Meng-Ching Chung1, Ming-Hsien Tsai2, Danielle E. Que3, Sayre J. Bongo4, Wen-Li Hsu1,5, Lemmuel L. Tayo4, Yi-Hsien Lin6, Sheng-Lun Lin7,8,9, Yan-You Gou1, Yi-Chyun Hsu10, Wen-Che Hou3, Kuo-Lin Huang1, How-Ran Chao This email address is being protected from spambots. You need JavaScript enabled to view it.1,11

Emerging Compounds Research Center, Department of Environmental Science and Engineering, College of Engineering, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan
Department of Child Care, College of Humanities and Social Sciences, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan
Department of Environmental Engineering, National Cheng Kung University, Tainan 70101, Taiwan
School of Chemical, Biological and Materials Engineering and Sciences, Mapúa University, Intramuros, Manila 1002, Philippines
Research Institute for Life Support Innovation, Research Organization for Nano and Life Innovation, Waseda University, Shinjuku, Tokyo 162-8480, Japan
Department of Plant Medicine, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan
Department of Civil Engineering and Geomatics, Cheng Shiu University, Kaohsiung 83347, Taiwan
Center for Environmental Toxin and Emerging-Contaminant Research, Cheng Shiu University, Kaohsiung 83347, Taiwan
Super Micro Mass Research and Technology Center, Cheng Shiu University, Kaohsiung 83347, Taiwan
10 Department of Environmental Engineering, Kun Shan University, Tainan 71003, Taiwan
11 Institute of Food Safety Management, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan


 

Received: March 21, 2019
Revised: April 23, 2019
Accepted: April 24, 2019

Download Citation: ||https://doi.org/10.4209/aaqr.2019.03.0127  

  • Download: PDF


Cite this article:

Chung, M.C., Tsai, M.H., Que, D.E., Bongo, S.J., Hsu, W.L., Tayo, L.L., Lin, Y.H., Lin, S.L., Gou, Y.Y., Hsu, Y.C., Hou, W.C., Huang, K.L. and Chao, H.R. (2019). Fine Particulate Matter-induced Toxic Effects in an Animal Model of Caenorhabditis elegans. Aerosol Air Qual. Res. 19: 1068-1078. https://doi.org/10.4209/aaqr.2019.03.0127


HIGHLIGHTS

  • C. elegans is a sensitive animal model to evaluate PM2.5 toxicity.
  • Low PM2.5 levels shortened the lifespan of C. elegans.
  • Low PM2.5 levels caused reproductive toxicity of C. elegans.
  • Low PM2.5 levels delayed locomotion behavior of C. elegans.
 

ABSTRACT


Research has been focused on the health hazards of ambient PM2.5 related to humans. Many PM2.5 toxicity assessments using in vitro studies have focused on PM2.5-bounded hazardous pollutants. However, PM2.5 toxicity assessment by in vivo studies allow for better observation of the overall effects of PM2.5 exposure on entire organisms, making in vivo PM2.5 toxicity assessment relevant. The toxic effects of outdoor PM2.5, collected from National Pingtung University of Science and Technology (NPUST) and Linluo Junior High School (LJHS), Pingtung, Taiwan, on nematode Caenorhabditis elegans (C. elegans) were investigated. PM2.5 from NPUST and LJHS were found to be 4.5 and 2.5 μg Nm–3, respectively, which did not meet the standard. This levels of PM2.5 in Taiwan. For acute toxicity, no significant PM2.5 lethality on C. elegans was observed between NPUST and LJHS. PM2.5 from NPUST exhibited greater toxicity to lifespan (ageing), locomotion (head thrash), and reproduction (brood size) in the C. elegans animal models than that from LJHS; therefore, adverse effects could be correlated with PM2.5 concentrations. Prolonged exposure to PM2.5 led to more severe toxicity in nematodes as compared to acute exposure. In conclusion, this study suggests that the long-term adverse effects of ambient PM2.5 on environmental organisms should be carefully considered even when PM2.5 is at low levels. C. elegans is a sensitive animal model for the evaluation of PM2.5 ecotoxicity.


Keywords: C. elegans; PM2.5; Lifespan; Locomotion; Reproduction; Ageing.


INTRODUCTION


Particulate matter 2.5 (PM2.5) is a type of fine particles with an aerodynamic diameter of less than 2.5 µm. It is produced through processes including natural, indoor, and outdoor combustion processes, as well as other anthropogenic activities (Li et al., 2018a; Martins and da Graça, 2018). Some of the identified major sources of PM2.5 emissions into the atmosphere are power plants (Mari et al., 2016; Dodla et al., 2017), waste incineration facilities (Yan et al., 2016), and industrial processes, such as the manufacture of steel stacks in the steelmaking industry (Gibson et al., 2013; Owoade et al., 2015). In addition, the large surface area of PM2.5 facilitates easy adhesion of toxic compounds such as polycyclic aromatic hydrocarbons (PAHs), polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins/furans(PCDD/Fs). on its surface (Pandey et al., 2013; Chao et al., 2016; Lee et al., 2018). The World Health Organization (WHO) established guidelines on air quality that include limitations on exposure to PM2.5 of not more than 25 µg m3 day1 and 10 µg m-3 year1 (Hopke et al., 2018). In Taiwan, the allowable level of exposure to PM2.5 per year is 35 µg m3, and for 24 hours, it is 15 µg m3, as regulated by the Taiwan Air Quality Monitoring Network (TAQMN) of the Environmental Protection Administration, Taiwan (TEPA).

Due to the extremely fine size of PM2.5 particulates, they have been reported to be able to penetrate the blood brain barrier (Feng et al., 2016; Lin et al., 2017). Upon exposure, particulate matter induces overexpression of macrophages in the immune system and oxidative stress resulting in atherosclerotic plaques (Brook and Rajagopalan, 2010) and endothelial and vascular dysfunctions (Feng et al., 2016). PM2.5 also affects the respiratory tract, particularly tiny sacs in the lungs called alveoli, where carbon dioxide and oxygen exchange takes place during respiration (Pinkerton et al., 2000). This can further result in the activation of a series of inflammatory responses in the lungs (Chao et al., 2018; Zhao et al., 2019). PM2.5 is ubiquitous in the environment (Salako et al., 2012; Mohammadyan et al., 2017; Seneviratne et al., 2017), and it has been reported that people suffering from cardiopulmonary and respiratory diseases are at high risk because their exposure to fine particles can cause short-term health effects such as eye, nose, throat and lung irritation, coughing, sneezing, runny nose, and shortness of breath (Kim et al., 2018; Maciejczyk et al., 2018; Polezer et al., 2018). Several in vivo studies utilizing mice models have reported that exposure to airborne PM2.5 prolongs lung tumour progression (Yang and Xiao, 2018), promotes abdominal aortic aneurysms (Jun et al., 2018), increases inflammatory cell infiltration (Hu et al., 2017), and causes lipid accumulation and hepatic function loss (Xu et al., 2019). In vitro studies have associated PM2.5 exposure to induced cytotoxicity and autophagy in human endothelial cells (Zhou et al., 2018), as well as telomere lengthening (Miri et al., 2019). Most importantly, epidemiological studies have shown that both short-term and long-term exposure to PM increases the risk of developing neurological disorders such as stroke (Fu et al., 2019), Parkinson’s disease (Hu et al., 2019), and congenital heart disease as a result of prenatal exposure (Huang et al., 2019),and generally stronger associations have been found with mortality and hospitalization (Karimi et al., 2019). Recently, Lo et al. (2017) reported that PM2.5 was involved in deaths resulting from ischemic heart disease, stroke, lung cancer, and chronic obstructive pulmonary disease, wherein a fraction of 18.6% of the specified diseases was linked to PM2.5 in Taiwan. Therefore, PM2.5 is a major risk factor in Taiwan (Lung et al., 2016). Currently, there is no known established standard PM2.5 exposure level that may be referenced to avoid its adverse health effects (Kiesewetter et al., 2015). Similarly, there is no singular in vivo animal model that can completely represent the human system. Therefore, there is a need for more research using other available model organisms to further understand the effects of PM2.5 on human health (Feng et al., 2016).

Caenorhabditis elegans (C. elegans) was established by Sydney Brenner as a model organism in 1965 (Brenner, 1974). Compared with single-cell based in vitro assays, C. elegans-based assays allow for the evaluation of multiple toxicity endpoints (Kaletta and Hengartner, 2006). At the genetic level, more than half of the conserved signaling regions in humans are found in the nematode (Aoki and Mori, 2015). Numerous studies have reported the sensitivity of C. elegans in terms of pollution from different types of environmental media (Clavijo et al., 2016; Zuo et al., 2017; Rai et al., 2019). However, only a few studies have reported the sensitivity of C. elegans to air pollutants, specifically, to PM2.5 (Zhao et al., 2014; Sun et al., 2015, 2016; Wang et al., 2019). In a study by Sun et al. (2015), exposure to coal combustion-related PM2.5 induced lower response in all organism-level endpoints, specifically, prolonged exposure caused more severity than acute exposure to the nematodes. In addition, when Sun et al.(2016) determined the contribution of heavy metals found in coal combustion-related PM2.5, he found that the combined exposure to specific metals caused greater toxicity than single exposure of said metals. Most importantly, transgenerational effects after exposure to diesel- and traffic-related PM2.5 were discovered by Wang et al. (2019) and Zhao et al. (2014) wherein reproductive disability had been transferred to later generations of C. elegans.

C. elegans is an inexpensive, bioethical, and easy model animal to maintain, with attractive physiological characteristics that can be used to assess the toxicological effects of environmental pollutants (Leung et al., 2008). In this study, the toxicity of acute exposure to ambient outdoor PM2.5 samples collected from Southern Taiwanese schools such as National Pingtung University of Science and Technology (NPUST) and Linluo Junior High School (LJHS) was assessed using the different C. elegans toxicological endpoints, which include survival (lethality and lifespan), reproduction (brood size), developmental (growth), and locomotion (head thrash and body bend) endpoints.


MATERIALS AND METHODS


 
Chemical Reagents

The wild-type N2 strain of C. elegans was acquired from the Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University (Tainan, Taiwan) and was maintained in nematode growth medium (NGM) plates containing bacteriological agar and bactopeptone (Laboratories Conda, S.A., Spain) and NaCl (Honeywell Fluka™, New Jersey, USA). Additional NGM plate components such as CaCl2, K2HPO4, and cholesterol were obtained from Sigma-Aldrich (St. Louis, MO, USA), and MgSO4 was acquired from Avantor Performance Materials, ltd. (Gyeonggi-do, South Korea). OP50 Escherichia coli (E. coli) cultures were acquired from the Bioresources Collection and Research Center (Hsinchu, Taiwan) and Luria-Bertani broth was obtained from Sigma-Aldrich (St. Louis, MO, USA). For the bleaching solution, NaOCl was obtained from J.T. Baker (Central Valley, PA), and KOH was obtained from Duksan Pure Chemicals (Gyeonggi-do, South Korea). KH2PO4 used for the phosphate buffer was acquired from Avantor Performance Materials, LLC (Radnor, PA, USA), and Na2HPO4 used for the M9 buffer was obtained from Honeywell Fluka™ (New Jersey, USA). All physiological observations were done under a dissecting microscope (Olympus, SZX10, Waltham MA, USA). For the K-medium ingredients, NaCl was obtained from Honeywell Fluka™ (New Jersey, USA), and KCl was acquired from Avantor Performance Materials, ltd. (Gyeonggi-do, South Korea). Dehydroethidium (DHE) for oxidative stress detection was purchased fromInvitrogen™, Thermo Fisher Scientific (Waltham, MA USA).

 
Sample Collection

Outdoor ambient PM2.5 samples were collected from two southern Taiwanese schools, NPUST and LJHS, located in Neipu, Pingtung County which is in the rural area. The air sampling site in NPUST was set up at the top floor of the College of Engineering in the NPUST campus. The other site in LJHS is nearby Linlou System Interchange of Formosa Highway. Each air sample was collected for 48 hours (2 days, 1 m3 min1) using a high-volume air sampler (SIBATA HV-1000R, Japan) following US EPA Reference Method TO9A. The PM2.5 samples were collected onto quartz fibre filters that were pre-heated before sampling at 600°C for 2 hours. A balance with an accuracy of 0.1 mg was used to weigh the filter paper conditioned in an electronic desiccator before and after the sample collection for 24 h. The loaded filters were stored in a refrigerator at–20°C before extraction to limit the possible evaporation of volatile components.

 
Extraction, Purification, and Sample Preparation

After the collection of air samples, the mixture was extracted using a Soxhlet extractor with dichloromethane (DCM) for 24 h. The extracts were eluted with 15 mL DCM during the alumina column clean-up. The eluate was concentrated to approximately 1 mL and transferred to a vial. The concentrate was further concentrated to near dryness using a stream of nitrogen. For exposure concentrations, PM2.5 was serially diluted 10X with 1% DMSO. The toxic effects in the study were limited in the organic fraction.


 C. 
elegans Age Synchronization for Exposure Experiments

Wild-type N2 C. elegans were maintained on NGM plates seeded with E. coli as food and incubated at 22°C until the plates had a high density of eggs and gravid nematodes. These were then washed off the plates, placed into centrifuge tubes, and the gravid nematodes were lysed with a bleachingmixture leaving only the eggs to obtain age synchronized populations of L1 nematode larvae in preparation for the exposure experiments and toxicity assays.


Acute Exposure

Synchronized L1 larvae were seeded onto NGM plates and incubated until L3 or young L4 stage. The L3 and young L4 worms were rinsed from the plate with K-medium, gently washed, and centrifuged at 2500 x g for 4 minutes to remove the E. coli in the supernatant by aspirating without disturbing the nematode pellet. After washing, the pellet was re-suspended in K medium through continuous pipetting, and a seeding volume (worm per µL) was calculated by pipetting two 5 µL drops on a glass slide and manually counting the worms in each drop. Approximately 200 L3/young L4 worms were dispensed into each well of a 12-well plate each containing 1 mL of the different exposure concentrations for the two PM2.5 samples (one set for NPUST and another for LJHS) diluted with K-medium. Acute (24-hour) exposure was performed at 22°C without the presence of food.

 
Lethality Assay

The lethality assay was performed by transferring 50 exposed nematodes (per exposure concentration) to NGM plates without food (OP50). These worms were gently prodded using the worm picker to assess viability. Nematodes that were non-responsive to stimulus were scored as dead. The assays were done in triplicates for each of the concentrations.

 
Lifespan Assay (Ageing Assay)

Fifty L3/young L4 worms from the acute exposure treatment were transferred to NGM plates with food (OP50). The worms were transferred to fresh NGM plates every day for the first 4 to 5 days (egg-laying period), and after that, the worms were no longer transferred. The plates were incubated at 22°C. Alive, censored (lost), and dead nematodes were recorded every 2 days within a span of 24 days (general lifespan of C. elegans). The survival plot or the Kaplan-Meier plot was constructed to evaluate the effects of the different concentrations of PM2.5 on the lifespan or ageing of the nematodes. Triplicates were done for each of the exposure concentrations.

 
Reproductive or Brood Size Assay

L3/young L4 worms from the acute exposure treatment were assessed for 4 to 5 days of egg-laying. One L3/young L4 worm was transferred to each of the wells of a 12-well NGM plate with a fresh OP50 lawn. Each worm was transferred to a new plate every 2 days within the duration of egg-laying period. The old plates containing the eggs were hatched and incubated to L4 worms for easier counting of progeny. The total progeny for each of the worms was recorded. Twelve worms were evaluated for each of the exposure concentrations.

 
Growth Measurement Assay

L3/young L4 worms exposed to each of the PM2.5 concentrations for 24 hours with prolonged exposure were measured for their length to assess the effects of PM2.5 on the growth of the nematodes. Ten L3/young L4 nematodes exposed to each of the exposure concentrations were transferred to NGM plates with OP50 lawns and were incubated at 22°C for 48 hours until they reached the old L4 stage. The nematodes from each of the concentrations were exposed to a high temperature of 66°C in a dry bath for 5 minutes. This process straightens the body of the C. elegans, making it easier to measure the length. An optical microscope (Olympus) was used, and ImageJ software was utilized for the measurement of the worm length.

 
Locomotion Assay (Head Thrash and Body Bend Assay)

The endpoints of head thrash and body bending were used to evaluate the locomotion of the exposed L3/young L4 C. elegans by allowing the nematodes swim in K-medium. A head thrash was defined as a change in the direction of the mid body wherein the head and tail falls in the same direction as it moves. Body bending was regarded as a change in direction of the part of the nematodes corresponding to the posterior bulb of the pharynx along the y axis, assuming that the nematode was traveling along the x axis. Head thrashing was counted for 1 minute, and body bending was counted for 20 seconds. Thirty nematodes were examined per treatment, and three replicates were performed.

 
Statistical Analysis

The locomotion, brood size, and length measurement data for the nematodes did not meet the normal distribution using a normality test such as the Shapiro-Wilk test. The Kaplan-Meier plots for the survival rates were used to evaluate the effects of the different PM2.5 concentrations on the lifespan or ageing of the nematodes. The nonparametric Kruskal-Wallis H test was used to examine the differences between each the concentrations in comparison to the control. The survival plots or the Kaplan-Meier plot were derived for the lifespan data using GraphPad Prism 6 (San Diego, California, USA). The significance of each of the days were determined with the Kruskal-Wallis H test. Days with significance were chosen and tested further. Each of the concentrations from the chosen days was also compared to the control using the Mann-Whitney U test. The statistical analyses were tested using SPSS version 12 (International Business Machines Corp., New York, USA).

 
RESULTS AND DISCUSSION


 
Lethality Effects of PM2.5 on C. elegans

Age-synchronized L4 worms were subjected to 24 hours of exposure to serially diluted samples of PM2.5, after which lethality was investigated. No lethality was observed in any of the PM2.5 sample concentrations of NPUST (0.0014443–1444.3 mg L1) and LJHS (0.0011518–1151.8 mg L1) (Fig. 1) collected from NPUST and LJHS. Similarly, in a study conducted by Sun et al. (2015), coal combustion-related PM2.5 with exposure concentrations ranging from 0.01 to 100 mg L1 did not show any lethal effects on nematodes.


Fig. 1. Dose-dependent lethality of PM2.5 from (Left) NPUST and LJHS after 24-hour exposure.Fig. 1.
 Dose-dependent lethality of PM2.5 from (Left) NPUST and LJHS after 24-hour exposure.


Life Span of C. elegans after PM2.5 Exposure

In this study, the toxicological endpoint lifespan was also evaluated since exposure of the nematodes to PM2.5 concentrations greater than 1 µg L1 was observed to cause toxicities. The concentrations of PM2.5 determined from NPUST and LJHS were 4.5 and 2.5 µg Nm3 respectively. The detected levels of PM2.5 from both schools were lower than the allowable levels of outdoor PM2.5 in Taiwan, as regulated by the TAQMN. However, prolonged exposure showed that the lifespan of C. elegans was significantly reduced for all sample concentrations from the NPUST sampling site. Specifically, the lowest and highest concentrations (1.4443 × 103 mg L1 and 1.4443 × 103 mg L1) from NPUST caused the sharpest decrease in lifespan starting on post-exposure day 10 (Fig. 2). Additionally, a significant reduction in lifespan was observed on an earlier post-exposure day for LJHS for all concentrations. Concentrations of 1.5518 × 103 and 1.5518 × 102 mg L1 caused a rapid decrease in the population of the nematodes.


Fig. 2. Effects of PM2.5 from NPUST and (Bottom) LJHS on C. elegans lifespan after 24-hour exposure.Fig. 2.
 Effects of PM2.5 from NPUST and (Bottom) LJHS on C. elegans lifespan after 24-hour exposure.

Lastly, C. elegans death occurred on post-exposure day 22, which is two days earlier than the usual lifespan of the nematode. In a similar report by Sun et al. (2015), coal combustion-related PM2.5 at 100 mg L1 significantly reduced the lifespan of C. elegans. However, there was no significant effect on the lifespan when the nematodes were exposed to concentrations 0.01–10 mg L1, which is inconsistent with the results of this study. Bases on the previous description of the Sun’s study (Sun et al., 2015), it was little concern for their study deign. It was difficult to prevent from aggregation of PM2.5 after air sampling, but they didn’t show and explain how to maintain PM2.5-type particulate with diversely and homogenously widespread in the nematodes’ medium. The finding of our studying is valuable due to our result indicating low levels of PM2.5 exposure in nematodeswas still significantly linked into lower lifespan compared with the control. According to the previous reports and the present study (Zhao et al., 2014; Sun et al., 2015, 2016; Wang et al., 2017), PM2.5 exposure decreased lifespan in the in-vivo and epidemiological studies including coal combustion in C. elegans (Sun et al., 2015, 2016), traffic-related pollutants in the C. elegans (Zhao et al., 2014), ambient air in Drosophila (Wang et al., 2017), ambient air in newborns (Martens et al., 2017), and ambient air in elderly (Li et al., 2018b). Therefore, the level of outdoor air PM2.5from the two schools, although lower than the national standard in Taiwan, was still shown to have a significant impact on microorganisms and ultimately may pose a threat on human health. To this end, additional endpoints (such as reproductive and locomotion) were investigated to further confirm this effect.

 
Growth Measurement Assay

No lethality was observed when the nematodes were subjected to acute exposure with the different concentrations of PM2.5 from either sampling site (NPUST and LJHS). Also, specified concentrations of samples from both the NPUST (1.4443 × 103 to 1.4443 × 103 mg L1) and LJHS (1.5518 × 103 to 1.5518 × 103 mg L1) sampling sites did not lead to the alterations in body length (Fig. 3). Results showed that in all concentrations, the body length of the nematodes remained at 1 mm, the standard length of C. elegans (Leung et al., 2008). A similar study by Sunet al. (2015) reported that acute and prolonged exposure of C. elegans to PM2.5 of concentrations 0.01 to 10 mg L1 had no significant effect on the body lengths of the nematodes under study; however, acute exposure to PM2.5 at a concentration of 100 mg L1 and prolonged exposure to PM2.5 at concentrations of 10 to 100 mg L1 significantly reduced body length. Our result was inconsistent with that discussed in Sun’s study (Sun et al., 2015).


Fig. 3. Effects of PM2.5 from (Left) NPUST and LJHS on C. elegans growth, observed as changes in body length, after 24-hour exposure.Fig. 3. 
Effects of PM2.5 from (Left) NPUST and LJHS on C. elegans growth, observed as changes in body length, after 24-hour exposure.


Effect of Outdoor Air PM2.5 Levels on the Reproductive System of C. elegans

The secondary targeted organs of environmental pollutants include the neurons and the reproductive system (Leung et al., 2008; Aoki and Mori, 2015). In the C. elegans model, the reproductive system is one of the major targets of toxicants in the body, next to the neurosensory system (Aoki and Mori, 2015). In order to understand the effects of PM2.5 on its translocation in C. elegans, the brood size was also assessed for each of the worms exposed to different PM2.5 concentrations (Fig. 4). The total number of eggs, or brood size, was determined twice per concentration of samples from NPUST and LJHS. After acute exposure, the first measurement (Fig. 4(A)) showed that brood size was significantly reduced as the concentration of samples from NPUST increased. Specifically, brood size at 1.4443 × 103mg L1 was reduced to 18 eggs laid per worm, and concentrations from 1.4443 × 102 to 1.4443 × 103 mg L1 reduced the brood size to 21, 11, 10, 10, 10, and 3 eggs laid per worm, respectively. Similarly, the second assessment of brood size (Fig. 4(B)) showed that at concentrations ranging from 1.4443 × 103 to 1.4443 × 103 mg L1, the eggs laid per worm decreased to 13, 6, 5, 2, 6, 3, and 1, respectively. For samples gathered from LJHS, the first and second assessment of brood size (Figs. 4(C) and 4(D)) showed no significantly different number of eggs laid per worm at concentrations ranging from 1.5518 × 103 to 1.5518 × 103 mg L1. In this study, the results showed that exposure to high ambient PM2.5concentrations had a significant effect on the reduction of brood size of C. elegans. This result is analogous to the study conducted by Wang et al.(2019), wherein diesel particulate matter (DPM) caused reproduction deficits in C. elegans, as shown by a decrease in brood size during the first generation of offspring. Moreover, in the same study, it was observed that consecutive generation exposure to DPM led to severe limitations in brood size.


Fig. 4. Effects of PM2.5 from NPUST and (Bottom) LJHS on C. elegans brood size, observed (A and C) two days and (B and D) four days after 24-hour acute exposure.Fig. 4.
 Effects of PM2.5 from NPUST and (Bottom) LJHS on C. elegans brood size, observed (A and C) two days and (B and D) four days after 24-hour acute exposure.

Currently, studies have shown that the self-progeny of C. elegans is an effective parameter that can be used to determine the extent of reproductive disability due to the toxicity of various substances (Wang et al., 2018; Moon et al., 2019). In general, the effect can be observed in a dose-dependent manner (Yang et al., 2018). This was not the case for this study. The control group generated a relatively low brood size of 18. Dietary restriction (DR) can significantly lower the progeny number in C. elegans (El-Hajj and Newman, 2015). In addition, for the size, PM2.5 is greater than E. coli OP50 (2.5 µm > 2 µm, general food source of C. elegans) (Khan et al., 2018). Therefore, toxicity of PM2.5 is related to the size of the exposed organism.

 
Effects of Ambient PM2.5 on Locomotion Behavior of C. elegans

Locomotion assays used to analyze the head thrashing and body bending movements of the nematodes were conducted to assess the neurological toxicity brought about by their exposure to different concentrations of PM2.5 (Fig. 5). A significant reduction in head thrashing movement and body bending were observed for nematodes exposed to PM2.5 sampled from NPUST as compared to nematodes exposed to PM2.5 sampled from LJHS. These results imply that levels of PM2.5 may be correlated to the greatly disrupted functions of the reproductive and neurosensory systems in C. elegans. Additionally, prolonged exposure to PM2.5 was observed to cause more sensitivity to C. elegans. Zhao et al. (2014) established that acute exposure to high concentrations of PM2.5 causes adverse effects on both the reproduction and neurosensory functions of C. elegans. Moreover, prolonged exposure to low concentrations of PM2.5 further significantly decreased the locomotion behavior of the nematodes. However, this was true for exposure to pollutant concentrations in the range of µg L1. This is because of the greater dispersion of PM2.5 in DMSO, which increased the bioavailability of the particulate matter to the nematodes (Artifon et al., 2019). The main route of uptake of pollutants in C. elegans is through the alimentary system; the worms actively ingest particulates while feeding on bacteria. The presence of bacterial food causes the adsorption of a proportion of the particles and enhances the oral uptake, resulting in higher toxicity (Gonzalez-Moragas et al., 2015). Therefore, the dispersion of particulate matter in the media of choice is important in assessing their effects on in vivo systems.


Fig. 5. Effects of PM2.5 from NPUST and (Bottom) LJHS on C. elegans locomotion and movement after 24-hour acute exposure. A and C for head thrashing and B and D for body bending.Fig. 5. 
Effects of PM2.5 from NPUST and (Bottom) LJHS on C. elegans locomotion and movement after 24-hour acute exposure. A and C for head thrashing and B and D for body bending.

Several toxic endpoints were examined in the present study. Low PM2.5 levels were found in our sampling areas. To make our amazement, the low levels of PM2.5-bounded pollutants induced several adverse effects including accelerated aging process, reduced reproduction, and delayedlocomotion. The present study was limited based on the air sampling technique and pretreatment protocol. The standard methods of PM2.5 air sampling and PM pretreatment were used in our study. This study excluded in the toxicity in inorganic fraction due to use of the Soxhlet extractors. The present study only considered the toxic effects of PM2.5-bounded hazardous pollutants after the high-volume PM2.5 air samplers mainly due to occurrence of aggregation during the air sampling. Future work will be encouraged to gather airborne PM2.5-type particulate directly into nematodes’ medium before aggregation.


C
ONCLUSIONS


Low PM2.5 levels were obtained at NPUST and LJHS in this study. It was found that ambient PM2.5 levels at NPUST were higher than the PM2.5level at LJHS. In addition, the in-vivo model of C. elegans was utilized to evaluate the possible health risks associated with differing PM2.5 levels through the investigation of multiple toxic endpoints namely, lethality, lifespan, development, locomotion, and reproduction. The acute toxicity and growth measurement of C. elegans was unaffected by PM2.5 collected from NPUST and LJHS, but its lifespan was significantly reduced in the case of airborne PM2.5 at NPUST starting on post-exposure day 10. For the reproductive toxicity and locomotion, reduced brood size and the frequency of head thrashes for NPUST-PM2.5 was observed, but there were no significant differences in lifespan, brood size, head thrashes, and body bends between the control and LJHS-PM2.5. Our significant findings were to indicate the potential toxic effects of long-term exposure to low PM2.5 on C. elegans models, even though the measurements were lower than PM2.5 standards. This study implored that the long-term adverse effects of ambient PM2.5 on environmental organisms should be carefully considered even though PM2.5 levels didn’t meet the standards.


A
CKNOWLEDGMENTS


This study was supported by a grant from the Ministry of Science and Technology (MOST 106-2221-E-020-001-MY3). We acknowledge Mr. Ying-Jhih Syu from National Pingtung University of Science and Technology for assisting us to maintain and culture C. elegans. We also want to thank Miss Chia-Jung Yen from Kaohsiung Medical University for assisting us with the C. elegans experiments. We would also like to thank Dr. Chang-Shi Chen at National Cheng Kung University for his advice and help in attaining the C. elegans culture. The authors thanked the teachers and students in Linluo Junior High School for providing the air sampling site.


D
ISCLAIMER 


The authors declare no conflicts of interest.



Share this article with your colleagues 

 

Subscribe to our Newsletter 

Aerosol and Air Quality Research has published over 2,000 peer-reviewed articles. Enter your email address to receive latest updates and research articles to your inbox every second week.

5.9
2020CiteScore
 
 
81st percentile
Powered by
Scopus

2020 Impact Factor: 3.063
5-Year Impact Factor: 2.857

Aerosol and Air Quality Research (AAQR) is an independently-run non-profit journal that promotes submissions of high-quality research and strives to be one of the leading aerosol and air quality open-access journals in the world. We use cookies on this website to personalize content to improve your user experience and analyze our traffic. By using this site you agree to its use of cookies.