 20 and 40 mg/kg bw, the frequency of polychromatic erythrocytes micronucleated (PCEMN) in bone marrow cells was assessed in 2000 polychromatic erythrocytes (PCE). The potential prevention of ZEN-induced effects by 25 microM Vitamin E (Vit E) was also evaluated.In vivo, doses of 10, 20 and 40 mg/kg bw ZEN representing, respectively 2, 4 and 8% of the LD50 (LD50 of ZEN in mice is 500 mg/kg bw), were administered to animals either with or without pre-treatment with Vit E (216.6 mg/kg bw) in order to evaluate its preventive potential.ZEN was found to induce micronuclei (MN) in a dose-dependent manner in cultured Vero cells as well as in mouse bone marrow cells. The present data emphasise the likely clastogenic pathway among the molecular mechanisms that underlay the ZEN-induced genotoxicity. Vit E was found to prevent partially-from 30 to 50%-these toxic effects, most likely acting either as a structural analogue of ZEN or as an antioxidant.     

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.