A combined testing protocol approach for mutagenicity testing.

The antischistosomal agent, hycanthone methanesulfonate (HMS), was employed to illustrate the utility of carrying out several mutagenicity tests in a single concurrent animal experiment. Several commonly used procedures that were successfully integrated into a multiple testing protocol included (1) metaphase analysis in bone marrow, (2) micronucleus test in bone marrow, (3) analysis of the urine for mutagenic constituents, and (4) the host-mediated assay using Salmonella typhimurium. In addition to these animal studies, in vitro mutagenicity testing with and without activation was carried out using S. typhimurium. HMS produced positive, dose--response effects in in vitro tests, metaphase analysis, micronucleus test, and urine analysis, but not in the host-mediated assay. The results of these integrated techniques suggest that such a protocol may be a benefit to those concerned with mutagenicity testing of chemicals.     

Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test

As part of a larger literature study on transgenic animals in mutagenicity testing, test results from the transgenic mutagenicity assays (lacI model; commercially available as the Big Blue(R) mouse, and the lacZ model; commercially available as the Mutatrade markMouse), were compared with the results on the same substances in the more traditional mouse bone marrow micronucleus test. 39 substances were found which had been tested in the micronucleus assay and in the above transgenic mouse systems. Although, the transgenic animal mutation assay is not directly comparable with the micronucleus test, because different genetic endpoints are examined: chromosome aberration versus gene mutation, the results for the majority of substances were in agreement. Both test systems, the transgenic mouse assay and the mouse bone marrow micronucleus test, have advantages and they complement each other. However, the transgenic animal assay has some distinct advantages over the micronucleus test: it is not restricted to one target organ and detects systemic as well as local mutagenic effects.     

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 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.     

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.     

Technical aspects of automatic micronucleus analysis in rodent bone marrow assays

The mouse bone marrow micronucleus assay is anin 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 analyis 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.Abbreviations MNE NCE containing micronuclei - MPE PCE containing micronuclei - NCE normochromatic erythrocyte - PCE polychromatic erythrocyte deceased on 25 May 1994     

Fetal liver micronucleus assay in mice of 5-fluorouracil and related compounds

The cytogenetic effects of 5-fluorouracil (5-FU), 1-hexyl-carbamoyl-5-fluorouracil (HCFU) and 1-(2-tetrahydrofuryl)-5-fluorouracil (TF) were examined with the fetal liver micronucleus assay in mice. The frequencies of micronucleated polychromatic erythrocytes (MNPCEs) in fetal liver peaked at 27, 24 and 27 h, respectively, after single intraperitonealinjections into pregnant mice on day 13 of gestation. The highest frequency of MNPCEs by 5-FU treatment in fetal liver was 13.6%, whereas the frequency in maternal bone marrow was only 0.4%. The micronucleus frequency and the number of micronuclei per individual polychromatic erythrocyte were clearly dose-dependent.These results suggest that the micronucleus test in fetal liver has particular advantages compared to maternal bone marrow for evaluating the cytogenetic effects of 5-FU and related compounds after a single treatment. The cytogenetic effect was ranked 5-FU = HCFU > TF, in both a time-course study and a dose-response study of micronucleus distribution.     

Stochastic modeling in toxicokinetics. Application to the in vivo micronucleus assay

A stochastic model for the in vivo micronucleus assay is presented. This model describes the kinetic of the rate of micronucleated polychromatic erythrocytes induced by the administration of a mutagenic compound. For this, biological assumptions are made both on the erythropoietic system and on the mechanisms of action of the compound. Its pharmacokinetic profile is also taken into account and it is linked to the induced toxicological effect. This model has been evaluated by analyzing the induction of micronuclei is mice bone marrow by a mutagenic compound, 6-mercaptopurine (6-mp). This analysis enabled to make interesting remarks about the induction of micronuclei by 6-mp and to put to light an unsuspected wavy kinetic by optimizing the experimental design of the in vivo micronucleus assay.     

The assessment of micronucleated polychromatic erythrocytes in rat bone marrow. Technical and statistical considerations

Traditionally, the mouse is the species of choice for the rodent bone marrow micronucleus assay (MN). However, the rat is used for most other toxicological studies. The suitability of the rat as a test species for the MN was therefore investigated. In this paper, the methodological aspects of the assay have been considered. The distribution and incidence of micronucleated polychromatic erythrocytes (MPEs) on bone marrow slides prepared by two techniques, the conventional smear and the paint-brush technique, were assessed in control and cyclophosphamide-dosed male and females rats. MPEs were shown to be homogeneously distributed when assessed over a large number of PEs on slides prepared by both techniques, but when viewed over a few hundred PEs (less than 500 PEs), the incidence of MPEs on the same slides was seen to vary considerably (0-10 MPEs/500 PEs). Variability was within acceptable limits when at least 1000 PEs/animal were analysed. The spontaneous incidence of MPEs in the AP rat is low (0-2 MPEs/1000 PEs). Cyclophosphamide increased the incidence markedly and there was a wide inter-animal variability in the response (10-40 MPEs/1000 PEs). The paint-brush technique is considered technically simpler and recommended over the smear technique. This study shows that MPEs can be accurately scored in the bone-marrow of the rate provided due consideration is given to staining and sample size of PEs analysed per animal.     

Simplified mouse peripheral reticulocyte micronucleus test with dimethylnitrosamine.

The induction of micronuclei by treatment with dimethylnitrosamine was evaluated and compared in peripheral blood and bone marrow cells of male CD-1 mice. Peripheral blood preparations were made on acridine orange (AO)-coated slides and scanned by fluorescence microscopy. A significant increase in micronuclei was observed 24 h after treatment in bone marrow polychromatic erythrocytes, and 24-48 h after treatment in peripheral reticulocytes. The peak frequency of micronuclei in peripheral reticulocytes was delayed by about 24 h relative to bone marrow polychromatic erythrocytes. This micronucleus test using peripheral blood was shown to be easy to do and as sensitive as the test using bone marrow cells. From this result, it is concluded that the method with AO-coated slides and peripheral blood is as suitable as bone marrow cells for the micronucleus assay.     

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.     

Rapid method for improving slide quality in the bone marrow micronucleus assay; an adapted cellulose column procedure

Micronuclei are routinely scored in anucleate erythrocytes in bone marrow smears stained with acridine orange. Intense fluorescence from the many nucleated cells in the preparations can interfere with micronucleus detection and cause fatigue in the reader. A method for removing nucleated cells by filtering bone marrow through cellulose packed in syringes was developed by Romagna some ten years ago, but has not been used routinely because of the excessive time needed to prepare columns. We have modified the method very simply by filling chromatography columns by pipet with a cellulose suspension. We show here that column filtration of bone marrow does not affect the numbers of micronucleated polychromatic erythrocytes (MN-PCEs) scored from mice treated with the chromosome breaking agents mitomycin C and cyclophosphamide, or the aneuploidy-inducing spindle poisons, colchicine and vinblastine. The extra preparation time is only about half an hour for a full scale micronucleus assay, and results in better slides and faster scoring.     

Comparison of single-, double- or triple-exposure protocols for the rodent bone marrow/peripheral blood micronucleus assay using 4-aminobiphenyl and treosulphan

Two human carcinogens, 4-aminobiphenyl (4AB) and treosulphan (Treo), were tested in male B6C3F1 mice for the induction of micronuclei in bone marrow and peripheral blood cells by 1-, 2- and 3-exposure protocols. Both compounds tested positive. The magnitude of response with respect to the incidence of micronucleated polychromatic erythrocytes by 2- and 3-exposure protocols was considerably higher than by the single-exposure protocol. The peripheral blood results for Treo were as typically seen with a 24-h delay when compared to the bone marrow. The peripheral blood results for 4AB, however, differed from those expected. The incidence of MN-PCE in peripheral blood of animals exposed to 4AB was significantly greater than seen in the bone marrow in 2- and 3-exposure protocols. There was also an increase in the % PCE at the 60 mg/kg dose level as a function of time. Based on these studies, it is concluded that a step-wise scoring scheme may be the best protocol for rodent micronucleus assay, involving a 3-exposure protocol with single sampling of bone marrow (24 h after the last treatment) and two samplings of peripheral blood (24 h and 48 h after the first treatment). This approach is cost-effective, it limits the number of animals required and provides maximum sensitivity.     

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.     

Comparison of single and multiple treatment regimens in the mouse bone marrow micronucleus assay for hydroquinone (HQ) and cyclophosphamide (CP)

Three experiments are reported as a contribution to the validation of the multiple treatment protocol presently under discussion. (1) In the single treatment experiment with hydroquinone (HQ) the time of maximum micronucleus response was determined to be 24 h with a dose of 75 mg/kg given intraperitoneally. In the subsequent dose-response study at the 24-h interval the lowest positive dose was 50 mg/kg of HQ (P less than 0.01). The increase in micronucleus frequencies was non-linear. (2) Daily treatments with 15 or 75 mg/kg of HQ and bone marrow sampling on days 2-4 after the start of treatment resulted in an increased micronucleus yield for the lower dose (P less than 0.05) and a decrease in micronucleus frequencies at the higher dose (P less than 0.01) with increasing numbers of treatments. At the same time no change in the composition of the erythrocyte population was observed. (3) With 25 mg/kg of cyclophosphamide (CP) the micronucleus yields increased from 1 to 3 daily doses but dropped significantly from 3 to 4 daily doses (P less than 0.01). The polychromatic to normochromatic erythrocyte ratio was significantly decreased after single (P less than 0.01) and marginally lower than the control value after 4 daily treatments with CP. The present data suggest that the response of the bone marrow micronucleus assay to multiple treatments may depend on the dose employed and may differ from chemical to chemical. The specific clastogenic action or other cellular effects of a chemical may influence the micronucleus yields of an individual chemical and it may be difficult to generate a strict protocol recommendation.     

In vivo erythrocyte micronucleus assay III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single dose-level limit test

The in vivo micronucleus assay working group of the International Workshop on Genotoxicity Testing (IWGT) discussed new aspects in the in vivo micronucleus (MN) test, including the regulatory acceptance of data derived from automated scoring, especially with regard to the use of flow cytometry, the suitability of rat peripheral blood reticulocytes to serve as the principal cell population for analysis, the establishment of in vivo MN assays in tissues other than bone marrow and blood (for example liver, skin, colon, germ cells), and the biological relevance of the single-dose-level test. Our group members agreed that flow cytometric systems to detect induction of micronucleated immature erythrocytes have advantages based on the presented data, e.g., they give good reproducibility compared to manual scoring, are rapid, and require only small quantities of peripheral blood. Flow cytometric analysis of peripheral blood reticulocytes has the potential to allow monitoring of chromosome damage in rodents and also other species as part of routine toxicology studies. It appears that it will be applicable to humans as well, although in this case the possible confounding effects of splenic activity will need to be considered closely. Also, the consensus of the group was that any system that meets the validation criteria recommended by the IWGT (2000) should be acceptable. A number of different flow cytometric-based micronucleus assays have been developed, but at the present time the validation data are most extensive for the flow cytometric method using anti-CD71 fluorescent staining especially in terms of inter-laboratory collaborative data. Whichever method is chosen, it is desirable that each laboratory should determine the minimum sample size required to ensure that scoring error is maintained below the level of animal-to-animal variation. In the second IWGT, the potential to use rat peripheral blood reticulocytes as target cells for the micronucleus assay was discussed, but a consensus regarding acceptability for regulatory purposes could not be reached at that time. Subsequent validation efforts, combined with accumulated published data, demonstrate that blood-derived reticulocytes from rats as well as mice are acceptable when young reticulocytes are analyzed under proper assay protocol and sample size. The working group reviewed the results of micronucleus assays using target cells/tissues other than hematopoietic cells. We also discussed the relevance of the liver micronucleus assay using young rats, and the importance of understanding the maturation of enzyme systems involved in the processes of metabolic activation in the liver of young rats. Although the consensus of the group was that the more information with regard to the metabolic capabilities of young rats would be useful, the published literature shows that young rats have sufficient metabolic capacity for the purposes of this assay. The use of young rats as a model for detecting MN induction in the liver offers a good alternative methodology to the use of partial hepatectomy or mitogenic stimulation. Additional data obtained from colon and skin MN models have been integrated into the data bases, enhancing confidence in the utility of these models. A fourth topic discussed by the working group was the regulatory acceptance of the single-dose-level assay. There was no consensus regarding the acceptability of a single dose level protocol when dose-limiting toxicity occurs. The use of a single dose level can lead to problems in data interpretation or to the loss of animals due to unexpected toxicity, making it necessary to repeat the study with additional doses. A limit test at a single dose level is currently accepted when toxicity is not dose-limiting.     

Prior bleeding enhances the sensitivity of the in vivo micronucleus test

It has been reported that the sensitivity of the in vivo mouse bone marrow micronucleus test can be increased by inducing erythropoiesis with exogenous erythropoietin prior to treatment (Suzuki et al., 1989). In these studies we demonstrate that removing approximately 0.5 ml of blood from an adult male BDF1 mouse, another method for increasing the rate of erythropoiesis, synergistically increased the frequency of bone marrow micronucleated polychromatic erythrocytes induced by mitomycin C, with maximal enhancement occurring when the mutagen was given 24 h after bleeding. This enhancement response was also demonstrated for benzo[a]pyrene and dimethylnitrosamine but not for 2-acetylaminofluorene. These results indicate that bleeding mice prior to chemical treatment is a simple method for increasing the sensitivity of the micronucleus assay.     

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

A micronucleus assay using mouse peripheral blood and supravital staining with acridine orange (AO) was validated by two laboratories on triethylenemelamine-treated mice. Dose- and time-dependent increases in micronucleated peripheral reticulocytes were observed. This new method can be used as an alternative to the conventional bone marrow micronucleus assay.     

The mouse peripheral blood micronucleus test with 2-acetylaminofluorene using the acridine orange supravital staining method.

The peripheral blood micronucleus test using the acridine orange (AO) supravital staining method was validated with the potent bone marrow clastogen 2-acetylaminofluorene (2-AAF). 2-AAF induced micronuclei in peripheral blood reticulocytes dose-dependently as well as in bone marrow polychromatic erythrocytes. The incidence of micronucleated reticulocytes (MNRETs) peaked 48 h after a single treatment in both CD-1 and BDF1 mice, and the incidence of micronucleated polychromatic erythrocytes (MNPCEs) peaked 24 or 48 h after treatment. The maximum incidences of MNRETs were always higher than those of MNPCEs in both mouse strains treated once. In the double-treatment regime, the maximum incidence of MNRETs was observed at 24 h after the second treatment in each strain. The incidences of MNRETs in BDF1 mice were higher than in CD-1 mice after a single treatment but were comparable after double treatment. These results indicate that the peripheral blood micronucleus test using AO supravital staining is as sensitive as the conventional bone marrow assay. The new staining method can be performed more easily than the original smear method using either bone marrow or peripheral blood cells. Thus, the peripheral blood method using AO supravital staining is a possible 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.