This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.
Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.
It has been estimated that exposure to particulate matter (PM) causes over 3 million excess deaths annually, primarily from cardiopulmonary disease and lung cancer1. Indeed, PM was recognized as carcinogenic to humans by the International Agency for Research on Cancer (Group 1), as an increased risk of lung cancer with increasing levels of exposure to PM has been shown2. Interestingly, almost all the cancer cells harbor numerical and/or structural chromosomal abnormalities. Particulate organic carbon is a variant, complex, and heterogeneous mixture, whose composition and size distribution depends on emissions as well as physical and chemical transformations. It accounts from 35-55% of urban PM2.5 mass (PM 2.5 µm in size and smaller) and more than 60% of rural and continental background PM2.5 mass3, 4. The water soluble fraction accounts for 30-90% of organic aerosol. A large number of organic compounds have been identified, including aliphatic hydrocarbons, polycyclic aromatic hydrocarbons, and their oxygenated and nitrated derivatives, aliphatic aldehydes and alcohols, free fatty acids and their salts, di-carboxylic acids, multifunctional compounds, proteins, and humic-like macromolecules (HULIS) using chromatographic methods coupled with mass spectrometric techniques5-8. These compounds represent less than 10-20% of particulate organic carbon, thus most of organic carbon is unknown9.
Experimental evidence suggests that cytotoxicity, oxidative stress, and inflammation are involved in the development of PM-associated pathological states. It has been recently shown, however, that exposure to PM also results in a number of epigenetic alterations, including alterations in DNA methylation of repetitive elements, in both experimental in vitro systems and in human subjects10-12. Of particular interest are the effects of PM on satellite DNA — major and minor satellites — which are found in the heterochromatic region around the centromere of chromosomes. It was shown that these effects may be persistent by nature, as they can be detected for at least 72 hr after exposure12. Alterations in DNA methylation, particularly around centromeres, may lead to accumulation of satellite DNA mRNA transcripts, compromise chromosomal integrity during cell division and, subsequently, result in the development of a variety of pathological states13.
Adaptation of cytogenetic approach for the analysis of CAs as an end-point of epigenetic alterations caused by PM, thus, is of high importance. Here, we report the approach for the ambient PM collection and preparation, in vitro exposure and analysis of CAs using the murine macrophage RAW264.7 model system. Macrophages comprise the first line of defense against the inhaled foreign objects and, therefore, this cell line serves as an established and the most frequently used model in particle toxicology11, 12, 14, 15.
1. Particle Collection and Preparation
2. Particle Exposure
3. Cytogenetic Assay
Great care should be taken in choosing the location of the TSP sampler, as well as the time of the year when collection is performed. The chemical composition as well as the size of particles may substantially influence the results. The material collected should be visible against the white filter. A normal mouse metaphase spread will have 40 acentric chromosomes. The goal of the technique is to demonstrate a change (or absence thereof) in the proportion of abnormal chromosomes in treated cells versus controls. That change can then be quantified (number of abnormal chromosomes) and qualified (type of abnormalities).
Normal mouse metaphase spread will have 40 acentric chromosomes (Figure 3A). Treatment with particulate matters induces various CAs as shown in Figure 3, such as chromatid break (3B), acentric fragment (3C), ring chromosome (3D), dicentric chromosome (3E), double min (3F), Robertsonian translocation (3G), and endoreduplication (3H). For full results, please refer to our published study12.
Figure 1. The high volume total suspended particles (TSP) sampler. The picture shows the TSP sampler installed in a roof in an urban area to collect ambient particles. Please click here to view a larger version of this figure.
Figure 2. Loaded quartz fiber filter following collection using the high volume TSP sampler. Close-up on the filter installed on the TSP sampler, after use. Note that the color of the filter changed from white to gray. Please click here to view a larger version of this figure.
Figure 3. Photomicrograph showing representative examples of different types of structural aberrations induced in RAW264.7 cells after treating with different concentrations of particulate matter. The aberrations are indicated by arrows. (A) normal metaphase spread with 40 acrocentric chromosomes, (B) chromatid-type break (CTB), (C) acentric fragment (acentrics), (D) ring chromosome, (E) dicentric chromosome (Dic), (F) double min (Dimn), (G) Robertsonian translocation, and (H) endoreduplication (duplicated chromosomes are held together). Original magnification 100X, scale bar = 5 µm. Please click here to view a larger version of this figure.
Cytogenetic study or microscopic analysis of the numbers or structures of chromosomes, primarily in metaphase spreads, provides information crucial for prognosis, risk assessment, and treatment for various diseases. It is now well-established that cytogenetic abnormalities are linked with the progression and development of several diseases, including cancer. To date, CAs have been found in all major tumor types. CAs may arise spontaneously or by either external or internal stimuli, such as ionizing radiation (IR) — one of the major external risk factors that can induce various types of cytogenetic alterations16. Analysis of CAs aids in assessment of the absorbed radiation dose (called cytogenetic radiation biodosimetry). Moreover, the analysis of frequency and type of specific aberration also provides information important for determining the quality of radiation being absorbed17, 18. Cytogenetic study provides a direct image of the impact of damaging agents on DNA. This is in contrast to indirect techniques of such as the comet assay. However, cytogenetic study has been under-utilized in fields outside of radiation, partly because of the numerous hands-on steps involved.
Although exposure to organic carbon present in the PM is known to adversely affect our health, few studies reporting biological effects include their composition9. This is due to the costly and cumbersome chromatographic methods that require labor-intensive extraction, processing, concentrations, and derivatization protocols, while they only provide insights for a small subset of a very specific type of compounds each time. Moreover, the protocols result in the substantial manipulation or destruction of the original sample, thus, additional testing cannot be performed. Advantages of nuclear magnetic resonance (NMR) analysis include that NMR is a non-destructive method; it requires a very limited number of steps (extraction, concentration) prior to analysis, and high-frequency NMR instruments (from 400 MHz to 900 MHz) are available in many, if not all, universities within the core facility. A comprehensive review of atmospheric aerosol NMR studies has been recently published19. Finally, because of its ability to provide structural information and bonds between groups, NMR is superior to other spectrometric methods such as FT-IR, UV-Vis, and Raman spectrometry that only provide data on the type of functional groups. If needed, morphological characterization of PM can be performed in addition to NMR spectrometry with the use of scanning electron microscopy.
Even from a single location, the composition of the PM collected may vary depending on the time of the year. A large enough amount of material should be processed and kept at -80 °C for repeat experiments or complementary assays. Steps 2.1.1 through 3.1.3 should be performed aseptically in a biosafety cabinet. If contamination or compromised cellular morphology is observed, do not proceed with the rest of the protocol. The trypsin treatment time needs to be adjusted based on cell type. Here, macrophages are very adherent and we have found that a combination of 1 min trypsin and scraping works best, but trypsin alone for 5 min works for most cell types. Hypotonic treatment time also varies depending on cell type and needs to be standardized. The dose and treatment time of colcemid are the most important parameters for chromosome preparation; an over-dose can kill the cells or stall the cell cycle. We describe here conditions for RAW264.7 cells, and optimization is required for other cell types. Failure to find ideal conditions in this step may adversely affect the mitotic index as well as chromosome morphology, affecting the results. It is also recommended to assess the levels of cytotoxicity of the PM prior to cytogenetic analysis and select the doses accordingly.
Conceptually, in vitro toxicological assessments are limited by the type of used cells, exposure magnitude, and particle concentrations. Furthermore, in this study, we utilized the water soluble extract of PM and, therefore, the potential of water-insoluble species to cause cytogenetic aberrations may not be accounted. Other limitations are associated with the presence of "polymorphic variants" – variations in the centromeric regions and short arms of chromosome; some submicroscopic or cryptic rearrangements that may be misinterpreted and the presence of complex karyotypes.
Here, we demonstrate that the introduction of cytogenetic analysis may aid in the identification of PM-induced damage to DNA and may potentially serve as a predictive endpoint in assessment of the health effects caused by exposure to ambient PM. Our study is, to our knowledge, the first one to indicate CAs in response to PM. Replication of these results is desirable to confirm our findings. This protocol could also be adapted to assess almost any type of suspected DNA damaging agent.
The authors have nothing to disclose.
The work was supported, in part, by the National Institute of Health Center of Biological Research Excellence [grant number 1P20GM109005], the Arkansas Space Grant Consortium through National Aeronautics and Space Administration [grant number NNX15AK32A], and the National Institute for Occupational Safety and Health (NIOSH) [grant number 2T420H008436]. The authors would like to thank Christopher Fettes for proofreading and editing this manuscript.
Total suspended particulate sampler | Tisch Environmental | TE-5170 | |
Bruker Avance III NMR spectrometer | Bruker | NA | |
TopSpin 3.5/pl2 software | Bruker | NA | |
ACD/NMR Processor Academic Edition | ACD/Labs | NA | |
RAW264.7 murine macrophages | ATCC | ATCC TIB-71 | |
High glucose DMEM GlutaMAX media | ThermoFisher | 10569010 | Warm in a 37°C waterbath before use |
Fetal Bovine Serum | ThermoFisher | 16000044 | Warm in a 37°C waterbath before use |
Penicillin-Streptomycin (10,000 U/mL) | ThermoFisher | 15140163 | Warm in a 37°C waterbath before use |
Trypsin-EDTA (0.25%) | ThermoFisher | 25200056 | Warm in a 37°C waterbath before use |
PBS, pH 7.4 | ThermoFisher | 10010049 | Warm in a 37°C waterbath before use |
KaryoMAX Colcemid Solution in PBS | ThermoFisher | 15212012 | Warm in a 37°C waterbath before use |
KaryoMAX Potassium Chloride Solution | ThermoFisher | 10575090 | Warm in a 37°C waterbath before use |
Methanol (HPLC) | Fisher Scientific | A452N1-19 | |
Acetic Acid, Glacial | Fisher Scientific | BP1185-500 | |
Decon Contrad 70 Liquid Detergent | Fisher Scientific | 04-355-1 | |
Wright and Wright-Giemsa Stain Solutions | Fisher Scientific | 23-200733 | |
Permount Mounting Medium | Fisher Scientific | SP15-100 | |
Axio Imager 2 | Zeiss | NA |