key: cord-1005364-ry4b7ael
authors: Chen, Haoxuan; Li, Xinyue; Yao, Maosheng
title: Rats Sniff Off Toxic Air
date: 2019-09-01
journal: bioRxiv
DOI: 10.1101/739003
sha: 9504b1ba87ea78e28d2d1b77924e0b883bf44845
doc_id: 1005364
cord_uid: ry4b7ael

Breathing air is a fundamental human need, yet its safety, e.g., when challenged by various harmful or lethal substances, is often not properly guarded. Currently, air toxicity is monitored only for single or limited number of known toxicants, thus failing to fully warn against possible hazardous air. Here, using a photoionization detector (PID) or GC-MS/FID we found that within minutes living rats emitted distinctive profiles of volatile organic compounds (VOCs) via breath when exposed to various airborne toxicants such as endotoxin, O3, ricin, and CO2. Compared to background indoor air, when exposed to ricin or endotoxin aerosols breath-borne VOC levels, especially that of carbon disulfide, were shown to decrease, while elevated levels were observed for O3 and CO2 exposures. Principal component analysis (PCA) revealed a clear contrast in breath-borne VOCs profiles of rats among different toxicant exposures. MicroRNA regulations such as miR-33, miR-146a and miR-155 from rats’ blood samples also suggested varying mechanisms used by the rats in combating different air toxicant challenges. By integrating living rats, breath sampling, and VOC online detection, we pioneered a system that can real-time monitor air toxicity without the need of detecting specific species. Importantly, rats were shown to be able to sniff off toxic air. Abstract Graphic

Toxic Air

Rats Sniff off Toxic Air (RSTair) VOC Sensor

Breathing air is a fundamental human need, however its safety is often not properly 57 guarded. Common pollutants in the air include particulate matter (PM), biologicals, and 58 also gaseous substances such as O3 and NOx. Inhaling these pollutants can cause a 59

variety of health problems such as respiratory, cardiovascular diseases, and even death 60 monitoring methods have been previously developed or tested for individual pollutants 81 such as the PM, and other chemicals [13] [14] [15] [16] . While for bioaerosols, the adenosine 82 triphosphate (ATP) bioluminescence technology, surface-enhanced Raman 83 spectroscopy (SERS), bioaerosol mass spectrometry (BAMS), ultraviolet aerodynamic 84 particle sizer (UVAPS) as well as silicon nanowire (SiNW) biosensor were investigated 85 and attempted over the years [17] [18] [19] . It is well known that these existing or developed 86 technologies are mostly restricted to either single agent or overall microbial 87 concentration levels without identifying species. In addition, airborne pollutants and 88 toxicity could vary greatly from one location to another 20-21 , thus presenting location-89 specific air toxicity and health effects. Current epidemiological or toxicological 90 methods involving data analysis or animal and cells experiments cannot provide in situ 91 air toxicity information, accordingly failing to represent the response at the time of 92 exposure because biomarker levels evolve over time 22 . In addition, under certain 93 scenarios (high profile meetings or locations) a rapid response to air toxicity needs to 94 be in place in order to protect the interest. However, the response time is very 95 demanding for an immediate effective countermeasure, for example, usually several 96 minutes can be tolerated 10, 23 . In many air environments, multiple hazardous pollutants 97 could also co-exist even with unknown ones at a particular time, which makes 98

protecting the air rather difficult, if not impossible, using current technologies of 99 species level detection and warning. 100 101 Previously, olfactory receptors of mouse cells for odors 24 , immune B cells 25 for 102 pathogen detections, and silicon nanowire sensor arrays for explosives were studied.

8 USA). Detailed information about ricin preparation and exposure can be also found in 156 our previous work. Here, ozone was generated by an ozone generator (Guangzhou 157

Environmental Protection Electric Co., Ltd., China) using corona discharge method. 158

The ozone was further diluted with indoor air for rat exposure experiments, and the 159 ozone concentration in the exposed chamber was approximately 5 ppm. Carbon dioxide 160 was purchased from Beijing Haike Yuanchang Utility Gas Co., Ltd., and diluted 20 161 times with indoor air to a concentration of about 5%. 162 163

To investigate whether we can use breath-borne VOCs from living systems to real-165 time monitor toxic air, we have developed the system named as RSTair (Rat Sniffs off 166 Toxic Air). As shown in Figure 1 and Figure S1 (Supporting Information), the system 167 is composed of four major parts: toxicant generator, exposure chamber, exhaled breath 168 sampling and online VOC analysis. Indoor air was used as carrier gas for generating 169 toxicant aerosols (ricin and endotoxin) using a Collison Nebulizer (BGI, Inc., USA) or 170 diluting toxicants gas (ozone and carbon dioxide). The toxicant aerosol or toxicant gas 171 was introduced into the exposure chamber at a flow rate of 1 L/min. As shown in Figure  172 S1, a metabolic cage was used as the exposure chamber which can allow rat's feces and 173 urine to fall from below quickly so as to reduce their influences on VOC analysis. In 174 addition, teflon tubes and vales were also applied to reducing adsorption loss of VOCs. 175

Indoor air was first introduced into the chamber for 10 minutes, then followed by each 177 of tested toxicants for about 10 minutes to conduct exposure tests. Before and after the 178 exposure, bloods of the rats were taken. As for the exhaled breath sampling and VOC pump was used to real-time monitor TVOC at a flow rate of 0.6 L/min. Besides, the 181 breath samples in the chamber were also collected by using a Silonite canister (Entech 182

Instruments, Inc., USA), and VOCs species were analyzed by a gas chromatograph-183 mass spectrometry/flame ionization detection (GC-MS/FID) system (Agilent 184

Technologies, Inc., USA). Each of the experiments were conducted with three rats and 185 repeated three times independently. 186 were diluted with indoor air before introduced into the chamber. The metabolic cage 190 was used as the chamber for exposure and air sampling. After placing rats into the 191 chamber, the VOCs in the chamber before and after the toxicant exposure were 192 analyzed by the PID directly and also by GC-MS/FID system after being collected by 193 using the Silonite canister. During the VOC measurements, the toxicant exposure was 194 terminated. Each time only one rat was placed into the chamber. 195 196

In this work, total VOCs (TVOC) level in the exposure chamber was real-time 198 monitored by a PID sensor during all the experiments: 1) when the rats were not in the 199 chamber (background air); 2) rats in the chamber (before toxicant exposure) and rats in 200 the chamber (after toxicant exposure). The working procedure of the PID sensor is to 201 first ionize the VOCs gas with a high-energy UV lamp, and then the ionized fragments 202 are collected by the ion chamber to generate a current signal. The signal in general is 203

proportional to the concentration of the target VOCs. In this study, the PID sensor is 204 assembled with the oil-free pump and pre-filter and a signal encoder, and the signal is 205 transmitted and displayed in real time. The sensor was calibrated using 1 ppm 206 isobutylene prior to use. Technology Co., Ltd, and is briefly described as follows: the Entech 7200 system was 216 used to pre-concentrate the sample, including enrichment of the sample, and removal 217 of water and carbon dioxide from the sample. Then, an Agilent 5975C/7890A gas 218 chromatography mass spectrometer was used to qualitatively analyze sample according 219

to the standard mass spectrometry library and the mixed gas standard compound. The Table S1 (Supporting Information) . 251 252

In this study, the TVOC levels for all samples detected by the PID sensor were not 254 normally distributed, so the Mann-Whitney rank sum test was used to analyze the 255 differences in TVOC levels before and after each toxicant exposure. The t-test was used 256

to analyze the differences in TVOC change rates between each toxicant exposure group 257 (PID instrument failure for one rat in each group) and the control group (indoor air). 258

analyze differences for each VOC species before and after the exposure. The software 260

Canoco 4.5 was used to visualize the VOC profile distance and relatedness between the 261 samples of different groups using the principal component analysis (PCA). Besides, the 262 concentrations of micro-RNAs in blood samples from different rat groups were 263 determined by RT-qPCR. For the group exposed to carbon dioxide, blood samples were 264 only taken from two rats (before and after the 10-min exposure) because of catheter 265 blockage for the other two. For the other three groups, blood samples were obtained for 266 all four rats. The outliers were examined and eliminated by a Grubbs test. The 267 differences between micro-RNA levels in blood samples before and after the exposure 268 in one group were analyzed using a paired t-test (data exhibited a normal distribution) 269

or Wilcoxon signed rank test (data did not follow a normal distribution). All the 270 statistical tests were performed via the statistical component of SigmaPlot 12.5 (Systat 271 Software, Inc., USA), and a p-value of less than 0.05 indicated a statistically significant 272 difference at a confidence level of 95%. 273

As described in the experimental section, four toxicants (ricin, endotoxin, ozone 278 and carbon dioxide) and indoor air (as a background control) were used for inhalation 279 exposure in rats. Before and after the exposure, the TVOC level in the exposure 280 chamber was monitored by the PID sensor. For each group, the TVOC levels of only 3 281 rats (PID instrument failure for one rat) were shown in Figure 2 . The indoor air 282 background TVOC was found to be less than 0.02 ppm. After one rat was placed in the 283 exposure chamber, the TVOC level in the cage was shown to first gradually increase as 284 shown in Figure 2 , then reach a relatively stable level after about 500 seconds. The 285 TVOC level before the exposure (indoor air) when one rat was in the chamber was 286 about 2 ppm as shown in Figure 2 , except for the CO2 group of which was about 0.5-287 0.8 ppm (These background differences, if any, applied to both control and exposure 288 tests, thus presenting no influences on the same experiments). The air pump of the PID 289 sensor was then turned off, and air in the exposure chamber was sampled using a 290

Silonite canister. After the sampling (about four minutes) was completed, each of the 291 tested toxicants was then introduced into the exposure chamber. After the exposure (ten 292 minutes) was completed, the original indoor air supply was provided again to each rat, 293 and the TVOC monitoring by the PID sensor was resumed. As shown in Figure 2 , the 294 differences in TVOC levels for indoor air exposures (different times: "before" and 295 "after", but the same indoor air) were small (the average change rate was about -4%±1.4% 296 (95% confidence interval)), although the Mann-Whitney Rank Sum Test showed that 297 for each of the rats, the difference (over some time for the indoor air) was significant 298 when rats were exposed to different toxicants via inhalation for 10 mins: Indoor air, 307 ricin, endotoxin, O3 and CO2. During the exposure processes, the PID sensor was turned 308 off. Data lines (measurement time was 1000 s) represent results from three individual 309 rats (#1, #2, #3) before or after exposure to each of the air toxicants (aerosolized 310 amounts described in the experimental section) tested. Each exposure test was 311 independently repeated with four rats from the same group (PID instrument failure for 312 one rat). 313

In contrast, TVOC levels were shown to vary greatly with different toxicant 315 exposures as shown in Figure 2 . For example, as shown in Figure 2 , when rats were 316 exposed to the ricin, the TVOC level was observed first to increase slightly, then 317 decreased to a level comparable to that of before exposure with an average change rate 318 of -3%±1.6 (Mann-Whitney Rank Sum Test, all p-values<0.001). Compared to the 319 control group (indoor air) shown in Figure 2 , the difference of the TVOC change rate 320 was not significant for the ricin exposure (t-test, p-value=0.426). For ricin exposure, its 321 concentration (40 μg/mL aerosolized) might be too low in aerosol state after the 322 aerosolization from the liquid to produce a detectable response from the rats. This 323 suggests that ricin, given the amount aerosolized here, presented no additional health 324 challenge compared to the indoor air at the time of the experiment. Compared to the 325 ricin exposure, however we observed a different phenomenon for the endotoxin (50 326 ng/mL aerosolized) tests as shown in Figure 2 . Upon the endotoxin exposure, the TVOC 327 level was observed to first increase slightly, and then surprisingly decreased to a level 328 that was about 21-46% below the pre-exposure level after four minutes (Mann-Whitney 329

Rank Sum Test, all p-values<0.001). Compared to the control group (indoor air), the 330 difference of the TVOC change rate was statistically significant for endotoxin (t-test, 331 p-value=0.0147). The observed differences from the ricin and endotoxin exposures 332 could be due to different mechanisms initiated by different substances involved. Ricin 333 is derived from plant, while endotoxin is from Gram-negative bacterial membrane. 334

They could interact differently with relevant human respiratory or other body cells. 335

After exposure to gaseous toxicants such as ozone and carbon dioxide, the levels 337 of TVOCs in the exposure chamber with rats were observed to have increased 338 significantly, as observed in Figure 2 . As can be seen from the figure, the TVOC levels 339

has increased for about 44-110% for ozone and about 109-265% for carbon dioxide 340 exposure (Mann-Whitney Rank Sum Test, all p-values<0.001). The t-test showed that 341 differences of the TVOC change rates of both ozone and CO2 exposures compared to 342 the control group (indoor air) were statistically significant (p-value=0.0219 and 0.0296, 343 respectively). These data indicated that rat exposure to both ozone and CO2 has resulted 344 in significant elevations of TVOCs, suggesting rats were actively responding to the 345 exposure challenges. The behavior observation from a video also indicated that rats 346 after the exposure to O3 seemed to be suffering from the challenge (Video S1, 347

Supporting Information). 348 349

In order to determine the changes of VOC species exhaled out by rats before and 351 after the exposures to four different substances, the GC-MS/FID method was used to 352 qualitatively and quantitatively analyze the VOCs in the exposure chamber. The 353 background indoor air without and with rat in the chamber were first analyzed. A total 354 of 31 different VOCs were detected and shown in Figure S2 (Supporting Information). 355

Among detected VOCs as shown in Figure S2 (Supporting Information), the VOCs with 356 the highest concentrations in indoor air were n-hexane, ethyl acetate and acetone, which 357 all come from the laboratory air. When rats were placed in the exposure chamber (one 358 rat at each time), the most abundant VOC species was detected to be acetone, which 359 was about 4 times more than that of the indoor air background. Statistical tests found 360 that the concentrations of ethylene and ethane in the chamber containing one rat were significantly lower than that of the background (paired t-test, p-value<0.05), which in 362 part could be due to the air dilution by the rat's breath. 363 toxicants exposures were also shown in Figure 3 . There were no significant differences 373 in the concentrations of any VOCs before and after the exposure for the control group, 374

i.e., indoor air (t-test, p-value=0.05). This suggests that indoor air is relatively less toxic 375 to a level that is unable to detect a VOC change. In contrast, specific VOC species had 376 experienced significant changes when rats were exposed to ricin, endotoxin, O3 and 377 CO2 as observed from Figure 3 . For example, exposure to ricin caused significant 378 higher concentration of ethyl acetate (183% higher), while lower concentration of 379 carbon disulfide (22% lower). As shown in Figure 3 , after the endotoxin exposure 380 process, concentrations of five VOC species: ethane, acetone, cyclopentane, carbon 381 disulfide and methylcyclopentane were shown to be significantly different with those 382 of before the exposure (t-test, all p-values<0.05). As can be seen from the results of the 383 ozone exposure group in Figure 3 , the concentrations of propionaldehyde, pentane, 2-384 butanone, hexane and 2-methylpentane exhibited significant differences before and 385 after exposure (t-test, all p-values<0.05), in which all the VOCs except 2-386 methylpentane were elevated. In comparison, rat exposure to CO2 resulted in acetone 387 level increase by 34% (t-test, p-value=0.0016). These data suggest that exposure to 388 different toxicants had led to production of different VOC species in addition to their 389 level changes. 390 391

To further explore the VOC response mechanism of rats to toxicants exposure, 393 microRNAs (miRNA) in the blood samples were examined by an RT-qPCR assay. Fold-394 changes in microRNA levels after toxicants exposure were shown in Table S2 395 (Supporting Information). The level of miR-33 in the blood of rats was shown to be significantly lower than that before ricin exposure (p-value<0.05); after exposure to 397 ozone, miR-146a in the blood samples of rats were significantly higher than those 398 before the exposure (p-value <0.05), while miR-155 was significantly lower than that 399 before the exposure (p-value <0.05). For other microRNAs as listed in Table S4 , the 400 changes seemed to be insignificant (t-test, p-values>0.05). 401 As shown in Figure 3 , the acetone level increased by 34±9% as a result of CO2 exposure, 446 suggesting CO2 caused hypoxia in rats, and led to increased respiration from rats. These 447 increases in acetone level corresponded to TVOC level increase as determined by the 448 PID sensor after the exposure to CO2. However, when exposed to endotoxin, the 449 acetone level in the exposure chamber decreased by about 10±6%, indicating that the 450 respiration of the rats may be attenuated by the exposure of endotoxin. Clearly, the 451 involved mechanisms by which endotoxin and CO2 cause health effects to rats could be 452 very different. In addition to these biologicals, we have also shown that exposure to chemicals 469 such as ozone and CO2 also resulted in in-vivo changes in VOC levels. From the fold 470 changes of various VOC species such as propionaldehyde, N-pentane, 2-Butanone, and 471

Hexane, the ozone exposure has resulted in an overall increase of VOCs in rats' exhaled 472 breath, which agreed with the TOVC monitoring shown in Figure 2 by the PID sensors. 473

It was previously indicated that increase in propionaldehydes, further products of lipid 474 peroxidation, indicated more severe oxidative damage in rats following exposure to 475 ozone . Ozone was described as a strong oxidizing agent, and can cause intracellular The experimental results here showed that the indoor air supply to the rats in the 505 exposure chamber did not cause obvious changes in the TVOC level. This indicated 506 that the indoor air exhaled by the rats were relatively less toxic, for example, unable to 507 induce a detectable TOC change from exhaled breath. When exposed to aerosolized ricin of the tested amount for 10 mins, the TVOC did not change significantly; while 509 the endotoxin exposure resulted in lower TVOC levels. In contrast, when exposed to 510 ozone or CO2 the TVOC levels were shown to have increased by more than 200%. 511

Clearly, these results indicate that when rats are exposed to toxic substances their 512 certain metabolic activities are immediately affected, i.e., these exposures promoted or 513 inhibited specific VOC productions. Based on the results we obtained from this work, 514 the following VOC emission mechanisms of rats when exposed to different toxicants 515 are proposed and illustrated in Figure 5 . Previously, it was suggested that VOCs are 516 produced during the normal metabolism in the body; while pathological processes, such 517 as metabolic disorders, can also produce new species of VOCs or alter the levels of 518 existing VOCs . Therefore, cell or tissue injuries caused by external toxicants exposure 519 also can alter the exhaled VOCs profile by disturbing the normal process. The exact 520 toxic effect mechanism as observed from this work could vary from one toxicant to 521 another. For some pollutants such as endotoxin and ricin, there are specific receptors to 522 recognize them and then start the chain of responses or reactions . Among these various 523 mechanisms, the ROS (reactive oxygen species) and oxidative stress are recognized to 524 be the central and the common mechanism in various forms of pathophysiology, as well 525

as the health effect of various air pollutants including ambient particulate matter (PM) . 526

Oxidative stress is essentially a compensatory state of the body and can trigger redox-527 sensitive pathways leading to different biological processes such as inflammation and 528 cell death . For example, the strong oxidants such as ozone might cause oxidative stress 529 through direct effects on lipids and proteins , which mostly caused the generation and 530 release of hydrocarbons and aldehydes, such as ethane, ethylene, and propionaldehyde . 531

While carbon dioxide tends to make the redox balance tilted toward the reduction side 532 by reducing oxygen supply and thus influencing the energy metabolism in cells . For ricin and endotoxin exposure, the underlying mechanisms seem to be different from 534 ozone and CO2, and they could cause oxidative stress indirectly through the activation 535 of intracellular oxidant pathways. Nonetheless, all toxicants share a common effect of 536 disrupting the redox balance, and thus interfere with normal biochemical reactions or 537 cause material damage in cells, accordingly changing the VOC profile and releasing 538 into the breath. As discussed above, in this work, the VOCs profile of rats changed 539 significantly after exposure to different toxicants. Therefore, regardless of toxicant 540 types, breath-borne VOCs from the rats seem to be capable of serving as a proxy for 541 real-time monitoring air toxicity. 

Recently, exhaled VOCs have increasingly been used as non-invasive samples for 552 exposure studies as well as clinical diagnosis. In this study, we examined the possibility 553 of using living animals' exhaled VOCs in real-time monitoring air toxicity. Our 554 experimental data showed that when the rats were exposed to air containing various 555 toxicants, characteristic VOC profiles were in vivo produced in the body within 10 556 minutes or shorter, and further emitted via exhaled breath. In addition, different toxicant 557 exposures were shown to have caused productions of distinctive profiles of VOC 558 species from the living rats. Therefore, the developed RSTair system, i.e., by integrating 559 inhalation exposure, living rats, breath sampling and online VOC sensor, was capable 560 of real-time monitoring toxic air. The RSTair system can detect a breath-borne VOC 561 change when the air is becoming toxic to rats. In doing so, the system can real-time 562 alter people of possible air toxicity change or hazardous air. Nonetheless, the PID sensor 563 used in this system only reports the TVOC level without classifying individual VOC 564 species. It should be also noted that the PID sensor responds to different VOCs 565 differently. For example, the PID could respond to one VOC with stronger signal, but 566 to another with weaker one even for the same concentration level. However, this can be 567 readily remedied by using portable direct-injection mass spectrum such as Proton 568

Transfer Reaction-Mass Spectrometry (PTR-MS). This study revealed that living 569 systems such as rats will in vivo alter specific VOC productions in response to external 570 toxicant exposure challenges from air. By using this discovered fundamental science, 571 the invented RSTair system here showed its great promise of revolutionizing the air 572 toxicity monitoring, and providing significant technological advances for air security in related fields such as military defense, customs, counter-terrorism and security 574 assurances for important events or special locations. 

The authors declare no competing financial interest. Table S2 Expression level changes (before and after the exposure) of microRNA levels 774 in blood samples from rats before and after exposure to ricin, O3, endotoxin and CO2. 775

The blood samples were taken before exposure and 10 minutes after exposure. SD 776 represents a standard deviation value. "*" indicates significant difference at p-777 

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