Validation of a flow cytometric acridine orange micronuclei methodology in rats.

Our laboratory has previously reported a flow cytometric acridine orange method for detection of micronucleus (MN) in the rat using cyclophosphamide as a test compound. To replace the manual method of scoring and satisfy Good Laboratory Practice (GLP) requirements, an extensive validation of the flow method was required. Therefore, manual scoring and flow cytometric determination of MN were compared using vincristine, chlorambucil, methotrexate, and doxorubicin compounds known to induce MN formation with various mechanisms of action. 1,2-Dimethylhydrazine (1,2-DH), a compound with negative or equivocal MN findings also was evaluated. The flow method consistently demonstrated dose- and time-dependent responses for MN production at all concentrations of vincristine, methotrexate, clorambucil, and doxorubicin. In contrast, manual scoring of slides failed to detect an increase in MN at the lowest doses of doxorubicin (1mg/kg) at 24 or 48 h, and methotrexate at 48 h, or any dose of methotrexate (50, 100, or 250 mg/kg) at 24h. Additionally, a dose-response for methotrexate at 48 h, and chlorambucil at 24 h were missed using manual scoring. For 1,2-DH, the flow method showed a low level (< 1.4-fold) increase in MN at all doses and times. In contrast, the manual method showed five-seven-fold increases at 24 h, but a < two-fold increase at 48 h in the highest dose only. These data may suggest that the flow method has a greater sensitivity and possibly accuracy than manual scoring. Significant decreases in polychromatic erythrocytes (PCE) were seen using both methods at approximately the same dose for all compounds. However, absolute flow cytometric PCE values were consistently higher than manual. Additional cytotoxicity parameters obtained by the flow method allowed a more complete assessment of cytotoxicity than PCE alone. Furthermore, data reported here combined with improved throughput, shortened data turnaround and reporting times, and possibly better precision due to evaluation of much larger numbers of cells clearly demonstrate the usefulness of flow cytometry method in the routine micronucleus evaluation.

Historical vehicle and positive control micronucleus data in mice and rats.

The rodent bone marrow micronucleus (MN) assay has been widely used as part of an in vivo genotoxicity test battery in product safety evaluation. In this assay, the historical vehicle and positive control data form an important component in the assay performance and data interpretation. Also, in light of minimizing animal use in research and still obtain required data from a study, the routine use of positive control in every MN assay has been questioned by the scientific community, especially in laboratories which have demonstrated assay reproducibility and conduct studies under Good Laboratory Practice regulations. In this paper, mouse and rat vehicle and positive control MN data, collected manually, are described as a reference for a period of 12 years (1987-1998) in our laboratory. The vehicles generally included a variety of aqueous solutions and suspensions and cyclophosphamide dosed intraperitoneally at 20mg/kg (rats) or 40 mg/kg (mice) served as positive control, in all studies. Based on combined sex data (430 animals), for CD(1) mice, the vehicle control MN polychromatic erythrocyte (PCE) range was 0.9-3.1 with a mean of 1.75 per 1000 PCE and the positive control range (220 animals) was 8.8-42.1 with a mean of 23.1 MNPCE per 1000 PCE. Similarly, for Wistar rats, the vehicle control range (360 animals) was 1.3-5.3 with a mean of 2.6 MNPCE per 1000 PCE and the positive control range (240 animals) was 10.4-33.8 MNPCE per 1000 PCE. Vehicle control ranges reported here are comparable to the literature database and the positive control response was > or = 4-fold over vehicle control, in all studies. These data demonstrate the reproducibility of positive control response in MN assay in our laboratory and support the MN Assay Expert Panel's view that the use of positive control may not be necessary in every study.

Principles and practices of integrating genotoxicity evaluation into routine toxicology studies: a pharmaceutical industry perspective.

In this article, an integrated in vivo genotoxicity testing philosophy and a practical approach, as applied to pharmaceuticals, are described. Recently, there has been an effort to integrate the rodent (primarily rat) micronucleus assay with routine 2-4-week toxicokinetic studies. This approach has several advantages: 1) it utilizes the general principles of toxicology that govern the overall toxicity profile of a test substance; 2) factors such as the dose and/or route of drug administration, drug metabolism, principles of toxicokinetics, and saturation of defense mechanisms are considered in evaluating genotoxicity; 3) it uses the concept of administering multiple tolerable doses aiding in achieving steady state plasma drug levels, which is more relevant for risk assessment compared to high acute doses; and 4) it helps minimize the amount of drug, number of animals used, and other resources. This integration approach can be extended to other toxicology studies and other relevant genotoxicity endpoints may be assessed. Based on the experience in our laboratory, integrating micronucleus assessment in routine toxicology testing is promising and should be utilized when practical.

Use of acridine orange in: flow cytometric assessment of micronuclei induction.

The micronucleus assay is a widely accepted method for evaluation of clastogens and aneugens. In the current study, acridine orange (AO) supravital staining was adapted for flow cytometric usage to assess micronucleated cells in rat bone marrow and spleen. Cyclophosphamide was used as a positive control test compound and results were compared to manual scoring in Wright-stained slides. In bone marrow, both manual and flow cytometric methods demonstrated positive dose response-trends for micronucleated polychromatic erythrocytes (MNPCE). Significant elevations in MNPCE were observed at all doses of cyclophosphamide, and comparisons between methods in bone marrow were not statistically different. The flow cytometric method was more sensitive in spleen samples, showing dose- and time-related increases in micronuclei compared with manual scoring. AO proved to be a sensitive discriminator of RNA and DNA, allowing distinct separation of polychromatic erythrocytes (PCE), normochromic erythrocytes (NCE), total nucleated cells (TNC), and micronucleated populations within both PCE and NCE regions. These results support the use of AO-based flow cytometry to provide a rapid and sensitive indicator of micronuclei inducers.

Use of acridine orange in: flow cytometric evaluation of erythropoietic cytotoxicity.

Cytotoxic insult to bone marrow frequently impairs the proliferating and maturational abilities of erythroid cells. Typically, a ratio of enucleated, immature polychromatic erythrocytes (PCE) to mature normochromatic erythrocytes (NCE) is used to assess cytotoxicity in the micronucleus (MN) assay. The effects of cyclophosphamide (CP) on PCE/NCE ratio in rat bone marrow and spleen were assessed by a newly developed flow cytometric procedure using glutaraldehyde-fixed, acridine orange (AO)-stained cells, and compared to manual scoring of PCE/NCE in Wright stained slides. Comparison of methods showed that manual and flow cytometric determination of PCE were not statistically different. Several other parameters of cytotoxicity could be simultaneously assessed because the method allowed use of unfractionated whole bone marrow/spleen cell samples. Absolute numbers of total nucleated cells (TNC), a ratio of TNC to total erythrocytes (TE), and determination of RNA content within the PCE population demonstrated dose- and time-dependent effects with CP treatment. Shifts in RNA content were particularly sensitive, correctly identifying all CP-treated from control specimens, even in those samples where PCE/NCE ratio was similar. The AO methodology provided a more rapid, statistically-superior, and thorough approach in the assessment of bone marrow and spleen cytotoxicity than the conventional manual method of scoring PCE/NCE ratio alone.

Genotoxicity evaluation of selenium sulfide in in vivo and in vivo/in vitro micronucleus and chromosome aberration assays.

Selenium monosulfide (SeS) was reported to be carcinogenic to livers of male and female rats and livers and lungs of female mice. However, its genotoxicity profile in short-term assays is somewhat equivocal. A multiple endpoint/multiple tissue approach to short-term genetic toxicity testing has been developed in our laboratory. In the present paper, the effect of SeS in in vivo and in vivo/in vitro micronucleus and chromosome aberration assays in rat bone marrow and spleen are reported. In the in vivo assay, small but statistically significant increases in bone marrow micronucleated polychromatic erythrocytes (MNPCEs) were observed 24 h after treatment of rats with 50 mg/kg SeS and 48 h after treatment with 12.5 mg/kg. A significant decrease in the PCE/total erythrocyte (TE) ratio, indicative of cytotoxicity, was observed at the 50 mg/kg dose at the 24-h timepoint. In spleen, no increases in MNPCEs or decreases in the PCE/TE ratios were observed. No evidence of a significant increase in aberrations was observed in bone marrow or spleen. In the in vivo/in vitro assay, no increase in micronucleated binucleated cells or cells with aberrations was observed in SeS-treated rats. The small but statistically significant increases in MN observed in the in vivo study are considered likely not to be biologically significant since no dose-response was observed and all the values obtained were within historical control range in our laboratory. Given the overall genetic toxicity profile of SeS, it appears that SeS may be a weak mutagen and that differences between testing protocols may be very important in determining whether or not it is found to be negative or positive. Histological evidence was obtained in this study that suggests that the liver is the acute target organ of SeS in rats. Given the fact that SeS is selectively hepatocarcinogenic, we are currently testing the hypothesis that the genotoxicity of SeS in rats may be more readily detectable in liver than in bone marrow or spleen.

Simultaneous evaluation of dexamethasone-induced apoptosis and micronuclei in rat primary spleen cell cultures.

Apoptosis or programmed cell death is a biological event that is biochemically and morphologically distinct from cellular necrosis. Nonetheless, its relationship has not been studied in terms of a cytogenetic endpoint such as micronucleus formation. In the present study, based on cytological observations, the incidence of dexamethasone-induced apoptotic cells was related to the frequency of micronucleated cells in vitro. Rat primary spleen cells were grown in 6-well plates with RPMI 1640 media using concanavalin A and lipopolysaccharide as mitogens. At culture initiation, the test agent dexamethasone (10, 20 or 40 microM) and a cytokinesis inhibitor cytochalasin B (3 micrograms/ml) were added. Cultures were harvested 18 h and 40 h later. Slides were prepared and stained with Diff-Quik stain. Frequencies of apoptotic cells and micronucleated binucleate cells were enumerated cytologically based on 500 cells per treatment from the same slides. The results showed a dose-dependent increase in the number of apoptotic cells in rat spleen cultures treated with dexamethasone. At 18 h, the percentages of apoptotic cells were 0.8, 1.6, 3.4 and 4.4 with 0, 10, 20 and 40 microM dexamethasone, respectively. The corresponding percentages of apoptotic cells at 40 h were: 2.8, 2.6, 5.6 and 10.4. However, at the same concentrations of dexamethasone, the micronucleus frequency in binucleate cells remained relatively unchanged. The phenomenon of apoptosis induced by dexamethasone was confirmed biochemically based on a characteristic DNA 'ladder' pattern by gel electrophoresis. These data suggest that dexamethasone at the concentrations which induced apoptosis did not produce cytogenetic damage. Also, these findings indicate that micronucleus formation and nuclear changes leading to apoptosis are separate events and these endpoints may not be closely correlated for dexamethasone.

An in vivo/in vitro method for assessing micronucleus and chromosome aberration induction in rat bone marrow and spleen. 1. Studies with cyclophosphamide.

The mouse micronucleus assay has long been used as an indicator of in vivo genotoxicity. Recently, it was shown that no single protocol is adequate to detect all clastogens. As a first step in developing a potentially more sensitive assay, micronucleus induction by cyclophosphamide (CP) was assessed in an in vivo/in vitro system using rat bone marrow and spleen cells. In each of two independent experiments, two rats/dose were treated i.p. with 0, 20, or 40 mg CP/kg and killed 6 h later. Cultures were then established in the presence of growth stimulants (interleukin-3 and granulocyte-macrophage colony stimulating factor for bone marrow; lipopolysaccharide and concanavalin A for spleen) and cytochalasin B, a cytokinesis inhibitor. Bone marrow cells were harvested and slides prepared 24 h after initiation, while spleen cells were harvested at 48 h. One thousand cells/tissue/group were scored for cell cycle kinetics and 1000 binucleate (BN) cells were scored for micronuclei. In addition, spleen cells were concurrently assayed for chromosome aberrations. A dose-related cell cycle delay was observed in both tissues in both experiments. Bone marrow showed a 6% average background frequency of micronucleated BN cells, while the low dose induced an average of 20%, and the high dose 31%. For spleen, the average control frequency of micronucleated BN cells was 3%, the low dose induced a 40% average frequency, and the high dose 65%. Also in splenocytes, a dose-dependent increase in chromosome aberrations was observed, with an almost 40-fold increase observed over the control value at the high dose. Thus, the in vivo/in vitro approach described here shows great potential in detecting drug induced genotoxicity. Also, spleen appears more sensitive than bone marrow to CP.

An in vivo/in vitro method for assessing micronucleus and chromosome aberration induction in rat bone marrow and spleen. 2. Studies with chlorambucil and mitomycin C.

An in vivo/in vitro system using rat bone marrow cells and spleen cells to assess micronucleus (MN) and structural chromosome aberrations (SCA) simultaneously (Moore et al., 1995) was further developed. In two separate experiments, two rats/dose/experiment were treated i.p. with 0, 5, 10 and 15 mg chlorambucil (CA)/kg or with mitomycin C (MMC) at 0, 1, 2, 4 mg/kg (experiment 1) or 0, 4, 6, and 8 mg/kg (experiment 2) and killed 6 h later. Cultures were then established in the presence of growth stimulants (interleukin-3 and granulocyte-macrophage colony stimulating factor for bone marrow; lipopolysaccharide and concanavalin A for spleen) and cytochalasin B, a cytokinesis inhibitor. Bone marrow cells were harvested 24 h after establishment of cultures, while spleen cells were harvested at 48 h. In addition, spleen cells were concurrently assayed for chromosome aberrations. With the MN endpoint, spleen cells appeared more sensitive than bone marrow cells to the effects of CA due both to a lower background and an increased response. For MMC, bone marrow cells exhibited both a higher background of MN and a greater numerical response than did spleen cells. However, on the basis of a fold-increase over control values, spleen cells were more sensitive than bone marrow cells. In general, the MN endpoint appeared more sensitive than the SCA in spleen cells after treatment with CA or MMC. Thus, the approach described here shows greater potential in detecting genotoxicity.

Immunofluorescent and confocal laser cytometric analyses of centromeres in V79 cells.

Previously, a modified anticentromere antibody (ACA) technique was established in the V79 Chinese hamster lung cells to simultaneously analyze chromosome damage and aneuploidy induced by various agents. Using this method, cyclophosphamide (CP) was evaluated further in the presence and absence of S9 activation for micronucleus/aneuploidy induction. The specific binding nature of ACA to the centromeric region was also analyzed using a confocal scanning laser cytometry. The results indicated that CP was primarily a clastogen and S9 activation was required for its activity. Vinblastine, the positive control for aneuploidy, produced predominantly centromere containing micronuclei and the addition of S9 was not required for its activity. X-radiation, the positive control for clastogenicity, predominantly produced centromere negative micronuclei confirming its clastogenicity. An evaluation of centromeric region under the standard fluorescence microscope indicated that ACA generally binds to most centromeric regions in a cell. However, by confocal imaging it was found that ACA binds to the central core proteins of the centromere region and not to the peripheral proteins.

Comparative mouse micronucleus evaluation in bone marrow and spleen using immunofluorescence and Wright's Giemsa.

Bone marrow and spleen toxicity, clastogenicity and aneugenicity were analyzed in the CD1 mouse using an antikinetochore antibody (AKA) procedure (Krishna et al., Mutation Res., 282, 159-169, 1992). Further, to verify the fluorescence micronucleus (MN) analysis, additional slides were stained with Wright's Giemsa and results were compared. 5 mice per sex were treated with cyclophosphamide (CP) (40 mg/kg) or vincristine (VC) (0.1 or 0.2 mg/kg). Slides were prepared 24 h postdose using a column fractionation procedure. Per animal, 400 total erythrocytes (TEs) for toxicity and 2000 polychromatic erythrocytes (PCEs) for MN per tissue were analyzed. In the fluorescent method, the clastogen, CP, produced MNPCEs predominantly devoid of kinetochores (K) and the aneugen, VC, produced mostly MNPCEs containing K. The MNPCE frequency did not differ significantly between tissues; however, it differed statistically between sexes. On an overall basis, spleen had significantly lower PCE to TE ratios compared to bone marrow. In general, CP and VC caused a small, but statistically significant decrease in PCE frequencies compared to controls, suggesting possible toxicity to these tissues at the given doses. The data on Wright's stain indicated that the proportion of PCEs and MNPCEs in general, were comparable to those using fluorescent stain. This study further confirms the usefulness of an AKA-staining technique in a multiple genetic endpoint evaluation under a single set of microscopic conditions.

Comparative micronucleus quantitation in pre- and post-column fractionated mouse bone marrow by manual and flow methods.

In the present study, cellulose-column fractionation methodology which has been used to eliminate nucleated cells in bone marrow was verified for its usefulness in micronucleus analysis and compared to standard smear methodology using cyclophosphamide as the test compound. Also, the possibility of using column-fractionated cells in the evaluation of micronucleus frequency by flow cytometry has been explored and comparative results are reported. The results indicated that column fractionation was effective in removing nucleated cells from mouse bone marrow and provided clean preparations of polychromatic and normochromatic erythrocytes (PCEs and NCEs). An initial comparison of manual scoring of cyclophosphamide-induced (10, 20 or 40 mg/kg) micronucleus frequency between standard whole bone-marrow smear and column-fractionated cytospun smears from the same animals showed comparable results. In a definitive study, manual scoring of micronuclei in whole bone marrow was compared with the column-fractionated cell preparations quantified manually and using flow cytometry. Statistically significant positive dose-related trends were detected with all 3 methods, with each treatment group having significantly elevated micronucleated PCEs (MNPCEs) compared to the control group. The 3 methods provided comparable MNPCE values for the lower dose groups but diverged somewhat for the high dose group. The flow method yielded similar individual animal variability in the data when compared to the other two methods. These results support the use of column fractionation in the enumeration of MNPCEs and indicate that coupling this technique with flow cytometry may provide a rapid and sensitive method for the conduct of mouse bone-marrow micronucleus studies.

Inhibition of postbinding target cell lysis and of lymphokine-induced enhancement of human natural killer cell activity by in vitro exposure to ultraviolet B radiation.

In vitro exposure of human peripheral blood mononuclear cells (PBMC) to ultraviolet B (uvB) radiation has been shown to inhibit natural killer (NK) cell-mediated cytotoxicity in a dose-dependent fashion. The purpose of this study was to examine the manner by which uvB produced these deleterious effects. Inhibition of NK activity was not due to lethal injury to NK cells since the viability of cell populations enriched for NK activity was greater than 90% with the uvB doses employed. uvB appeared to directly affect NK cells since procedures which removed suppressor mechanisms, such as removal of monocytes and pharmacologic inhibition of the cyclooxygenase pathway, failed to reverse the response. Furthermore, no suppression of activity of unirradiated NK cells could be produced by coincubation of unirradiated NK cells with uv-irradiated NK cells. When the single cell assay for binding and killing was employed to determine at which stage in the lytic sequence inhibition occurred, it was found that binding was normal but lysis of bound targets and the recycling capacity of active NK cells were markedly reduced. At uvB doses above 50 J/m2, both interferon alpha (IFN-alpha) and interleukin 2 (IL-2) were ineffective in augmenting NK cell-mediated cytotoxic reactions after cells had been irradiated with uvB. Furthermore, incubation of NK cells with IFN-alpha prior to irradiation failed to protect against the inhibitory effects. These studies provide evidence that in vitro exposure of NK cells to uvB radiation inhibits their function by a direct nonlethal effect and that this inhibition occurs selectively at the postbinding stage of target cell lysis.

Atopic asthma: T lymphocyte subpopulations.

Atopy is associated with diminished cell-mediated immunity and increased amounts of IgE, both of which may be caused by imbalances of T lymphocyte subsets. We compared the composition of highly purified peripheral-blood T cells of fifteen atopic asthmatics with ten non-atopic control subjects. Each subject was examined on five separate occasions. Indirect immunofluorescence using monoclonal antibodies was used to define T cell subsets. We examined the proportion of T cells with T3 (most T cells), T4 (helper/inducer), T8 (suppressor/cytotoxic), M1 (natural killer), and Ia (activated T cells) surface antigens. Blood was obtained at the same time of day to eliminate the effects of circadian rhythm. Subjects were taking no medications. We found no difference between the groups of the percentage of T cells with T4, T8, M1, and Ia antigens, nor the ratio of T4+ (helper) to T8+ (suppressor) cells. T3 percent was slightly (94.3 vs 92.5%) higher in the atopic group. We conclude that atopic asthma is not associated with imbalances of peripheral-blood T cell subsets.

Atopic asthma: T-cell response to corticosteroids.

Atopic asthma is associated with diminished cell-mediated immunity and elevated levels of IgE, both of which may be caused by imbalances of T-lymphocyte subsets. We analyzed the response of peripheral blood T-cell subsets to two commonly used corticosteroid preparations as a probe of T-cell subset regulation. We administered prednisone (P) 60 mg or 20 mg, beclomethasone dipropionate (BDP) aerosol, 336 micrograms, placebo, or BDP vehicle in a double-blind protocol to 15 atopic asthmatic patients and ten nonatopic subjects. No difference was found between the groups of the baseline number of T-cells with T4, T8, M1, and Ia antigens, nor the ratio of T4+ (helper) to T8+ (suppressor) cells. Five hours after administration of BDP aerosol, BDP vehicle, and oral placebo, there was no change of these values in either the atopic or in the nonatopic group. In contrast, P, 20 and 60 mg, caused a fall of T4/T8 ratio in the atopic, but not in the nonatopic population. Atopic asthma is not associated with baseline imbalances of peripheral blood T-cell subsets, but is associated with an abnormal response to systemic, but not inhaled corticosteroid.

Prednisone and T-cell subpopulations.

Alteration of T-cell subset relationships may cause many of the anti-inflammatory and immunoregulatory effects of glucocorticosteroids. The effect of oral administration of lactose or 60 mg of prednisone on peripheral blood T-lymphocyte subset profile and total eosinophil count (TEC) was examined. A purified T-cell peripheral blood population was obtained and the proportion of T cells with T3, T4, T8, M1, and la surface antigens was determined before and five hours after ingestion of lactose or prednisone. Lactose caused no change of any of the measured values. Prednisone caused a large (72%) decrease of the total lymphocyte number and the TEC (97%) but no change of the proportion of T cells with the previously mentioned antigens. Administration of 60 mg of prednisone does not acutely selectively deplete subclasses of T lymphocytes from peripheral blood.

Elastolysis of insoluble elastin.

We developed an assay for measurement of elastolytic activity using insoluble 3H-labelled particulate elastin adherent to plastic that is capable of detecting 150 picograms of pancreatic elastase. This equals or exceeds the sensitivity of the most sensitive previously reported systems, without requiring sodium dodecyl sulfate treatment of the elastin. Elastin digestion is dependent upon substrate and elastase concentration, but is not linearly related to time. This is partially attributable to elastase denaturation or autolysis under the assay conditions. The assay could easily detect elastase secreted by either peritoneal or alveolar macrophages. Compared to previously described assays using substrates that closely resemble the physiologic substrate, this represents a considerable increase of sensitivity of detection of elastolytic activity of enzymes.