Comparative Toxicity of Lunar, Martian Dust Simulants, and Urban Dust in Human Skin Fibroblast Cells
Article Category: Research Article
Publicado en línea: 01 jul 2015
Páginas: 51 - 58
DOI: https://doi.org/10.2478/gsr-2015-0005
© 2015 James T.F. Wise et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
NASA has plans to further their manned space exploration to Mars and possibly beyond (Chatterjee et al., 2010). One big health challenge in these endeavors is the exposure to lunar and Martian dust. The toxicity of lunar dust is a major health concern for NASA, as it posed a significant health hazard to astronauts during the Apollo program and, thus, there is also concern about the toxicity of Martian dust. Lunar dust particles adhere to spacesuits and they enter astronaut living quarters in spacecraft, resulting in direct bodily contact (Wallace et al., 2009; Rehders et al., 2011; Wagner, 2006). During several Apollo missions, irritation of the eyes, respiratory system, and skin were reported, and it is believed these effects were a result of direct contact with lunar dust (Wagner, 2006).
The overall toxicity of extraterrestrial dusts (Martian, lunar, and other celestial bodies [e.g., asteroids]) is poorly understood. Martian dust is highly oxidized (Allen et al., 1998); however, its formation, composition, and physical properties have not been fully characterized. Current data show Martian dust contains variable components, including known human carcinogens — such as chromium, nickel, and iron-rich silicate particles (Yen et al., 2005). Lunar dust is an ultra-fine, extremely abrasive substance with electromagnetic properties (Liu and Taylor, 2011) that cause the dust to stick to astronauts’ suits, boots, and equipment and makes them extremely difficult to clean. Lunar dust contains many different components, including chromium and silicates (McKay et al., 1994). The chemical and physical analysis of lunar and Martian dust suggests these dust particles could potentially contribute to the development of the disorders some of the astronauts presented during Apollo missions (Wagner, 2006).
There are only a few studies that have focused on the toxicity of extraterrestrial dust and their indicated toxic effects. One study reported lunar dust simulant causes a regeneration delay in cultured human keratinocyte monolayers (Rehders et al., 2011). The same study also showed Martian dust simulant induces cytotoxicity in human keratinocytes and CHO-K1 fibroblasts (Rehders et al., 2011). Latch et al. (2008) reported lunar and Martian dust simulants decrease the viability of human alveolar macrophages. Animals studies have shown that rats exposed to high doses of lunar dust via inhalation exhibit inflammation and lesions in the lung (Lam et al., 2013). Following intratracheally instilled exposure to lunar and Martian dust simulants, and then sampled at 7 or 90 days post exposure, mice lung tissues showed signs of inflammation and fibrosis at 7 days, but not 90 days (Lam et al., 2002a). It was also reported following acute exposure (24 h) to lunar and Martian dust simulants, mouse lung tissue showed signs of inflammation, while 4 h exposures had no effects (Lam et al., 2002b). Lunar dust was shown to cause minor ocular irritancy
Despite the fact some of the species found in lunar dust are known human carcinogens (McKay et al., 1994), the genotoxicity and carcinogenicity of extraterrestrial dust have not been investigated. A primary route of extraterrestrial dust exposure is through skin contact; however, only one study considered the cytotoxicity in skin cells (Rehders et al., 2011). Accordingly, the objective of this study was to determine the cytotoxicity and genotoxicity of lunar and Martian dust in human skin fibroblasts and compare it to urban dust. Since urban dust toxicity is better understood, it thus provides a good toxicity comparison.
BJhTERT cells are hTERT-immortalized human skin fibroblasts. The cells exhibit a diploid karyotype and normal growth parameters. They were a generous gift from Jerry Shay of the University of Texas, Southwestern Medical Center. The cells were cultured in a 50:50 mixture of Dulbecco’s minimal essential medium and Ham’s F12 medium, plus 15% cosmic calf serum, 1% L-glutamine, 1% penicillin/streptomycin, and 0.1% sodium pyruvate. Cells were maintained in a 37°C, humidified incubator with 5% CO2.
We used extraterrestrial dust simulants developed by NASA from volcanic ash found in Arizona (for lunar) and Hawaii (for Martian) to facilitate testing of toxicity and system requirements for lunar exploration. We obtained two size fractions of lunar dust simulant, fine and very fine (JSC-1A-f and JSC-1A-vf, respectively), a Mars dust simulant (JSC Mars-1A, referred to as Mars-1A), and a representative urban dust (UPM) from St. Louis, Missouri. Stock solutions of the above compounds were prepared by suspending them in a 50:50 mixture of Dulbecco’s minimal essential medium and Ham’s F12 medium. The stock solutions were sonicated prior to treatment. Cells were treated from the stock solutions at concentrations of 25, 50, 100, 200, or 400 μg dust/cm2 dish surface area for 24 h or 120 h.
Cytotoxicity was determined using a clonogenic assay, which measures the reduction in plating efficiency of treatment groups compared to controls, as previously described (Wise et al., 2002). Each experiment was performed at least three times with four dishes per treatment group. Cells were treated directly with the intact dust particles (described above in “Chemical Preparations”) and a metal particle as a positive control. We found our positive control behaved as expected.
Dust-induced clastogenicity was measured using the chromosome aberration assay, as previously described (Wise et al., 2002). One hundred metaphases per concentration were analyzed per experiment. Each experiment was repeated at least three times. Metaphases were analyzed for chromatid breaks, isochromatid breaks, chromatid exchanges, dicentrics, double minutes, acentric fragments, fragmented chromosomes, and centromere spreading. Cells were treated directly with the intact dust particles (described above in “Chemical Preparations”) and a metal particle as a positive control. We found our positive control behaved as expected.
Dynamic light scattering (photon correlation spectroscopy) was used to measure the mass median diameter (MMD) of the four dusts in this study.
Where mentioned, values are shown as mean ± SEM (standard error of the mean). Since the percentages calculated in repeated experiments at each treatment level are considered to be independent binomial measurements that can be approximated by a normal distribution, the standard independent two sample t test is valid to test the significance of differences between groups. It is expected that the variances of measurements at different treatment levels are different. We choose to use Satterthwart’s approximated t test, which assumes unequal variances between the two groups (p<0.05 was considered significant).
To determine the size of the dust compounds used, the mass median diameter of the four dust compounds was measured using dynamic light scattering (DLS). Table 1 reports the mass median diameters for JSC-1A-f, JSC-1A-vf, UPM, and Mars-1A; they are: 1.470, 0.726, 0.169, and 0.133 μm, respectively.
| Chemical | MMD (µm) |
|---|---|
| JSC-1A-f | 1.470 |
| JSC-1A-vf | 0.726 |
| Urban dust | 0.169 |
| Mars-1A | 0.133 |
All compounds induced a concentration-related decrease in relative survival after 24 h exposure, plateauing in response after concentrations 100 μg/cm2 (Figure 1). Mars-1A, JSC-1A-f, and UPM dusts induced similar cytotoxicity after 24 h exposure, while JSC-1A-vf was slightly less cytotoxic. For instance, concentrations of 25, 50, 100, 200, and 400 μg/cm2 Mars-1A resulted in 100, 76, 68, 39, and 32% relative survival, respectively. The same concentrations induced 83, 87, 62, 67, and 62% relative survival for JSC-1A-f, respectively; 76, 62, 54, 44, and 26% relative survival for JSC-1A-vf, respectively; and 90, 69, 59, 52, and 42% relative survival for urban dust, respectively (Figure 1).
Figure 1.
All the dust, except JSC-1A-f, also induced a concentration-dependent decrease in relative survival after 120 h exposure and a higher cytotoxicity compared to 24 h. Mars dust simulant had the highest toxicity. For example, concentrations of 25, 50, and 100 μg/cm2 Mars-1A induced 54, 12, and 0% relative survival, respectively. JSC-1A-f at concentrations of 25, 50, 100, 200, and 400 μg/cm2 induced 78, 78, 64, 60, and 51% relative survival, respectively. The same concentrations of JSC-1A-vf induced 78, 52, 24, 6, and 2% relative survival, respectively. Concentrations of 50, 100, and 200 μg/cm2 UPM induced 52, 31, and 21% relative survival, respectively (Figure 2).
Figure 2.
Next, we compared the genotoxicity of these four dusts. We used chromosomal aberrations as a measure of large-scale DNA damage. We only investigated the chromosome aberration induced from 120 h exposure to the dust because our preliminary data show no chromosome aberrations were observed after 24 h exposure. 120 h exposure to JSC-1A-f, JSC-1A-vf, Mars-1A, and UPM did not cause significant chromosome damage at tested concentrations. For example, at concentration of 50 μg/cm2, JSC-1A-vf, Mars-1A, and UPM induced 3, 12, and 5% of metaphases with damage, respectively (Figure 3.A.); and 3, 13, and 5 total chromosome aberrations in 100 metaphases, respectively (Figure 3.B.). Mars-1A caused cell cycle arrest at concentrations of 100 μg/cm2, and JSC-1A-vf caused cell cycle arrest at 50 and 100 μg/cm2. Data were unable to be generated from higher concentrations due to abundant dust on the slides, which made the scoring inaccurate.
Figure 3.
The risks of dust exposure are a major health concern for NASA as astronauts on planned lunar and Mars missions will encounter dusts of a variety of sizes and compositions. The present study aims to investigate the possible genotoxicity and cytotoxicity of lunar and Martian dust simulants, and urban dust to human skin fibroblast cells. Our study is important, as it is the first to investigate the potential carcinogenicity of lunar and Martian dust.
We found lunar dust simulants (JSC-1A-f, JSC-1A-vf), Mars dust simulant (Mars-1A), and urban dust are cytotoxic to human skin cells at concentrations of 25-400 μg/cm2 (97-1, 565 μg/ml). All three dust simulants and urban dust caused a similar level of cytotoxicity after 24 h exposure, and JSC-1A-f is less cytotoxic than the other three dusts after 120 h exposure. Our data are consistent with other studies. Latch et al. (2008) reported lunar (JSC-1A) and Martian (Mars-1) dust simulants decrease the viability of human alveolar macrophages with a concentration-dependent increase in apoptosis (100-500 μg/ml). Another study found Mars-1A reduces viability of HaCaT keratinocytes and is more cytotoxic than lunar dust simulants (JSC-A/B-1). At concentrations of 570-11, 360 μg/cm2 lunar dust simulant induced only slight cytotoxicity in HaCaT keratinocytes (Rehders et al., 2011). The difference in toxicity could be due to the size difference. The size of JSC-A/B-1 (⩽1 mm) used in their study (Rehders et al., 2011) is much bigger than those in the present work. We found Mars dust simulant, which has the smallest size, induces the highest cytotoxicity in skin cells among the four dusts after 120 h exposure. Interestingly, despite the similar particle size, urban dust is much less cytotoxic than Mars dust simulant, suggesting some chemical component of Mars dust simulant also played an important role in its toxicity. The most likely toxic components of Mars dust would be the chromium, titanium, or manganese compounds, or a combination of all three. We noticed JSC-1A-f did not cause an increase in cytotoxicity with increasing time, as the other three dusts did. The reason for this difference is not clear. A previous study reported lunar dust simulant (JSC-1A) at concentrations of 50-2000 μg/ml induced enhanced expression of inducible nitric oxide synthase (iNOS) in Murine Raw 264.7 Macrophage Cells (Chatterjee et al., 2010), indicating dust-induced reactive oxidation species may contribute to its cytotoxicity.
This study is the first to investigate the genotoxicity of lunar and Martian dust simulants. We found neither lunar, Martian dust simulants, nor urban dust, were clastogenic. Mars-1A and JSC-1A-vf caused cell cycle arrest at 100 μg/cm2, indicating some DNA damage may have occurred, leading to the arrest. Studies have shown urban particulate matter induced DNA damage, including chromosome aberrations, DNA adducts, and strand breaks (Chen et al., 2013; Gutiérrez-Castillo et al., 2006; Healey et al., 2005). It should be noted the urban particulate matter used in those studies were collected at roadside with high-density of passing traffic, or in polluted areas containing a large number of genotoxic substances, and thus may explain why we don’t see any chromosomal aberrations in our urban particulate matter exposures.
In summary, our study shows extraterrestrial dust simulants are cytotoxic but not clastogenic to human skin cells. Their toxic effect is similar to the urban dust. Prolonged exposure to very fine lunar dust and Martian dust increases their cytotoxicity and induces some DNA damage. Given the extended time required for extraterrestrial exploration, the astronauts would be more likely chronically exposed to the dusts present there. Our data are important for understanding the potential health hazards astronauts may experience during planetary exploration. The data suggest prolonged exposure to extraterrestrial dusts may be dermally harmful to humans. It should be noted that many of the exact parameters encountered during a space mission were not replicated in these experiments (e.g., low gravity and microgravity, radiation), and these may consequently aggravate the effects of the dust. Furthermore, some of the physical properties of the actual extraterrestrial dusts are lost when using simulants (e.g., oxidized particle surface of Martian dusts; jagged and porous surface of lunar dusts).