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.     

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.     

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 reticuiocytes dose-dependently as well as in bone marrow polychromatic erythrocytes. The incidence of micronucleated reticuiocytes (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.     

Micronucleus test with vincristine sulfate and colchicine in peripheral blood reticulocytes of mice using acridine orange supravital staining.

The induction of micronuclei in mouse peripheral blood reticulocytes (RETs) was studied with the spindle poisons vincristine sulfate (VINC) and colchicine (COL) using acridine orange (AO) supravital staining. Each chemical was studied independently in two laboratories using the same protocol. Blood samples were prepared at 0, 24, 48, and 72 h after a single intraperitoneal treatment with VINC (0.0625, 0.125, and 0.25 mg/kg) or COL (0.25, 0.5, 1.0, and 2.0 mg/kg). Both VINC and COL induced micronucleated RETs (MNRETs) significantly and dose-dependently with a peak at 48 h after treatment. Maximum frequencies of micronucleated polychromatic erythrocytes (MNPCEs) were observed 24 h after treatment with VINC; thus, the transition time from MNPCEs to MNRETs was about 24 h. Both spindle poisons gave comparable results in the paired laboratories, indicating that the present AO supravital staining method is highly reproducible.     

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.     

Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining: The summary report of the 5th collaborative study by CSGMT/JEMS · MMS

The main goal of the Collaborative Study Group for the Micronucleus Test (CSGMT) was to validate a new method for the micronucleus test, recently introduced by Hayashi et al. (1990), using mouse peripheral blood cells stained supravitally with acridine orange (AO). The micronucleus tests were performed on CD-1 mice using 23 chemicals with various modes of action. As a rule, one chemical was studied by two participants. Peripheral blood sampled from the same animal was examined 0, 24, 48, and 72 h (or longer) after treatment. The frequencies of micronucleated peripheral reticulocytes (MNRETs) were recorded based on observation of 1000 reticulocytes per mouse.All chemicals induced MNRETs dose-dependently. Interlaboratory differences in the induction of MNRETs were in an acceptable range for most chemicals tested. Although differences were observed with some chemicals, there were no discrepancies in qualitative judgment. Most chemicals gave the greatest response 48 h after treatment, which was less variable than in the bone marrow assay (greatest response, 24–48 h). These results suggest that the peripheral blood assay using the AO supravital staining technique generates reproducible and reliable data to evaluate the clastogenicity of chemicals. This makes the peripheral blood micronucleus assay an attractive alternative to the conventional bone marrow assay.     

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

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

A staining procedure for micronucleus test using new methylene blue and acridine orange: specimens that are supravitally stained with possible long-term storage

The micronucleus test has been widely used as an in vivo cytogenetic test. It employs two different kinds of supravital staining methods which use either new methylene blue (N) and Giemsa (G) or acridine orange (AO). We have developed a new staining procedure for the preparation of specimens supravitally stained with possible long-term storage, using both N and AO. This N/AO-staining method involves three steps; (1) combination of the target tissue or target cells with an equivalent volume of 0.5% solution of new methylene blue (N-staining step), (2) immediate smear of the mixture, followed by treatment with methanol for 10 min for fixation and removal of N and drying (referred to as fixed-decolorized specimens), and (3) staining with 0.007% solution of AO for 3 min, followed by washing with Sörensen's buffer (pH 6.8) and covering of specimens before observation (AO-staining step). To examine whether the N/AO-staining method is useful for the micronucleus test, comparisons were made between N-, N/AO-, and AO-stained specimens prepared supravitally from peripheral blood of rats with and without treatment of cyclophosphamide. The results indicate that N/AO-stained specimens can be supravitally observed after long-term storage with the same coloration and comparable frequencies of micronucleated reticulocytes with a positive response as AO-stained specimens, if the staining process is temporarily stopped before AO-staining (as fixed-decolorized specimens), or if the AO-staining step is repeated. The results also showed that separated reticulocyte types are supravitally stained in a similar fashion to N-stained specimens but not to AO-stained specimens, indicative of the preservation of the supravital feature of N-staining. Taken together these results suggest that the N/AO-staining procedure could offer an additional useful staining tool for the micronucleus test.     

A staining procedure for micronucleus test using new methylene blue and acridine orange: specimens that are supravitally stained with possible long-term storage

The micronucleus test has been widely used as an in vivo cytogenetic test. It employs two different kinds of supravital staining methods which use either new methylene blue (N) and Giemsa (G) or acridine orange (AO). We have developed a new staining procedure for the preparation of specimens supravitally stained with possible long-term storage, using both N and AO. This N/AO-staining method involves three steps; (1) combination of the target tissue or target cells with an equivalent volume of 0.5% solution of new methylene blue (N-staining step), (2) immediate smear of the mixture, followed by treatment with methanol for 10 min for fixation and removal of N and drying (referred to as fixed-decolorized specimens), and (3) staining with 0.007% solution of AO for 3 min, followed by washing with S?rensen's buffer (pH 6.8) and covering of specimens before observation (AO-staining step). To examine whether the N/AO-staining method is useful for the micronucleus test, comparisons were made between N-, N/AO-, and AO-stained specimens prepared supravitally from peripheral blood of rats with and without treatment of cyclophosphamide. The results indicate that N/AO-stained specimens can be supravitally observed after long-term storage with the same coloration and comparable frequencies of micronucleated reticulocytes with a positive response as AO-stained specimens, if the staining process is temporarily stopped before AO-staining (as fixed-decolorized specimens), or if the AO-staining step is repeated. The results also showed that separated reticulocyte types are supravitally stained in a similar fashion to N-stained specimens but not to AO-stained specimens, indicative of the preservation of the supravital feature of N-staining. Taken together these results suggest that the N/AO-staining procedure could offer an additional useful staining tool for the micronucleus test.     

Snapper (Pagrus auratus) leucocyte proliferation is synergistically enhanced by simultaneous stimulation with LPS and PHA

Important channels of communication between mammalian leucocytes have long been recognised. Here, data are reported that suggest similar integrations may occur between snapper leucocytes upon mitogen stimulation. Cell surface immunoglobulin (IgM) expression was used in conjunction with intracellular fluorescence staining and flow cytometry to differentiate proliferating peripheral blood leucocyte subsets (PBLs). Independent activation using phytohaemagglutinin (PHA) or lipopolysaccharide (LPS) drove both mIg(-)and mIg(+)cells into cycle. It is not known if the proliferation of mIg(+)cells was mediated by a mutually exclusive effect of the mitogen on each cell population, cognate cellular interaction or a soluble growth factor. Simultaneous activation of PBLs with PHA and LPS consistently induced significantly more cells to proliferate than the sum of proliferating cells stimulated solely with PHA or LPS. Together, the results suggest that different leucocyte subsets have the capability to influence their respective responses to mitogenic stimulation. Therefore, like in the mammalian immune system, communication may occur between snapper leucocyte subsets.     

Role of XIAP protein, a human member of the inhibitor of apoptosis (IAP) protein family, in phytohemagglutinin-induced apoptosis of human T cell lines

A new family of human genes xiap, hiap-1 and hiap-2, which are homologous to the baculovirus iap (inhibitor of apoptosis) genes cp-iap and op-iap, has been recently cloned and shown to suppress apoptosis after serum withdrawal or exposure to a free radical inducer. In order to examine the role of one of these human genes, namely xiap, in lymphoid cells, we studied XIAP expression, after PHA stimulation in three different human T cell lines. We report here that stimulation with PHA resulted in the human T cell lines undergoing apoptosis, as assessed by DNA fragmentation and by propidiumiodide (PI) staining and flow cytometry. When XIAP protein expression was evaluated by Western blot, we observed that the induction of apoptosis by PHA was associated with a parallel decrease of XIAP expression. We also provide evidence that stably transfected Jurkat cells containing the xiap open reading frame became resistant to PHA-induced apoptosis. These data suggest a role for XIAP protein in the regulation of apoptosis in lymphoid cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Suppression of Interleukin-2 Receptor Expression on Mouse CD4+ T Cells by Bovine κ-Caseinoglycopeptide

Bovine K-caseinoglycopeptide (residues 106–169, CGP) completely inhibited the PHA-induced proliferation of mouse splenocytes when CGP was added simultaneously with PHA. The inhibitory effect, however, was reduced to about one-half when CGP was added after 24 h of cultivating the splenocytes with PHA or when anti-IL-lra antibody was added simultaneously with PHA and CGP. On the other hand, CGP bound to mouse CD4 ⁺ T cells but not to CD8⁺ T cells. CGP suppressed IL-2 receptor expression of the PHA-stimulated mouse CD4⁺ T cells.     

Different instability of nuclear DNA at acid hydrolysis in cancerous and noncancerous cells as revealed by fluorescent staining with acridine orange

Ehrlich cancer cells and inflammatory cells in mouse ascitic fluid were hydrolyzed and stained with acridine orange (AO). The AO hydrolysis curves for G1/G2 + M phase cancer cells and inflammatory cells were differentially determined using flow cytometry by monitoring the metachromatic red-shifted fluorescence of the fluorochrome bound to the single-stranded DNA produced by acid hydrolysis. By computer fitting of the Bateman function to the hydrolysis curves, the kinetic parameters k1 (rate constant for the production of single-stranded DNA), k2 (rate constant for the degradation of the produced single-stranded DNA), and y0 (theoretical value of the single-stranded DNA present initially) were determined. It was found that the k2 value, which reflects the degree of DNA instability, was much higher for cancer cells in both the G1 and G2 + M phases than for inflammatory cells. This finding led us to develop a method for the differential AO staining of cancer cells and non-cancerous cells utilizing the different degree of DNA instability at acid hydrolysis. AO staining after hydrolysis with 2N HCl at 30 degrees C for 8.5 min was found to be the optimal method. In the 60 cases of human malignant epithelial and nonepithelial tumors tested, all of the malignant tumor cells emitted metachromatic red fluorescence, while all of the nonmalignant tumor cells (5 cases of benign tumor) and normal cells emitted orthochromatic green fluorescence when observed with a violet excitation light under a fluorescence microscope. This new technique can be a useful tool for the screening of malignancy in exfoliative cytology and also for basic cancer research.     

The induction of the human T-cell growth factor receptor precedes the production of RNA and occurs in the presence of inhibitors of RNA synthesis

The receptor for T-cell growth factor (TCGF) is an activation antigen that is present in low amounts on a small fraction of resting T lymphocytes. The TCGF receptor on human T cells can be detected with the anti-Tac monoclonal antibody within 7-12 h of stimulating the cells with phytohemagglutinin (PHA). In the current studies, we examined human lymphocytes cultured alone, with PHA, or with PHA plus sufficient actinomycin-D to inhibit RNA synthesis. After varying intervals, aliquots of the lymphocytes were stained with acridine orange (AO) or pyronin-Y(PY) to measure RNA and/or with anti-Tac plus FITC goat anti-mouse Ig. Tac expression began to increase after 6-8 h incubation with PHA, whereas increases in PY or AO staining were not detected until 12 h or later. Furthermore, the initial increase in Tac expression was not affected by sufficient actinomycin-D to block all detectable nucleic acid synthesis. Therefore, it appears that the initial expression of TCGF receptors detected after lymphocyte activation does not require de novo production of RNA.     

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.     

Critical evaluation of techniques to detect and measure cell death--study in a model of UV radiation of the leukaemic cell line HL60.

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V-FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.     

Effect of multiplication stimulating activity (MSA) on intracellular cAMP levels and adenylate cyclase activity in chick embryo fibroblasts.

Stimulation of hamster lymph node cells, splenocytes and thymocytes by the mitogen phytohemagglutinin-P (PHA) was found to be greatly enhanced by addition of 1–10 mM LiCl to the cultures. Lithium enhanced stimulation, as determined by [³H]TdR incorporation, only if added within the first 24 h of culture. The enhancing effect of lithium was specific for this monovalent cation since equivalent concentrations of KCl or NaCl did not induce a similar effect on [³H]TdR incorporation. The divalent cations Mg²⁺ (1–10 mM) and Ca²⁺ (1-1.6 mM), also had an enhancing effect on PHA stimulation. However, addition of Li⁺ to cultures enhanced with Mg²⁺ and/or Ca²⁺ led to an additional potentiation of the response to PHA. These results suggest that Li⁺ modifies a unique early event during stimulation of lymphoid cells by this mitogen.     

Comparison of different staining procedures for the flow cytometric analysis of U-937 cells infected with different Leishmania-species.

The human macrophage cell line U-937 infected with different Leishmania species, Leishmania mexicana amazonensis (Lma), Leishmania donovani (Ld) and Leishmania infantum (Li), was analyzed by flow cytometry (FCM). Leishmania spp. were labeled with different stains prior to the infection of the U-937 cells (BCECF-Am, PKH2-GL and SYTO 17) or after the infection (AO, FITC-conjugated monoclonal antibodies, PI). Infected cells were analyzed by flow cytometry, fluorescence microscopy and in parallel microscopically after Giemsa staining. The data obtained by these two methods were compared to decide which method is mostly appropriate for detection and estimation of the infection rate. Three fluorescent stains were suitable: BCECF-Am, SYTO 17 and FITC-conjugated MoAb with 0.02% digitonin. None of the vital stains gave evaluable results after 3 days of incubation.     

Permeabilization of lymphocytes with polyethylene glycol 1000. Discrimination of permeabilized cells by flow cytometry

The toxicity of polyethylene glycol 1000 (PEG) used similarly as in cell hybridization experiments, has been studied by flow cytometry, measuring the light scattering and fluorescence distributions of PEG-treated human lymphocytes stained with propidium iodide, fluorescein diacetate and acridine orange (AO). The sensitivity of these tests to detect permeabilized, or potentially dead cells, was equal. In addition, PEG proved to interfere with AO staining most likely through the inhibition of its binding to nucleic acids. The decrease of AO fluorescence in cells killed by PEG was unexpected since intercalation of propidium iodide was the same as in alcohol fixed cells. Permeabilization of cells by PEG appears to be an all-or-none phenomenon, accompanied by entrance of PEG into the cells. The findings are described in the context of a review of the currently used flow cytometric techniques to discriminate viable and lethally affected cells; also, the problems of interpretation are discussed.