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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 15, 2004 as doi:10.1096/fj.04-2729fje. |
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,1
* Nutritional Genomics Center, Children’s Hospital Oakland Research Institute, Oakland, California, USA; and
Department of Nutrition & Food Management and Linus Pauling Institute, Oregon State University, Corvallis, Oregon, USA
2Correspondence: Nutritional Genomics Center, Children’s Hospital of Oakland Research Institute, Oakland, CA 94609, USA. E-mail: bames{at}chori.org
SPECIFIC AIMS
Exposure to environmental stresses such as radiation, poor nutrition, or smoking can cause hazardous lesions in DNA, including double-strand breaks. The micronucleus test is an established method to analyze in vivo chromosomal damage. Conventional micronucleus assays performed in peripheral blood lymphocytes apply a chemical to block cytokinesis after a single cell division, and micronuclei (MN) are manually counted and scored using microscopy. In the current study, we sought to develop a simple assay to detect small increases in MN frequencies in people at risk. To accomplish this, rapid scoring of large numbers of cells was required. Micronuclei are particularly apparent in red blood cells (RBCs), which otherwise lack DNA. In humans, in contrast to mice, micronucleated erythrocytes are soon filtered from the circulating blood by the spleen and so generally are not available for analysis. We combined immunomagnetic separation technique and single-laser flow cytometry to isolate and analyze immature reticulocytes in the peripheral blood for the presence of micronuclei before these cells are removed by the spleen. We used this method to quantitate the micronuclei frequency in smokers.
PRINCIPAL FINDINGS
1. Early reticulocytes can be enriched from whole blood samples using immunomagnetic separation techniques
Newly formed reticulocytes that still possess transferrin receptors (CD71) on their membrane surfaces were isolated from peripheral blood by magnetic separation and identified by FITC antibodies. As demonstrated in Fig. 1
A, a very low percentage of cells show increased CD71 fluorescence in an unenriched sample of peripheral blood from healthy volunteers. In contrast, when using the immunomagnetic separation enrichment for the immature CD71-positive reticulocytes (trf-ret), a significant increase in the percentage of trf-ret is seen (Fig. 1B
). Using this positive selection technique helps enrich the cell population of interest (trf-ret) and enables sensitive detection of micronuclei.
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2. Micronuclei can be detected in immature reticulocytes by flow cytometry
After fixation and permeabilization of trf-ret’s with ultra-cold methanol, we stained the cells for miconuclei using the DNA dye 7-aminoactinomycin D (7-AAD).
Figure 2
shows the distribution of micronucleated trf-ret (MN-trf-ret) after CD71 enrichment and staining with 7-AAD. Immature reticulocytes show increased CD71-FITC staining. Micronucleated reticulocytes also show an increase in 7-AAD fluorescence. To verify that the increase in 7-AAD fluorescence in blood samples was due to increases in DNA content (micronuclei), CD71-enriched samples from four healthy volunteers were preincubated with DNase before 7-AAD staining and FACS analysis. DNase treatment reduced the number of MN-trf-ret by 
90%, confirming that the increase in 7-AAD fluorescence is due to DNA content and is not an artifact.
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3. Micronucleus detection by flow cytometry has low variability and is reproducible
If samples are kept at 4°C and processed within 5 days after blood is drawn, variation among replicates is minimal. To test for assay variation and reproducibility, blood samples from healthy donors were drawn and the micronuclei assay was performed on 3 separate days. Variation was minimal (SD=0.008) between replicate samples processed on 3 consecutive days within 5 days after blood was drawn. To examine interassay differences, donors were sampled three times over 4 months. Little change in micronuclei frequency was seen over time, with values of (mean+SD) 0.096 ± 0.026 for a heavy smoker and 0.014 + 0.004 for a typical nonsmoker.
4. Increased micronuclei frequency can be detected in smokers
Micronuclei frequencies were compared between healthy nonsmokers and smokers. A significant increase in MN-trf-ret in smokers (n=12) vs. nonsmokers (n=15) was evident.
CONCLUSIONS AND SIGNFICANCE
We have developed a simple method to isolate and analyze immature reticulocytes in the peripheral blood to detect the presence of micronuclei. Applying single-laser flow cytometry enables rapid analysis of large numbers of cells. This method will help monitor human populations for genetic damage and should be particularly useful for studies of micronutrient deficiencies, which appear to be a major cause of chromosomal damage. This improved MN assay was used to show that tobacco smoking correlates with elevated micronuclei frequencies, as detected in peripheral blood of normal humans (i.e., with a functioning spleen).
In humans with functioning spleen, micronuclei will only be present in immature reticulocytes, which generally account for <1% of the cells in whole blood. Abramsson-Zetterberg et al. described a method to measure MN in an enriched peripheral blood reticulocyte population. Hoechst 33342 and thiazole orange were used to stain DNA and RNA, respectively. They showed that MN frequencies in reticulocytes approximate those observed in bone marrow. However, their method included multiple laborious steps and required a dual-laser flow cytometer with a UV laser to excite the Hoechst 33342 fluorochrome. This type of flow cytometer is more specialized and not widely available in common laboratory settings. Dertinger et al. further improved the scoring of micronucleated reticulocytes in humans to permit use of widely available bench top instruments, but their procedure did not include reticulocyte enrichment and required long data collection times. Dertinger et al. reported baseline MN frequencies in 10 healthy volunteers with an average of 0.09% MN/total reticulocytes. Their method enabled scoring of only 10,000 CD71-positive reticulocytes per sample. Their data showed a >10-fold range of variation. Unlike Abramsson-Zetterberg et al., our method to enrich immature reticulocytes in RBCs used BioMag Anti-CD71 beads, which do not require a prelabeling procedure. The 1 µm beads are compatible with flow cell analysis, obviating the need to release cells from beads by DNase treatment. The practical importance of this assay is emphasized by the projected statistical power for detection of an increase in 1 micronucleus per 1000 cells. For type I error = 5%, assuming an SD of 0.3 MN/1000 cells within a group (4 subjects per group), would yield a statistical power of 99% to detect an increase of 1 MN/1000 cells.
The present results show this assay can detect a small increase in MN frequency in peripheral blood of people exposed to environmental factors such as smoking. We found a significant increase in micronuclei frequency in smokers vs. nonsmokers. However, the population of smokers in this study included young healthy volunteers generally smoking fewer than 20 cigarettes/day, which may explain the relatively low frequencies of MN within the smokers group. Smokers who smoked more than 20 cigarettes had a trend of a proportional increase in micronuclei frequency.
An international collaborative study was recently undertaken to evaluate the cytokenesis block micronuclei assay (CBMN) and micronucleus frequencies in humans after genotoxic stresses. The Human MicroNucleus project, based on a pooled analysis of data from several laboratories, found a significant increase in MN frequencies only in individuals smoking >30 cigarettes per day. However, a major limitation to the CBMN assay is that it relies on in vitro culturing of lymphocytes and requires cell division. It is possible that while cigarette smoking induces formation of micronuclei in vivo, MN-containing cells may not divide and not be included in the MN scoring. To overcome this, scoring needs to be done in nondividing mononucleated cells. When evaluating micronuclei frequencies in young reticulocytes, no in vitro stimulation of cell division is necessary for the assay, so another advantage of this improved method is that it enables the capturing of in vivo formation of MN, and may be more accurate in representing genotoxic damage to cells.
Most MN assays do not distinguish between aneugenic and clastogenic effects resulting in lagging chromosomes or fragments, respectively. Since various genotoxic exposures may induce only one type of MN, a specific analysis of MN type may improve the sensitivity of detection. Micronuclei containing chromosomes can be distinguished from those containing acentric fragments by the presence of a centromere. Work is under way to develop centromere detection using flow cytometry in the enriched reticulocyte population.
The assay described is simple and enables short data acquisition times with the requirement of single-laser flow cytometer, readily available in most laboratory settings, and should be an excellent tool for genetic monitoring of human populations. Since many vitamin and mineral deficiencies lead to chromosome damage, the assay should be particularly useful when examining nutrition and cancer prevention. In view of the emerging new field of the nutritional genomics of genome stability, the human micronuclei assay may serve as a key biomarker to improve public health.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2729fje;
1 These authors contributed equally. The assay was developed and carried out in Oakland and the smoking study was done in Corvallis. 
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