Testing chemicals for the ability to induce numerical or structural chromosomal damage is easily accomplished by using the micronucleus assay. A "micronucleus" is literally a small nucleus. The nucleus is the organelle in the cell that contains the genetic material (DNA) that directs normal cellular function and cellular reproduction. In cells of eukaryotic organisms, the nucleus contains DNA packaged into chromosomes. Chromosome shape, size, and number are constant for a species. During cell division, the genetic material replicates and then divides equally between the two daughter cells that are produced. If the process is disrupted, or the chromosomes are broken or damaged by chemicals or radiation, then the distribution of genetic material between the two daughter nuclei during cell division may be affected and pieces or entire chromosomes may fail to be included in either of the two daughter nuclei. When this occurs, the genetic material that is not incorporated into a new nucleus may form its own "micronucleus" which is clearly visible with a microscope. Thus, in the micronucleus test, animals are treated with a chemical and then the frequency of micronucleated cells is determined at some specified time after treatment. If a treated group of animals shows significantly higher frequencies of micronucleated cells than do the untreated control animals, then the chemical is considered to be capable of inducing structural and/or numerical chromosomal damage.
The NTP is interested in determining a chemical's ability to induce chromosomal damage because it has been shown that most, if not all, cancers are characterized by chromosomal changes that are frequently specific to a particular tumor-type. Also, induction of aneuploidy (an abnormal chromosome count) or chromosomal rearrangements in germ cells (eggs and sperm) is a cause of birth defects, fetal deaths, and infertility in animals; therefore, a chemical that can produce chromosomal damage in somatic cells, perhaps presenting a risk of carcinogenicity, may also carry a risk of producing damage in germ cells, resulting in adverse reproductive outcomes.
The micronucleus test is performed in a variety of ways, depending upon the questions the investigator is attempting to answer, the test organism, the cell type that is assayed, and the mode of action of the chemical. The NTP conducts micronucleus tests in rats or mice, and the tissues most often assessed for frequency of micronuclei are bone marrow and peripheral blood. Erythrocytes (red blood cells) are the cells that are scored in the bone marrow or the blood for presence of micronuclei. Micronucleus tests must be performed on cells that are dividing. Erythrocytes arise from "stem cells" in the bone marrow and are produced by a series of divisions in a precursor cell population. The constant, rapid turnover of precursor cells makes erythrocytes an ideal cell type for a micronucleus test. Another unique feature of the erythrocyte is that in the cell division immediately prior to formation of the fully differentiated erythrocyte, the nucleus is pushed out of the cell: erythrocytes are the only mammalian cell type that does not contain a nucleus, and therefore, the differentiated erythrocyte cannot further divide. Thus the bone marrow stem cells are continuously producing new erythrocytes to replace the ones that eventually die. If a chemical damages a stem cell and a micronucleus is formed as a consequence of this damage, the micronucleus remains in the cell after the main nucleus has been pushed out and is very easy to observe microscopically.
Bone marrow assays
Rat and mouse bone marrow micronucleus tests typically employ 1 to 3 treatments of the chemical under study; treatments are administered at 24 hr. intervals, and there are normally 5 male animals per treatment group. Doses extend up to the maximum tolerated dose. The route of administration in these short-term tests is usually either intraperitoneal injection or oral gavage. Based on the cell cycle and maturation times of the erythrocytes, harvesting of the bone marrow usually occurs 24 hours after the last dosing; this interval is indicated in the data tables as "sample collection time." At that time, about 50% of the erythrocytes in the bone marrow are immature, newly formed erythrocytes, and these are the cell types that are checked for presence of micronuclei. The animals are euthanized by CO2 inhalation and the femurs are removed.
The bone marrow is flushed from the femurs and spread onto slides. The slides are air-dried, fixed, and stained with a fluorescent DNA-specific stain that easily illuminates any micronuclei that may be present. Typically, 2000 polychromatic erythrocytes (PCEs, reticulocytes; immature erythrocytes) are scored per animal for frequency of micronucleated cells in each of 5 animals per dose group. In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow is scored for each dose group as an indicator of chemical-induced toxicity. In non-treated healthy mice and rats, the %PCE in bone marrow is usually around 50-60%. If a chemical interferes with the production of erythrocytes in the bone marrow, then the %PCE in the bone marrow may decline from the typical normal level. Conversely, if erythrocyte production is stimulated by chemical exposure, then a higher percentage of immature erythrocytes may be observed.
As part of these bone marrow micronucleus tests measuring induction of chromosomal changes after short-term exposures to potentially mutagenic agents, peripheral blood samples are sometimes taken, usually about 48 hr after treatment. This sample collection time is based on cell cycle and erythrocyte maturation data, as well as the timing of erythrocyte translocation from the bone marrow compartment to the peripheral circulation. Thus, in these instances, although micronuclei are induced in erythrocytes in the bone marrow, it is the circulating erythrocyte population that is analyzed. In these cases, 2000 PCE are analyzed for frequency of micronucleated cells, as described above, but 1000 erythrocytes are scored for determination of %PCE, since the percentage of PCE in blood is only around 3-5% in a healthy animal.
Micronucleus analysis in NTP toxicity studies
Standard slide scoring procedure
The NTP also routinely conducts peripheral blood micronucleus tests on mice that are treated in the 13-week toxicity studies as part of the bioassay program. At the end of the 13-week exposure period (routes of exposure: inhalation, dosed-feed, drinking water, oral gavage, skin painting), a blood sample is obtained from male and female mice in each dose group (usually 10 animals per treatment group per sex) and slides are prepared, fixed and stained as for the bone marrow studies. Sample collection time is typically between 0 (in the case of continuous exposures) and 24 hours (in the case of single daily exposures). 1,000 to 10,000 mature erythrocytes (normochromatic erythrocytes or NCEs) are scored per animal for presence of micronuclei. These mature erythrocytes represent about 95% or more of the circulating erythrocytes. The percent PCE is determined in the blood as a measure of chemical-induced toxicity to the bone marrow. All data are analyzed separately for male and female mice.
The acridine orange staining procedure that is used for micronucleus slides allows the scorer to differentiate between the recently formed, immature erythrocytes (polychromatic or PCE) that are less than 48 hr old, and mature erythrocytes 2-35 days old (normochromatic or NCE) based on their staining characteristics. PCE contain residual RNA and thus they stain differently than the NCE that no longer have residual RNA. MN in PCEs arise from damage that occurred recently (within the past 48 hr) and the NCE population shows the result of damage accumulated over the past month, with the NCE population being in steady state equilibrium in the peripheral blood (newly damaged or undamaged erythrocytes are moving from bone marrow to blood at the same rate as old erythrocytes -- the NCEs-- are being removed from the blood). Thus, for longer-term peripheral blood MN studies, it is usually more appropriate to score MN in the NCE population. The mouse spleen is inefficient in removing damaged erythrocytes from circulation (thus permitting the achievement of steady state), but the rat spleen quickly eliminates micronucleated erythrocytes from circulation. Therefore, only mice can be used in a longer-term peripheral blood MN test that analyzes the NCE population. For acute studies, particularly those in which bone marrow tissue is analyzed, PCEs are scored.
Flow cytometric analysis
Recently, it has become possible to analyze peripheral blood erythrocytes for frequency of MN using flow cytometry. In studies using flow cytometry, blood samples are obtained within 24 hr of the last dosing from mice and/or rats that are treated in the 13-week toxicity studies as part of the bioassay program, or that are treated for shorter periods of time in acute assays conducted independently at the genetic toxicity testing laboratory.. The heparinized blood samples are fixed in ultracold methanol and analyzed for frequency of micronucleated erythrocytes using a flow cytometer; both the NCE and the PCE populations can be analyzed separately by employing special cell surface markers to differentiate the two cell types. Because the very young erythrocyte population can be targeted using this technique, rat blood samples can be analyzed for damage that occurred within the past 24-48 hr., before the action of the rat spleen appreciably alters the percentage of micronucleated reticulocytes in circulation. Thus, the use of flow cytometry permits the assessment of micronucleus induction using blood samples instead of requiring collection and processing of bone marrow (Witt et al., 2008).
With flow cytometry, approximately 20,000 PCEs (rats and mice) and 1,000,000 NCEs (mice only) are routinely scored for frequency of MN. %PCE determinations are also made. In addition, the DNA content of micronuclei is estimated, providing an indication of whether a MN arose through chromosome breakage (clastogenic event) or whole chromosome loss (aneuploidy).
A formal statistical analysis of the data is performed that includes a trend test, to determine if there is an overall increase across all doses in the frequency of cells containing micronuclei, and a pairwise comparison of each dose group to the corresponding control, to see if any one dose group is statistically different from the control group in frequency of micronucleated cells. Data are typically presented as the mean number of micronucleated cells per 1,000 cells for each treatment group. A positive trend test is one in which the P value is equal to or less than 0.025. For the slide-based micronucleus data, the micronucleus frequency in any dose group is considered significantly elevated over the control group if the P value is equal to or less than 0.025 divided by the number of chemical-treatment groups. Thus, if the number of treated groups is 3, then the required pairwise P value is 0.008. This adjustment in the pairwise P value is a correction for multiple comparisons of the same data. In the short-term studies, tests that give positive results are repeated to confirm the response. The 13-week micronucleus tests are not repeated, since they are included as part of the NTP subchronic toxicity tests which are not repeated.
For micronucleus data obtained through flow cytometric analysis of blood samples, approximately 1 x 106 erythrocytes and 20,000 reticulocytes are scored for presence of micronuclei. Based on prior experience with the large number of cells scored using flow cytometric scoring techniques (Kissling et al., 2007), it is reasonable to assume that the proportion of micronucleated reticulocytes is approximately normally distributed. The statistical tests selected for trend and for pairwise comparisons with the control group depend on whether the variances among the groups are equal. Levene's test at α = 0.05 is used to test for equal variances. In the case of equal variances, linear regression is used to test for a linear trend with dose and Williams' test is used to test for pairwise differences between each treatment group and the control group. In the case of unequal variances, Jonckheere's test is used to test for linear trend and Dunn's test is used for pairwise comparisons of each treatment group with the control group. To correct for multiple pairwise comparisons, the p-value for each comparison with the control group is multiplied by the number of comparisons made. In the event that this product is greater than 1.00, it is replaced with 1.00. Trend tests and pairwise comparisons with the controls are considered statistically significant at p = 0.025.
Factors that must be considered in analyzing micronucleus test data include number of animals per dose group (a minimum of 3 is required), dose levels and number of doses administered, route of administration, tissue and cell type analyzed, sample time (interval between last dosing and harvesting of cells for analysis), frequencies of micronucleated cells in the negative and positive controls, and the results of the statistical analyses. The final conclusion for a micronucleus test is determined by considering the results of statistical analyses, the reproducibility of any observed effects, and the magnitude and biological significance of those effects.
The micronucleus test provides information that is complementary to the gene mutation information that is obtained from a Salmonella assay. However, there does not seem to be an increased ability to define the carcinogenicity of a chemical by conducting a micronucleus test in addition to a Salmonella assay. Despite the lack of increased predictive value for carcinogenicity, the micronucleus test provides valuable information about the chemical's ability to disrupt mammalian chromosome structure and function. Most known human carcinogens are positive in mammalian micronucleus tests.
Kissling et al. reference:
Kissling GE, Dertinger SD, Hayashi M, MacGregor JT. Sensitivity of the erythrocyte micronucleus assay: dependence on number of cells scored and inter-animal variability. Mutat Res. 2007 Dec 1;634(1-2):235-40.
Witt et al. reference:
Witt KL, Livanos E, Kissling GE, Torous DK, Caspary W, Tice RR, Recio L. Comparison of flow cytometry- and microscopy-based methods for measuring micronucleated reticulocyte frequencies in rodents treated with nongenotoxic and genotoxic chemicals. Mutat Res. 2008 Jan 8;649(1-2):101-13.