The micronucleus test of methyl methanesulfonate with mouse peripheral blood reticulocytes using acridine orange-coated slides.

The usefulness of the micronucleus assay using mouse peripheral blood erythrocytes and acridine orange (AO)-coated slides was evaluated with methyl methanesulfonate (MMS). The micronucleus test was carried out at doses ranging from 20 to 80 mg/kg body weight in CD-1 mice by intraperitoneal injection. Peripheral blood cells were examined from 0 to 72 h after treatment at 12- or 24-h intervals. Bone marrow cells from other mice treated with 80 mg/kg MMS were also sampled at the same times. The frequency of micronucleated reticulocytes (MNRETs) increased dose-dependently at every sampling time except 72 h, and the maximum frequency of MNRETs was observed at about 36 h after treatment. Micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow after a dose of 80 mg/kg were significantly induced at 12 h to 36 h, and the maximum frequency of MNPCEs was observed at 24 h after treatment. The induction of MNRETs was delayed by about 12 h compared to that of MNPCEs in bone marrow, and the maximum frequencies of MNRETs were lower than those of MNPCEs, but the induction of MNRETs by MMS was significant and dose-dependent. It is concluded, therefore, that bone marrow cells could be replaced by peripheral blood cells as material for the micronucleus assay using AO-coated slides.     

DNA adduct formation in mouse testis by ethylating agents: a comparison with germ-cell mutagenesis

DNA adduct formation in various organs of mice was determined after i.p. injection with the ethylating agents N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), and diethyl sulfate (DES). The potency of the 3 chemicals to react either at the O6 position of guanine or at the N-7 position of guanine was related to their potency to induce mutations in the specific-locus assay of the mouse. ENU, which produces relatively high levels of O-alkylations (O6-ethylguanine), is primarily mutagenic in spermatogonia of the mouse, whereas EMS and DES, which produce relatively high levels of N-alkylations (7-ethylguanine) in DNA, are much more mutagenic in post-meiotic stages of male germ cells. The relationship between exposure to ENU and the dose, determined as O6-ethylguanine per nucleotide in testicular DNA, is non-linear. However, the relationship between dose and mutation induction in spermatogonia by ENU appears to be linear, which is expected if O6-ethylguanine is the major mutagenic lesion. The relatively high mutagenic potency of EMS and DES in the late stages of spermatogenesis is probably due to the accumulation of apurinic sites which generate mutations after fertilization. A comparison of mutation induction by ENU in spermatogonia and mutation induction in cultured mammalian cells indicates that about 10 O6-ethylguanine residues were necessary in the coding region of a gene to generate a mutation.     

The micronucleus test using peripheral blood reticulocytes from methotrexate-treated mice.

The induction of micronuclei by methotrexate (MTX) was examined in two laboratories using mouse peripheral blood reticulocytes. MTX was a weak inducer in the micronucleus test using bone marrow cells and single treatments, and was one of the few chemicals showing a multiple-treatment effect (CSGMT/JEMS.MMS, 1990). In our preliminary experiments, the ratio of reticulocytes to total erythrocytes decreased greatly after a single treatment with MTX at 100 mg/kg, so lower dose levels of MTX were selected to carry out the micronucleus test in peripheral blood. Full-scale tests were performed at dose levels of 0, 10, 20, 40, and 80 mg/kg, with five sampling times of 0, 24, 48, 72, and 96 h. Frequencies of micronucleated reticulocytes (MNRETs) increased dose-dependently at 72 h, to a maximum of approximately 1%; some preparations obtained from the animals at higher doses could not be examined because the ratio of reticulocytes to total erythrocytes had decreased severely. At doses of 0.5-4.0 mg/kg, the effect of multiple treatments vs. single treatments was not clear, nor was the maximum level of response much different. Since MTX induced a clear positive response in peripheral blood reticulocytes after a single treatment, the reticulocytes in peripheral blood seem a more sensitive target.     

The micronucleus test using peripheral blood reticulocytes from methotrexate-treated mice

The induction of micronuclei by methotrexate (MTX) was examined in two laboratories using mouse peripheral blood reticulocytes. MTX was a weak inducer in the micronucleus test using bone marrow cells and single treatments, and was one of the few chemicals showing a multiple-treatment effect (CSGMT/JEMS · MMS, 1990). In our preliminary experiments, the ratio of reticulocytes to total erythrocytes decreased greatly after a single treatment with MTX at 100 mg/kg, so lower dose levels of MTX were selected to carry out the micronucleus test in peripheral blood. Full-scale tests were performed at dose levels of 0, 10, 20, 40, and 80 mg/kg, with five sampling times of 0, 24, 48, 72, and 96 h. Frequencies of micronucleated reticulocytes (MNRETs) increased dose-dependently at 72 h, to a maximum of approximately 1%; some preparations obtained from the animals at higher doses could not be examined because the ratio of reticulocytes to total erythrocytes had decreased severely. At doses of 0.5–4.0 mg/kg, the effect of multiple treatments vs. single treatments was not clear, nor was the maximum level of response much different. Since MTX induced a clear positive response in peripheral blood reticulocytes after a single treatment, the reticulocytes in peripheral blood seem a more sensitive target.     

A comparison of the genotoxicity of ethylnitrosourea and ethyl methanesulfonate in lacZ transgenic mice (Muta™Mouse)

We compared the induction of gene mutations and chromosomal aberrations by ethylating agents in lacZ transgenic mice (Muta™Mouse). Chromosomal aberrations were detected by the peripheral blood micronucleus assay. Gene mutations were detected in the lacZ transgene. A small amount of blood was sampled from a tail vessel during the expression time for fixation of gene mutations in vivo; this enabled us to detect and compare clastogenicity and gene mutations in the identical mouse. Single intraperitoneal injections of ENU (50–200 mg/kg) and EMS (100–400 mg/kg) strongly induced micronucleated reticulocytes (MN) detectable in peripheral blood 48 h after treatment. The maximum MN frequencies induced were 6.6% and 3.3% for ENU (100 mg/kg) and EMS (400 mg/kg), respectively (the control value was 0.3%). lacZ mutant frequency (MF) was analyzed in bone marrow and liver 7 days after treatment. Spontaneous MFs were 2.0–4.6x10⁻⁶. MF in bone marrow was increased by ENU to 3.4x10⁻⁵ at 200 mg/kg and induced by EMS to 1.8x10⁻⁵ at 400 mg/kg. In liver, however, both chemicals at their highest doses induced only slight increases in MF. The induction of both micronuclei and lacZ mutations in bone marrow by both ENU and EMS correlated better with O⁶-ethylguanine adducts than with N7-ethylguanine adducts. The mutants (19 for ENU and 12 for EMS) were subjected to DNA sequence analysis. Among EMS-induced mutations, 75% were GC to AT transitions, which were probably caused by O⁶-ethylguanine. Among ENU-induced mutations, in contrast, 40% occurred as AT base pair substitutions (6 AT to TA transversions and 2 AT to GC transitions) (no such mutations were induced by EMS). These results, together with the known reactivity of ENU to thymine suggest that thymine adducts play a significant role in the ENU mutagenesis.     

Letinula edodes (Berk.) Pegler (Shiitake) modulates genotoxic and mutagenic effects induced by alkylating agents in vivo

We evaluated the antimutagenic effect of Letinula edodes (Berk.) Pegler (Shiitake) on the frequency of micronuclei in mice treated with N-ethyl-N-nitrosourea (ENU) or cyclophosphamide (CP). Mice were orally (gavage) pretreated for 15 consecutive days with solutions of Shiitake (0.6 ml per day, gavage) prepared at three different temperatures: 4, 21 (RT), and 60 degrees C. Then, the animals were intraperitoneally injected on day 15 with CP (25 or 50mg/kg) or ENU (50 mg/kg) and killed 24 or 48 h after treatment for evaluation of micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow and micronucleated reticulocytes (MNRETs). A mixture of L. edodes lineages (LE 95/016, 96/14, 96/17, 96/22, 96/23, 97/27, and 97/28) significantly decreased the frequencies of MNPCEs and MNRETs induced by CP (25 and 50mg/kg). When a single lineage from the mixture (LE 96/17) was tested we also found a significant reduction in the frequencies of MNPCEs and MNRETs induced by both CP or ENU (50mg/kg). The comet assay was also performed 3h after ENU treatment using mice pretreated with the single lineage (LE 96/17) of L. edodes. The results showed a high degree of variability with some indications of an antigenotoxic effect. Taken together, our data show that solutions from Shiitake inhibit in vivo mutagenicity of CP and ENU.     

Micronucleus assays on 5-fluorouracil and 6-mercaptopurine with mouse peripheral blood reticulocytes.

As part of the 5th collaborative study of the Collaborative Study Group for the Micronucleus Test (CSGMT), the sensitivity and advantages of the micronucleus assay using mouse peripheral blood cells were evaluated using 5-fluorouracil (5-FU) and 6-mercaptopurine (6-MP). The peripheral blood cells were collected from a tail vein of CD-1 male mice just before and 24-120 h after intraperitoneal injection. At 24-h intervals. The maximum incidence of micronucleated reticulocytes (MNRETs) at 50 mg/kg 5-FU was observed 96 h after injection; at 100 mg/kg, the peak was delayed to 120 h, and followed severe bone marrow depression. With 6-MP, maximum MNRETs were observed 48 h after treatment at all doses tested. At dose levels higher than 50 mg/kg, severe bone marrow depression was observed after maximum MNRETs. Though the appearance patterns of MNRETs and the bone marrow depression were different between 5-FU and 6-MP, the positive response of both chemical could be detected with this assay system as well as with the micronucleus test using femoral bone marrow cells.     

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.     

Micronucleus test with ethyl methanesulfonate in mouse peripheral blood reticulocytes stained supravitally using acridine orange-coated slides.

A new method for the micronucleus test using peripheral blood reticulocytes stained supravitally using acridine orange-coated slides was evaluated in male CD-1 mice treated with ethyl methanesulfonate (EMS) at doses of 100, 200, 300, and 400 mg/kg. Peripheral blood samples were taken 0, 24, 48, 72, and 96 h after treatment from each mouse without killing. The frequencies of micronucleated reticulocytes increased dose-dependently with the peak at 48 h after treatment. These results indicate that, at least for EMS, the new method used here can be an alternative to the conventional method using bone marrow polychromatic erythrocytes.     

Micronucleus test with ethyl methanesulfonate in mouse peripheral blood reticulocytes stained supravitally using acridine orange-coated slides

A new method for the micronucleus test using peripheral blood reticulocytes stained supravitally using acridine orange-coated slides was evaluated in male CD-1 mice treated with ethyl methanesulfonate (EMS) at doses of 100, 200, 300, and 400 mg/kg. Peripheral blood samples were taken 0, 24, 48, 72, and 96 h after treatment from each mouse without killing. The frequencies of micronucleated reticulocytes increased dose-dependently with the peak at 48 h after treatment. These results indicate that, at least for EMS, the new method used here can be an alternative to the conventional method using bone marrow polychromatic erythrocytes.     

The micronucleus assay with peripheral blood reticulocytes by acridine orange supravital staining with 1-beta-D-arabinofuranosylcytosine.

Dose-dependent induction of micronuclei with 1-beta-D-arabinofuranosylcytosine (ara-C) was clearly shown in CD-1 mouse peripheral blood reticulocytes (RETs) using an acridine orange (AO) supravital staining method, as well as in the conventional bone marrow assay. The maximum frequencies of micronucleated RETs (MNRETs) in peripheral blood and of micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow were comparable, as shown in two laboratories independently. The maximum frequencies of MNRETs in peripheral blood lagged about 24 and 12 h behind those of MNPCEs in bone marrow in experiments with 24- and 12-h sampling intervals, respectively. The proportion of each type of RET was examined periodically after treatment with ara-C at doses ranging from 6.25 to 50.0 mg/kg. The proportion of type I RETs among total RETs decreased 24 or 48 h after treatment according to the dose level. This suggest that this ratio could be a good indicator of the bone marrow cell toxicity of test chemicals.     

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.     

Quantitative comparative mutagenesis in bacteria, mammalian cells, and animal-mediated assays A convenient way of estimating genotoxic activity in vivo?

The accumulation of environmental compounds which exhibit genotoxic properties in short-term assays and the increasing lag of time for obtaining confirmation or not in long-term animal mutagenicity and carcinogenicity tests, makes it necessary to develop alternative, rapid methodologies for estimating genotoxic activity in vivo. In the experimental approach used here, it was assumed that the genotoxic activity of foreign compounds in animals, and ultimately humans, is determined among others by exposure level, organ distribution of (DNA) dose, and genotoxic potency per unit of dose, and that knowledge about these 3 parameters may allow to rapidly determine the expected degree of genotoxicity in various organs of exposed animals. In view of the high degree of qualitative correlation between mutagenic activity of chemicals in bacteria and in cultured mammalian cells, and their mutagenic and carcinogenic properties in animals, and in order to be able to distinguish whether mutagenic potency differences were due to differences in (DNA) dose rather than other physiological factors, the results of mutagenicity tests obtained in the present experiments using bacteria and mammalian cells were compared on the basis of DNA dose rather than exposure concentrations, with the following questions in mind: Is there an absolute or a relative correlation between the mutagenic potencies of various ethylating agents in bacteria (E. coli K12) and in mammalian cells (V79 Chinese hamster) after treatment in standardized experiments, and can specific DNA adducts be made responsible for mutagenicity? Is the order of mutagenic potency of various ethylating agents observed in bacteria in vitro representative of the ranking of mutagenic potency found in vivo? Since the answer to this last question was negative, a further question addressed to was whether short-term in vivo assays could be developed for a rapid determination of the presence (and persistence) of genotoxic factors in various organs of mice treated with chemicals. In quantitative comparative mutagenesis experiments using E. coli K12 and Chinese hamster cells treated under standardized conditions in vitro with 5 ethylating agents, there was no indication of an absolute correlation between the number of induced mutants per unit of dose in the bacteria and the mammalian cells. The ranking of mutagenic potency was, however, identical in bacteria and mammalian cells, namely, ENNG greater than ENU greater than or equal to DES greater than DEN congruent to EMS, the mutagenic activity of DEN being dependent on the presence of mammalian liver preparations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of benzene in a micronucleus test on peripheral blood utilizing acridine orange-coated slides.

A micronucleus test was conducted on the peripheral blood of mice and rats utilizing acridine orange-coated slides (AO-coated method) after oral administration of benzene. Blood was sampled at 0, 24, 48, and 72 h after administration of benzene at doses of 500, 1000, and 2000 mg/kg in both mice and rats. The highest occurrence of micronucleated reticulocytes (MNRETs) was observed at 48 h after administration in both species. Species differences was found in the frequency of MNRETs, with the number being lower in rats than in mice. The present results indicate that the micronucleus test can easily detect chromosome aberrations in peripheral blood induced by benzene administration in both mice and rats. Furthermore, the AO-coated method used in this study was simpler to perform and allowed for easier detection of the effect than the conventional method.     

Effects of benzene in a micronucleus test on peripheral blood utilizing acridine orange-coated slides

A micronucleus test was conducted on the peripheral blood of mice and rats utilizing acridine orange-coated slides (AO-coated method) after oral administration of benzene. Blood was sampled at 0, 24, 48, and 72 h after administration of benzene at doses of 500, 1000, and 2000 mg/kg in both mice and rats. The highest occurrence of micronucleated reticulocytes (MNRETs) was observed at 48 h after administration in both species. Species differences was found in the frequency of MNRETs, with the number being lower in rats than in mice. The present results indicate that the micronucleus test can easily detect chromosome aberrations in peripheral blood induced by benzene administration in both mice and rats. Furthermore, the AO-coated method used in this study was simpler to perform and allowed for easier detection of the effect than the conventional method.     

Micronucleus tests on N-ethyl-N-nitrosourea with mouse peripheral blood reticulocytes using acridine orange-coated slides.

The micronucleus test with peripheral blood using acridine orange-coated slides was validated in male CD-1 mice treated once with N-ethyl-N-nitrosourea (ENU) at doses of 6.25, 12.5, 25.0, and 50.0 mg/kg body weight. Peripheral blood preparations were made 0, 24, 48, and 72 h after treatment. The frequencies of micronucleated peripheral reticulocytes in the ENU-treated groups increased dose-dependently, peaking at 48 h after treatment. The results indicate that the method used in the present study can be an alternative to the method using bone marrow polychromatic erythrocytes.     

Mutagenicity and cytotoxicity of congeners of two classes of nitroso compounds in Chinese hamster ovary cells

The induction of mutation by certain nitrosamidines and nitrosamides has been quantitated utilizing the hypoxanthine--guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary cells. Dose--response relationships for cytotoxicity and mutagenicity are presented for N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-butyl-N-nitrosourea (BNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Based on the concentration of each agent required to kill 90% of the cells, the following order of cytotoxicity was observed: MNNG greater than ENNG greater than MNU greater than ENU greater than BNU. This is the same order of potency as observed for mutation induction per unit concentration of mutagen.     

Cross-resistance studies with V79 Chinese hamster cells adapted to the mutagenic or clastogenic effect of N-methyl-N′-nitro-N-nitrosoguanidine

When V79 cells are exposed to a single low dose of MNNG or MNU they acquire resistance to the mutagenic or to the clastogenic effect of the agents. Here the effect of MNNG pretreatment on mutagenesis (6-thioguanine resistance) and aberration formation in cells challenged with various mutagens/clastogens is reported. MNNG-adapted cells were resistant to the mutagenic effects of MNU and, to a lower extent, of EMS. No mutagenic adaptation was observed when MNNG-pretreated cells were challenged with MMS, ENU, MMC or UV.

Cells pretreated with a dose of MNNG which makes them resistant to the clastogenic effect of this compound were also resistant to the clastogenic activity of other methylating agents (MNU, MMS), but not so with respect to ethylating agents (EMS, ENU). Cycloheximide abolished the aberration-reducing effect of pretreatment. However, when given before the challenge dose of MNNG, MNU or MMS, it drastically enhanced the aberration frequency in both pretreated and non-pretreated cells. No significant enhancement of aberration frequency by cycloheximide was found for ethylating agents.

The results indicate that clastogenic adaptation is due to inducible cellular functions. It is concluded that mutagenic and clastogenic adaptation are probably caused by different adaptive repair pathways.     

The relationship between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems. IV. The effects of the excision-defective mei-9L1 and mus(2)201D1 mutants on alkylation-induced genetic damage in Drosophila

Repair-defective mutants of Drosophila melanogaster which identify two major DNA excision repair loci have been examined for their effects on alkylation-induced mutagenesis using the sex-linked recessive lethal assay as a measure of genotoxic endpoint. The alkylating agents (AAs) chosen for comparative analysis were selected on the basis of their reaction kinetics with DNA and included MMS, EMS, MNU, DMN, ENU, DEN and ENNG. Repair-proficient males were treated with the AAs and mated with either excision-defective mei-9L1 or mus(2)201D1 females or appropriate excision-proficient control females. The results of the present work suggest that a qualitative and quantitative relationship exists between the nature and the extent of chemical modification of DNA and the induction of of genetic alterations. The presence of either excision-defective mutant can enhance the frequency of mutation (hypermutability) and this hypermutability can be correlated with the Swain-Scott constant S of specific AAs such that as the SN1 character of the DNA alkylation reaction increases, the difference in response between repair-deficient and repair-proficient females decreases. The order of hypermutability of AAs with mei-9L1 relative to mei-9+ is MMS greater than MNU greater than DMN = EMS greater than iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9L1 females are plotted against those determined for control females, straight lines of different slopes are obtained. These mei-9L1/mei-9+ indices are: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4 and iPMS = ENU = DEN = ENNG = 1. An identical order of hypermutability with similar indices is obtained for the mus(2)201 mutants: MMS(7.3) greater than MNU (5.4) greater than EMS(2.0) greater than ENU(1.1). Thus, absence of excision repair function has a significant effect on mutation production by AAs efficient in alkylating N-atoms in DNA but no measurable influence on mutation production by AAs most efficient in alkylating O-atoms in DNA. The possible nature of these DNA adducts has been discussed in relation to repair of alkylated DNA. In another series of experiments, the effect on alkylation mutagenesis of mei-9L1 was studied in males, by comparing mutation induction in mei-9L1 males vs. activity in Berlin K (control). Although these experiments suggested the existence of DNA repair in postmeiotic cells during spermatogenesis, no quantitative comparisons could be made.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of micronuclei in peripheral blood of mitomycin C-treated mice using supravital staining with acridine orange.

The induction of micronuclei in peripheral blood from mitomycin C (MMC)-treated mice was examined using a supravital acridine orange staining method. Male ICR mice were intraperitoneally given MMC at a single dose of 0.25, 0.5, 1, or 2 mg/kg. Blood was sampled from the tail 24, 48, 72, and 96 h after treatment, and the frequency of micronucleated reticulocytes (MNRETs) was examined. The induction of MNRETs peaked at 48 h after treatment with MMC; there was a clear, dose-related increase in MNRETs. In a multiple-treatment study, mice were treated with 4 consecutive daily injections of MMC at a dose of 0.13, 0.25, 0.5, or 1 mg/kg. The frequency of MNRETs increased markedly 24 h after the second treatment as compared with the first treatment, and did not change significantly until 24 h after the fourth treatment. The frequency of MNRETs decreased to approximately control values 96 h after the last treatment. In addition, a slight but statistically significant increase in the number of micronucleated normochromatic erythrocytes in peripheral blood was detected by means of Giemsa staining 7 days after the last treatment. These results confirm the usefulness of the supravital acridine orange staining method to evaluate micronucleus induction in mouse peripheral blood.