Distinct area distribution differences of micronuclei induced by clastogenic and aneuploidogenic chemicals in the bone marrow of the CD-1 mouse.

To distinguish between aneuploidogenic and clastogenic effects of test chemicals, area distributions of micronuclei (MN) in polychromatic erythrocytes (PE) from the mouse bone marrow were measured using an image analysis system. Triethylenemelamine (TEM), cytosine-beta-D-arabinofuranoside (ara-C), urethane (URT), cyclosphamide (CP), mitomycin C (MMC), colcemid (COL) and tubulazole C (TUB) were investigated for the induction of micronucleus area distributions. The area distribution of micronuclei of untreated mice was also determined. Reproducible small differences between the clastogens and the aneuploidogens were observed after measuring 1100-1200 micronuclei. A common feature of the distribution curves was a shoulder region in the same area range for all clastogens. The aneuploidogens COL and TUB showed a plateau (= wide peak) in this clastogenic shoulder region. For all clastogens, the integrated area of shoulder over a fitted function (shoulder strength) was evaluated. MMC and CP, thought to have some aneuploidogenic potential, showed an increased shoulder strength compared to TEM, ara-C and URT. The control area distribution had no similarities to the area distribution of either clastogens or aneuploidogens. In a further experiment, we attempted to correlate the size of micronuclei determined after treatment with the aneuploidogenic chemicals to the size of whole chromosomes. Micronuclei found by image analysis which bear chromosome-like structures (judged by light microscopy) were manually identified. This selection of micronuclei was area-distributed to determine the mean size of these micronuclei. None of the peaks and plateaus in the area distributions obtained with the aneuploidogenic chemicals could be attributed to the size of a chromosome.

Technical aspects of automatic micronucleus analysis in rodent bone marrow assays.

The mouse bone marrow micronucleus assay is an in vivo test commonly used in the pharmaceutical industry to evaluate the genotoxic potential of new compounds. The test detects agent-induced chromosomal damage or damage of the mitotic spindle apparatus. In this paper the state-of-the-art in automated rodent micronucleus evaluation using computerized image analysis in combination with high-quality slides obtained by the cellulose column fractionation technique is reviewed. The latter allows the effective removal of nucleated cells from rodent bone marrow. It has been found that automatic micronucleus scoring with the Leitz MIAC image analyzer is substantially faster than labor-intensive manual analysis. Automatic scoring can be performed overnight for up to 16 slides. We have been successfully using automatic micronucleus analysis for the testing of new pharmaceutical drugs for more than 3 years.

In vivo rodent erythrocyte micronucleus assay.

The following summary represents a consensus of the working group except where noted. The items discussed are listed in the order in which they appear in the OECD guideline (474) for easy reference. Introduction, purpose, scope, relevance, application and limits of test. The analysis of immature erythrocytes in either bone marrow or peripheral blood is equally acceptable for those species in which the spleen does not remove micronucleated erythrocytes. In the mouse, mature erythrocytes are also an acceptable cell population for micronucleus analysis when the exposure duration exceeds 4 weeks. Test substances. Organic solvents such as DMSO are not recommended. Freshly prepared solutions or suspensions should be used unless stability data demonstrate the acceptability of storage. Vegetable oils are acceptable as solvents or vehicles. Suspension of the test chemicals is acceptable for p.o. or i.p. administration but not for i.v. injection. The use of any unusual solvent should be justified. Selection of species. Any commonly used laboratory rodent species is acceptable. There is no strain preference. Number and sex. The size of experiment (i.e., number of cells per animal, number of animals per group) should be finalized based on statistical considerations. Although a consensus was not achieved, operationally it was agreed that 2000 cells per animal and four animals per group was a minimum requirement. In general, the available database suggests that the use of one gender is adequate for screening. However, if there is evidence indicating a significant difference in the toxicity between male and female, then both sexes should be used. Treatment schedule. No unique treatment schedule can be recommended. Results from extended dose regimens are acceptable as long as positive. For negative studies, toxicity should be demonstrated or the limit dose should be used, and dosing continued until sampling. Dose levels. At least three dose levels separated by a factor between 2 and square root of 10 should be used. The highest dose tested should be the maximum tolerated dose based on mortality, bone marrow cell toxicity, or clinical symptoms of toxicity. The limit dose is 2 g/kg/day for treatment periods of 14 days or less and 1 g/kg/day for treatment periods greater than 14 days. A single dose level (the limit dose) is acceptable if there is no evidence of toxicity. Controls. Concurrent solvent (vehicle) controls should be included at all sampling times. A pretreatment sample, however, may also be acceptable only in the short treatment period peripheral blood studies. A concurrent positive control group should be included for each experiment.(ABSTRACT TRUNCATED AT 400 WORDS)

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

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

Micronuclei as an index of cytogenetic damage: past, present, and future.

The workshop was designed to present what is known about the production of micronuclei, what protocols are now accepted or proposed internationally, what new results have been obtained, and what new methods and protocols are likely to be forthcoming. This report is designed to convey the flavour of the workshop and to provide the essence of the new information. After the workshop an effort was made to determine what single protocol would satisfy the requirements set for the micronucleus test by as many regulatory agencies as possible. The result, reported here, includes the requirements of six regulatory authorities in Canada, the European Economic Community, the Organization for Economic Co-operation and Development, Japan, and the United States.

Oxidative stress induces DNA damage and inhibits the repair of DNA lesions induced by N-acetoxy-2-acetylaminofluorene in human peripheral mononuclear leukocytes.

Human mononuclear leukocytes were exposed to prooxidants such as H2O2, phorbol-12-myristate-13-acetate, and 4-nitroquinoline-N-oxide, and the effects on induction of DNA damage and repair were evaluated. ADP ribosylation was activated by prooxidant exposure and the response was bimodal with peaks of activation occurring at about 30 min and 4-5 h. Other evidence for prooxidant-induced DNA damage was provided by nucleoid sedimentation assays. Unscheduled DNA synthesis (UDS) was only slightly induced by prooxidant exposure which suggested that either the DNA lesions were repaired by a short patch mechanism involving little UDS, or the repair process was inhibited by prooxidant exposures, or some combination of both. This point was clarified by the fact that the repair of DNA lesions induced by N-acetoxy-2-acetylaminofluorene, an inducer of large patch DNA repair, was inhibited in a dose-dependent manner by exposure to H2O2 and the inhibition was dependent on ADP ribosylation. In contrast, the repair of DNA strand breaks induced by prooxidant exposures as identified above were complete within about 8 h and the repair was independent of ADP ribosylation. Both ADP ribosylation and N-acetoxy-2-acetylaminofluorene-induced UDS were shown to be up- and down-regulated by the redox state of human mononuclear leukocytes indicating a unique mechanism of cellular control over DNA repair.

DNA repair induced by various mutagens in rat hepatocyte primary cultures measured in the presence of hydroxyurea, guanazole or aphidicolin.

Guanazole and aphidicolin were chosen as candidates in the search for a selective, non-genotoxic inhibitor of DNA replication which could be used instead of hydroxyurea to measure DNA repair synthesis in rat hepatocyte primary cultures by liquid scintillation counting. The genotoxicity of these 3 chemicals was studied using the Salmonella/liver homogenate assay and the autoradiographic UDS test in hepatocytes. Hydroxyurea was positive in both of these assays. Guanazole and aphidicolin did not induce DNA repair in hepatocytes. Aphidicolin was not mutagenic for Salmonella typhimurium, whereas guanazole increased the revertant numbers of strain TA102 slightly. The incorporation of [3H]thymidine was measured by liquid scintillation to determine DNA repair induced by 2-acetylaminofluorene (2-AAF), aflatoxin B1, benzo[a]pyrene, cyclophosphamide, H2O2, 6-hydroxydopamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), 4-nitroquinoline-N-oxide and UV irradiation in the presence of either 10 mM hydroxyurea, 15 mM guanazole or 0.015 mM aphidicolin. Aphidicolin had an inhibitory effect on DNA repair. Except for the 3 chemicals mentioned below, the sensitivity of the DNA repair measurement was the same, no matter whether hydroxyurea or guanazole was used to inhibit replicative DNA synthesis. In the presence of hydroxyurea, DNA repair synthesis was found at lower concentrations in the case of aflatoxin B1, due to differences in the solvent control values, and in the case of H2O2, possibly due to a synergistic effect between hydroxyurea and H2O2. Guanazole allowed the detection of DNA repair induced by MNNG at lower concentrations, probably because of an antagonistic effect between hydroxyurea and MNNG. Based on these results, it was concluded that guanazole, but not aphidicolin, could be used instead of hydroxyurea to measure DNA repair synthesis by liquid scintillation in rat hepatocyte primary cultures. Although guanazole does not completely fulfill the criteria for an ideal DNA replication inhibitor, it has the advantage of being less genotoxic than hydroxyurea, and also appears to have a smaller potential to falsify the results by interacting with the test compounds.

Comparison of single/multiple-dose protocols using triethylenemelamine and procarbazine hydrochloride for the mouse bone marrow micronucleus test.

Treatment of mice with a single dose of either 4.8 mg/kg of triethylenemelamine (TEM) or 348 mg/kg of procarbazine hydrochloride (PC) induced higher frequencies of micronucleated polychromatic erythrocytes (MPE) after 48 h than after 24 h. The same observation was made when animals were treated with 1.6 or 8 mg/kg of TEM or 116 or 580 mg/kg of PC for 2 consecutive days (double-dose protocol). Surprisingly, the third dose of either 1.6 or 8 mg/kg of TEM caused lower MPE frequencies at the 72-h than at the 48-h sampling time. The observation that lower MPE frequencies after 72 h were also accompanied by reduced bone marrow toxicity might have reflected a drug-related adaptive reaction of the animals, for example the induction of detoxifying enzymes. Mean MPE frequencies as well as bone marrow toxicity were also slightly decreased after the third dose of either 116 or 580 mg/kg of PC, but statistical analysis showed no differences between the 48-h and the 72-h sampling times as regards the MPE frequencies and bone marrow toxicity. In addition to the high mean MPE frequency observed after 2 doses of 116 mg/kg of PC at the 48-h sampling time, a late increase in micronucleus induction was also seen after triple dosing at the 96-h sampling time. The present experiments with TEM and PC showed similar sensitivity for the multiple-dose assays when compared with the single-dose micronucleus test. In the case of the triple-dose assay, bone marrow toxicity proved to be a critical factor for appropriate dose selection. The computerized image analysis system was a convenient and time-saving tool for the automatic scoring of large quantities of cells for micronuclei as well as for the evaluation of bone marrow depression from the entire cell population analyzed for micronuclei.

The automated bone marrow micronucleus test.

A new technology is presented which offers high-quality slides enabling the fully automated scoring of large quantities of erythrocytic cells for micronuclei by computerized image analysis. The techniques are applicable to bone marrow specimens as well as to peripheral blood obtained from various species of laboratory animals as well as from man. The key steps leading to this improved slide quality are the total removal of nucleated hematopoietic cells and the production of 'flat' cells by cytocentrifugation on polylysine-coated slides. The new procedures also allow the quantitative elimination of artifact-producing leukocytic granules from the rat bone marrow, even for the Fischer-344 strain, thus making the rat micronucleus test an attractive system for routine purposes in genetic toxicology. In addition, the proportion of immature erythrocytes can, if desired, be increased to more than 90% by using a Percoll step-gradient. This greatly facilitates the peripheral blood micronucleus test in laboratory animals as well as in (splenectomized) humans. First results, using peripheral blood from 2 rat strains, indicate that the immature erythrocyte population is very useful for micronucleus analysis, which encourages the development of a rat peripheral blood micronucleus test. This is an interesting application because it allows repeated testing in the same animals, resulting in fewer rats being needed, as no separate control groups are necessary. A further advantage is the possibility of concomitantly using rats from an ongoing toxicological study for micronucleus testing. The present results demonstrate that the new methodology is a valuable tool for improved micronucleus testing. Possible consequences in the field of genetic toxicology are discussed.

Detection of repair of chemical-induced DNA damage in vivo by the nucleoid sedimentation assay.

The nucleoid sedimentation assay was used to study chemical-induced DNA repair in vivo. Nucleoid bodies were prepared from liver and lung of mice at various times after i.p. treatment with 1-methyl-1-nitrosourea or 4-nitroquinoline-1-oxide. Both carcinogens induced a dose-dependent loss in negative DNA supercoiling in liver and lung. The rate of DNA repair of 1-methyl-1-nitrosourea was similar in liver and lung whereas 4-nitroquinoline-1-oxide-induced DNA damage was repaired faster in lung than in liver. Results obtained by the nucleoid sedimentation technique corresponded to measurements of DNA repair by unscheduled DNA synthesis. The nucleoid sedimentation assay should be a useful tool to examine in vivo repair of chemical-induced DNA lesions in various tissues of laboratory animals.

Epoxide hydrolase activity in small biopsy samples of preneoplastic and neoplastic rat liver.

Epoxide hydrolase (EH) activity was measured in serial fine-needle aspiration biopsy (FNAB) samples of male rats chronically treated with the hepatocarcinogen, N-nitrosomorpholine (NNM, 10 mg/100 ml drinking water). The development of neoplasia was confirmed histopathologically at the end of the experiment. The enzyme activity was significantly increased after 2 days of NNM administration and reached a 3 to 4-fold increase after 2 weeks that remained stable throughout the experiment. Although EH is not related to the metabolism of NNM, the induction of enzyme as readily recognized with the FNAB method might be a useful marker in the attempt to characterize hepatocarcinogenic substances in rats.

Distribution of nuclear size and DNA content in serial liver biopsies of rats treated with N-nitrosomorpholine, phenobarbital and butylated hydroxytoluene.

Nuclear size distribution and DNA content distribution were determined in liver biopsies of rats using an electronic particle counter and an impulse cytophotometer, respectively. Nuclear size distribution and DNA content distribution were found to be highly correlated regardless whether peak areas or peak heights of the histograms were compared. Treatment with the hepatocarcinogen NNM caused a dose-dependent increase in octoploid nuclei and a shift of the 2 n : 4 n ratio in favor of the diploid nuclei. Treatment with Pb and BHT had minor effects on nuclear size and DNA content distribution. The paper demonstrates the usefulness of the fine-needle aspiration biopsy procedure for the evaluation of liver changes induced by chemical substances.