Meets Blood : In vivo Damages and Immune Defense

Recent evidence shows that inhaled PM2.5 can enter the blood circulatory system and even the brain. However, the damage of blood-borne PM2.5 is not clearly elucidated. This work aims to understand and characterize the toxicity, i.e., the acute health effects, of PM2.5 that is directly injected into the blood circulatory system. Rats were injected with different dosages (568, the equivalent of 1 year of inhalation for a rat; 93; and 9.3 μg) of PM2.5 sampled from Beijing via a sterile catheter injected into the jugular vein. The behaviors of the rats upon external interruptions were recorded. Blood samples were collected before exposure and 1 h, 3 days, 5 days, 7 days, and 9 days after the PM2.5 injection for analyzing serum interleukin-6 (IL-6), C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), endotoxin, and 8-hydroxydeoxyguanosine (8-OHdG) levels. After euthanization, the heart, lung, liver, kidney, and spleen were taken and processed for histopathological analysis. PM2.5 components were also analyzed. Acute inflammation with 102% and 90% increases for IL-6 and CRP, respectively, was observed 1 h after the 568 μg-PM2.5 injection, while oxidative DNA damage occurred only five or more days later, which was accompanied by significantly elevated endotoxin levels. Hemorrhage of lung alveoli and behavioral changes, including fear and non-responsiveness, were also observed. Surprisingly, all exposed rats seemingly survived the PM2.5 injection, behaving similarly to the control groups. The immune defense might have played an important role in combating the PM2.5 injection. The results showed acute health effects from directly injected PM2.5, including rapid inflammation, oxidative damage, and routine-behavioral changes. Further study about the long-term effects of injection and the immune defense is warranted. Nonetheless, the results here suggest that PM2.5 health effects may have to some extent been exaggerated in the literature.


INTRODUCTION
Air pollution has nowadays become one of major environmental problems facing mankind (Shah et al., 2013;Ierodiakonou et al., 2016;Geravandi et al., 2017;Khaefi et al., 2017), especially in east Asian countries (McGranahan and Murray, 2012).For example, it was ranked as the fifth high-risk factor, and estimated to have resulted in 5.5 million global deaths and 141.5 million global disability-adjusted life-years in 2013 (Forouzanfar et al., 2015).Among other ambient pollutants, fine particulate matter (PM 2.5 ) has been subjected to extensive investigations (Zhang et al., 2012;Yu et al., 2013).Both short-term and long-term health effects of PM 2.5 were observed in past studies (Yu et al., 2015;Kioumourtzoglou et al., 2016).Epidemiological and toxicological studies have demonstrated that exposure to PM 2.5 is strongly associated with cardiovascular and respiratory diseases (Weichenthal et al., 2016;Yang et al., 2016;Zhang et al., 2016b).The PM 2.5 -bounded species, such as oxidant gases, organic compounds, and transition metals (Krzyzanowski et al., 2005;Lai et al., 2012;Fujitani et al., 2017) have been observed to weaken the antioxidant defense system and fail to counterbalance the production of reactive oxygen species (ROS) by inflammatory cells (Liu et al., 2009;Bae et al., 2010;Zhang et al., 2016c).In general, it is believed that PM 2.5 provokes oxidative stress and inflammatory response, which correspondingly leads to the release of cytokines.
Toxicological studies of PM 2.5 are generally conducted in vitro or in vivo.Møller et al. (2014) reviewed the most relevant studies available in literature that have assessed health effects of air pollution particles, and found out 97.6% of in vivo studies focused on inflammation while those in vitro studies dealt with both oxidative damage and inflammation.For in vitro experiments, the most predominant cell types used for elucidating the mechanisms of pulmonary and cardiovascular damages attributed to PM 2.5 are bronchial epithelial cells (Crobeddu and Baeza-Squiban, 2015) and vascular endothelial cells (Hu et al., 2016).Particularly, cultivated cells are exposed to various extracts of PM 2.5 samples and then subjected to different types of analyses.
In vitro studies provide a convenience for exposure and detection, but they are different from the true exposure settings.On the other hand, studies in vivo mainly focused on mice or rats, and most of them selected inhalation and instillation (nasal or intro-tracheal) as their exposure pathways (Li et al., 2015b;Dai et al., 2016).Inflammation in animals has been assessed by increased number of leukocytes, predominantly polymorphonuclear (PMN), or elevated concentrations of pro-inflammatory cytokines in bronchoalveolar lavage fluid (BALF) (Møller et al., 2014).Despite of such a great number of efforts, the potential toxicity of PM 2.5 remains to be adequately characterized.
Inhaled PM 2.5 , especially nanoparticles (NPs), may be able to translocate into blood circulation (Jin et al., 2015).Once inhaled, NPs can pass through the epithelial barrier, and then they may successively cross the basement membrane, subepithelial connective tissue layer, endothelial cells and finally enter the blood stream (Blank et al., 2014).One of earlier studies focusing on this topic showed that inhaled carbon particles (< 0.1 µm) was able to pass rapidly into the systemic circulation (Nemmar et al., 2002).Two more recent in vivo studies using intratracheal instillation of gold NPs and graphene, respectively, observed the translocation of particles into blood circulation and their accumulation in liver and spleen (Lipka et al., 2010;Mao et al., 2016).In addition to the insoluble particles, the soluble components cleaved from particles also have the potential of translocating into blood (Mills et al., 2006).After entering the blood stream, foreign substances might affect the function and viability of endothelial cells lining in the capillaries (Blank et al., 2014).Since these cells play an important role in inflammation processes, pro-inflammatory stimuli may also be induced (Michiels, 2003).There are about 3700 proteins in the complete plasma proteome (Lynch et al., 2007), and 50 of them have been found to be positively associated with foreign substances such as NPs (Aggarwal et al., 2009).Some studies also demonstrated that ambient particles and NPs are toxic to cytochromes P450, which is a ubiquitous superfamily of enzymes for eliminating most hydrophobic compounds in tissues throughout the body (Reed et al., 2015), as well as blood cells such as leukemic HL60 cells (Rodhe et al., 2015) and primary erythroid cells (Rujanapun et al., 2015).Foreign substances can be removed from the circulation through pores and fenestrae in the vascular endothelium.And they subsequently reach and combine with potentially sensitive target sites such as bone marrow, lymph node and organs via mononuclear phagocyte system in the reticular connective tissue (Oberdörster et al., 2005;Casals et al., 2008).It is thus believed that PM 2.5 could be most harmful when its components enter the blood circulation that is directly linked to all the organs.
Here, this work was carried out to investigate the acute effects of PM 2.5 samples when directly injected into the blood circulation of a new rat model.The experimental rats were cultivated with an embedded venous catheter that is directly connected to their blood circulation.During the experiments, the rats were injected via the catheter with PM 2.5 collected from Beijing during clear and haze days.The injection doses for high, middle and low groups were approximately 568, 93 and 9.3 µg PM 2.5 , respectively.Five serum biomarkers (interleukin-6, 8-hydroxydeoxyguanosine, tumor necrosis factor-α, endotoxin, and C-reactive protein) linking to oxidative damage and inflammatory responses were studied before and after the injection.In addition, the heart, liver, spleen, lung and kidney from both control and PM-exposed groups were taken after the euthanization for anatomy and histopathological analysis.Simultaneously, we also recorded all behaviors and responses of those control and PM-exposed rats upon external disturbance.In this work, a different protocol was developed for studying the toxicity of specific PM 2.5 samples by directly injecting them into the blood circulation, and the results from this work provide a new understanding of PM 2.5 toxicity and the immune defense.

PM 2.5 Sampling and Preparation
In this work, we aimed to investigate how toxic when PM 2.5 samples are directly injected into the blood circulation.Samples of PM 2.5 were collected at the Peking University air monitoring station on Jan 11 (a clear day with a daily average PM 2.5 concentration, temperature and relative humidity of 23 µg m -3 , -3.4°C and 30.8%, respectively) and Jan 20, 2016 (a haze day with a daily average PM 2.5 concentration, temperature and relative humidity of 140 µg m -3 , -4.4°C and 62.5%, respectively).The sampling station (39 o 59'N, 116 o 18'E) is located on the rooftop of a 7-story building of the main campus of Peking University, which is within 500 meter of the fourth ring road.Samples were collected onto flexible Teflon filters (46.2 mm diameter, Whatman Inc., the U.S.A.) using a 4-Channel Particulate Matter Sampler (TH-16A, Wuhan Tianhong Instruments Co., Ltd., China) at a flow rate of 16.7 L min -1 for 24 hours.Before sampling, filters were kept for 24 hr at 20°C and 50% relative humidity.Weights of blank filters were measured 3 times by a Mettler-Toledo AX105 Delta-Range microbalance (10 µg sensitivity).After sampling, filters were brought back and conditioned for another 24 hr and then weighed 3 times at the same temperature and relative humidity conditions of the first weighing.Filter weights from an average of three independent measurements were recorded for use.
Once the weights of samples were determined, filters were subjected to a sonication extraction process.Loaded filters were placed into centrifuge tubes and 6 mL of phosphate buffer saline solution (PBS) was added.Then, filters underwent a 30-minute sonication-assisted water extraction at room temperature.For sonication, an ultrasonic instrument (KQ218, Kunshan Ultrasonic Instruments Co. ltd., China) with an ultrasonic frequency and effective power of 40 kHz and 100 W, respectively, was used.The obtained extracts were further used for animal injection and other content analysis.

Animal and Treatments
Male Sarague Dawley (SD) rats at an age of 10 weeks weighing 200-240 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.The SD rat was first produced by the Spargue-Dawley farms in Madison, Wisconsin in 1925.It is an outbred multipurpose breed of albino rat.Calmness and ease of handling are the main advantages of this breed of rat.All rats used in our experiments were performed with the jugular vein catheterization operation, by Beijing Vital River Laboratory Animal Technology Co., Ltd., to embed a flexible sterile catheter into the jugular vein.The outer end of the catheter is about 1 centimeter and fixed on the back skin with staples.Extracts injection and blood sampling were performed through the catheter using sterile syringes with 23G flat-end needles as seen in Fig. 1.All rats were kept and examined at laboratory animal center of the Academy of the Military and Medical Sciences, with 12:12 light-dark cycle and food and water ad libitum.All animal experiments were approved by the Animal Ethics Committee of the Academy of the Military and Medical Sciences, and were performed in accordance with the Guideline for Animal Experiments of the Academy of Military and Medical Science.
After 1 week of acclimation, the rats were randomly divided into groups right before the experiments: high-dose group (n = 3 for video recordings, and n = 2 for other analyses due to the blood blocking of the catheter for one rat), middledose group (n = 4), low-dose group (n = 3), and control group (n = 3).The high-dose group (equivalent of 1-year rat inhalation dose) rats were injected with the extracts from PM 2.5 sample collected from the haze day (Jan 20, 2016).Middle-dose group rats were injected with extracts from PM 2.5 sample on clear day (Jan 11, 2016).Low-dose group rats were injected with 10-fold diluted extracts for middledose group.And the control group rats were injected with extracts from blank filter (without PM 2.5 sample) using the same method.Assuming the PM 2.5 were completely extracted, the injection dose for high, middle and low groups would be 568, 93 and 9.3 µg PM 2.5 , respectively.All groups were injected with the same volume (1 mL) of extracts.The highdose is approximately equivalent of one-year inhalation does from the rats given the PM 2.5 concentration level under which air sampling was conducted.This was calculated using body-weight ratio of average human vs. rat by considering human adult breathing rate of 12.5 L min -1 .The Haze sample was used for the injection of high-dose group in this work.
Blood samples were taken (0.3 mL for each time) before the injection and 1 h, 3 days, 5 days, 7 days, 9 days after the PM 2.5 injection, respectively.When injecting extracts or taking blood samples, rats' eyes were covered using a piece of black cloth to keep them in calm.Subsequently, the subcutaneously cultivated catheter was withdrawn about 2-3 centimeters outward and the cover was removed for PM 2.5 injection.Following this, the blood sealing solution was taken out first using a syringe and extracts or blood samples were injected into or taken out using a second syringe.After this, 0.2 mL normal saline and 50-60 µL sealing solution using new syringes were injected one after another.Finally, the catheter was fully sealed and placed under the skin.The sealing solution used is 50% glucose solution with 500 IU mL -1 heparin, which was applied to preventing blood clotting and ensuring the catheter to be unblocked.When changing syringes, the end of the catheter Fig. 1.PM 2.5 extracts injection and blood sampling via the catheter cultivated in the jugular vein of SD rats.The injection doses for high-, middle-and low-dose groups were 568, 93 and 9.3 µg PM 2.5 dissolved in 1 mL PBS, respectively.The control group was injected with 1 mL PBS without the PM 2.5 , and 0.3 mL blood was taken from the rats each time.
was tightly closed to prevent air admission.Blood samples were kept at -20°C until analysis.After the injection, both control and exposed groups of rats were monitored using video camera.

Measurements of Biomarkers Using Enzyme-Linked Immunosorbent Assay
We chose five biomarkers to study oxidative damage and inflammatory responses after the PM 2.5 injection in this study.The biomarkers included interleukin-6 (IL-6), 8hydroxydeoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α), endotoxin (ET), and C-reactive protein (CRP).The relevant kits for rat (Shanghai Jianglai Biotechnology Co., Ltd., China) via enzyme-linked immunosorbent assay (ELISA) were used to measure the above-mentioned biomarkers in the sera according to the manufacturer's instructions.Briefly, solid-phase antibody was prepared by coating microliter plate wells with purified rat antibody.Samples and HRP enzyme labeled antibodies were added into coated wells to form antibody-antigen -"enzyme-antibody" complex.Then TMB substrate solution was added after washing the wells.TMB substrate solution became blue after catalysis by HRP enzyme.The reaction was terminated by an addition of a sulphuric acid solution and the suspension finally turned into yellow.The color change was measured spectrophotometrically at a wavelength of 450 nm and the concentrations of biomarkers in the samples were then determined by comparing the optical densities (OD) of the samples to the standard curve.The deionized water and blood samples collected from rats injected with PBS were used as our controls.The blood samples were analyzed in duplicates for each of the biomarkers using the kits mentioned above.

Anatomy and Histopathological Analysis of Rats' Organs
We euthanized all the rats by cervical spine dislocation after the final planned blood sampling.Heart, liver, spleen, lung and kidney from the rats were taken and subsequently washed using PBS.Then the organs were steeped using 4% formaldehyde solution for 72 hours.The fixed organs were subjected to routine dehydration and paraffin-embedded as the preparation of tissue section.After cutting, the cross sections were mounted onto the glass slides and deparaffinized.The slice thickness was about 5 µm for each organ.All the resulting slides were stained with hematoxylin and eosin (H&E) for histopathological analysis.

Metal and Biological Component Analysis of PM 2.5 Extracts
Elemental metal analysis was performed using an aurora M90 ICP-MS (Analytikjena, German).The ICP-MS was equipped with a two-channel atomizing chamber with controlled temperature at 3 ± 0.1°C and a quartz integration quarter with the central passage size of 2.5 mm in diameter.The concentrations of twenty elements, i.e., Ca, Ti, V, Cr, Co, Ni, Cu, Zn, Mo, Cd, Tl, Pb, Th, U, Mg, Al, K, Mn, Fe and Ba were analyzed.

Statistical Analysis
Here, the concentrations of IL-6, CRP, endotoxin, TNF-α and 8-OHdG in the sera of rats for different groups were analyzed using ELISA.For all experiments, at least three independent rats or samples were used.In some cases, due to the blood clogging of the catheter blood samples from only two rats were taken.Independent sample t-test was used via the software SPSS 16.0 to analyze the differences of the data.The individual variations were also considered in the analysis.To study the individual variations in biomarkers in rats, we purchased additional two more male SD rats with the same physiological characteristics from the Beijing Vital River.These two rats were also performed with jugular vein catheterization operation but received no PBS nor PM 2.5 injection.Blood samples with the same amount and time frequency were taken with other rats and subjected to analysis of the same five biomarkers.The measurements showed that concentrations of biomarkers in rats' sera were constantly changing under normal physiological condition.The concentrations levels of biomarkers varied over the time from a decrease up to 38% to an increase up to 49%.Accordingly, only those changes over than 50% were treated as statistically significant changes in this work.A p-value of less than 0.05 indicates a statistically significant difference between different treatment groups.

Video Recordings of Exposed and Control Rats
According to our video recordings, the rats from highdose group appeared to be drowsy and less responsive to an external interruption (e.g., knocking the cage) 1 h after the PM 2.5 injection; while the control groups remained to be alert in response to the same interruption (Fig. 2 and also videos in Supporting Info S1).This behavioral discrepancy between two different groups suggested that the PM 2.5 after injected into blood circulation system of the rats might have caused acute health effects on the rats within a short Fig. 2. Video snaps of rats from control and high-dose groups in response to the same external interruption; the control group appeared to be alert while the high-dose group appeared to be drowsy and less responsive.All related videos are provided in Supporting Info S1. period of time.Apparently, the injection seriously affected the rats' daily routine, e.g., the sensitivity response to an external stimulus.As seen in the video recordings, the nerve system of the rats or related function could have been somehow affected in the high-dose group after the first hour of the injection.However, over the course of the experiment, i.e., after 3-5 days, we surprisingly found that the high-dose group rats seemed to have recovered from the PM 2.5 injection, and they behaviorally appeared to be as normal as the control with respect to their routine movements according to the video shown in Supporting Info S1.A series of immune responses and self-defending mechanisms in response to the PM 2.5 injection exposure could have taken place.During the 9-day observation time period, no rat death was observed in all the exposed and control groups.The video recordings presented a direct observational evidence of the impacts of PM 2.5 injected into the blood circulation on rats' daily routines including the ability to respond to environmental stimuli.Overall, the video recordings indicated that some internal influences of the injected PM 2.5 on the rats must have taken place.

Oxidative Damage and Inflammatory Response
The concentrations of IL-6, CRP, endotoxin, TNF-α and 8-OHdG in sera of rats from different groups before and 1 h after the injection are shown as means ± standard deviations of three repeats in Fig. 3.The catheter in one of the rats from high-dose group was blocked after the PM 2.5 extract injection, and thus no blood sample was obtained from this rat as shown in Fig. 3, but its behavioral changes were observed to be the same as the other two.As observed in Fig. 3, the mean level of IL-6 for high-dose (568 µg PM 2.5 -approximately equivalent of one year inhalation dose under the PM 2.5 level) group increased by 102% (from 87.51 ng L -1 to 177.15 ng L -1 , p-value = 0.007) 1 h after the PM 2.5 injection.For low-and medium-dose groups, the IL-6 levels seemed not to be changed significantly (p-value = 0.487 and p-value = 0.734, respectively).There were variations observed for serum IL-6 levels among the control (PBS), the low-dose (9.3 µg PM 2.5 ) and the medium-dose (93 µg PM 2.5 ) groups 1 h after the PM 2.5 extracts injection as shown in Fig. 3.They were -11%, 7% and -12%, respectively.Apparently, the injection of 568 µg PM 2.5 had resulted in a significant increase of IL-6.The proinflammatory behavior of IL-6 in toxic effects, as well as its positive relationship with the level of ROS generation, have been well addressed in both in vivo and in vitro studies previously (Li et al., 2015a).Nazariah et al. (2013) observed a positive correlation between the ambient PM 2.5 concentration and IL-6 expression among school children.Besides, IL-6 is also suggested to be an important mediator of fever and acute phase response (Soares et al., 2012).IL-6 is capable of crossing into the hypothalamus and inducing the synthesis of PGE2, thus changing the body's temperature set point (Murthy et al., 2015).The remarkable change in the IL-6 level in highdose group thus could induce rapid inflammatory and fever responses via generation of ROS or serving as a mediator for certain toxic substance release.Insignificant changes among the other groups may be due to the low dose of PM 2.5 or less toxic components of PM 2.5 collected during clear day (23 µg m -3 ) compared to the high-dose group.The results here suggest that the acute effects of PM 2.5 depend on its dose, and the lower dose of PM 2.5 (9.3 µg) even injected into the blood circulation did not result in immediate acute toxic effects.As an inflammation marker, IL-6 might be used as a sensitive indicator for PM 2.5 exposure.
Similar to IL-6, we have also observed significant increases in C-reactive protein (CRP) levels from the high-dose group as shown in Fig. 3 (p-value = 0.033).The CRP is a protein that is produced mainly in the liver and has been widely used to indicate systemic inflammation (Rich et al., 2012).In this work, thethe levels of CRP were observed to increased about 40% and 90% for the medium-and high-dose groups, respectively.The CRP levels did not change much for the low-and middle-dose groups compared with the control as shown in Fig. 3 (p-value = 0.0914 and p-value = 0.143, respectively).This has suggest that there exists some nonlinear relatiosnhiip between PM 2.5 level and response.Hennig et al. (2014) found that long-term exposure to PM 2.5 were positively associated with serum CRP among subjects under high traffic load.The significant increase of CRP from the high-dose (568 µg PM 2.5 ) group indicated that PM 2.5 when injected directly in the blood circulation would provoke a rapid and acute inflammatory response.An earlier study indicated that an injection of Escherichia coli endotoxin the CRP in mice sera did not change after 6 h injection, but following 24 h its level was significantly increased (Patterson and Higginbotham, 1965).In their study, they also observed that the CRP, which was induced by the E. coli endotoxin, developed a resistance to the Staphylococcus aureus infection.Compared to the IL-6, CRP biomarker seems to be a more sensitive biomarker as its rapid increase can be observed even for the medium-dose group.
In addition to the increases in IL-6 and CRP, we have observed significantly higher endotoxin level (an increase of 70%) for the high-dose group as observed in Fig. 3. Endotoxin is an outer membrane component of Gramnegative bacteria and cyanobacteria.Endotoxin is a wellestablished activator of TLR4 via binding the CD14 and MD2 receptor complex in immune cells such as microglia, monocytes and macrophages, and further promotes the secretion of pro-inflammatory cytokines and elicit strong immune responses (O'Callaghan et al., 2015).Shang et al. (2012) pointed out that endotoxin is one of the critical factors present in PM in triggering pro-inflammatory cytokines release.Moreover, endotoxin is an exogenous pyrogen.Here, the PM 2.5 samples collected from Beijing certainly contain a large amount of endotoxin.A previous study shows that the geometric means for endotoxin in PM were 10.25 EU mg -1 PM 2.5 (range: 0.38-1627.29)in Beijing (Guan et al., 2014).Direct injection of PM 2.5 samples into the rat's blood circulation also introduced the PM 2.5 -bound endotoxin into the blood.A recent study shows that exposure to high level of endotoxin can induce rapid increase in blood pressure (Zhong et al., 2015).Behbod et al. (2013) also found out exposure to endotoxin in ambient PM 2.5 induced systemic inflammation to human, with an interquartile increase in endotoxin (5.4 ng m -3 ) was significantly associated with an increase in blood leucotytes 3 h post-exposure.The increase in endotoxin level might be also contributed by the death of Gram-negative bacteria in the PM 2.5 samples injected into the blood.Here, the observed increases in relevant biomarkers and also behavioral changes could be in part contributed by the endotoxin.Endotoxin in the blood could have played an important role in inflammatory responses and the release of IL-6 and CRP as observed in the exposed groups.
In contrast with IL-6 and CRP, we have observed a decrease in TNF-α level in the high-dose group as shown in Fig. 3 (p-value = 0.038).In the past, a broad range of studies revealed the central role of TNF-α in inflammatory responses (Tsai et al., 2012), but some studies suggest the other way.For example, many studies chose IL-6 and TNF-α as combined biomarkers for studying inflammatory responses.Li et al. (2015a) investigated the oxidative stress, calcium homeostasis and inflammation in hearts of rats exposed to PM 2.5 and simultaneous increases in both IL-6 and TNF-α levels were detected.However, some studies found that high concentration of TNF-α was detrimental as it may induce cell death, while low levels were protective and were believed to participate in normal lung function (Kleinbongard et al., 2011).Dobreva et al. (2015) found a low level of serum TNF-α but a high level of IL-10 in adolescents living in highly industrialized cities. Different from our observations, Xu et al. (2016) also found that serum TNF-α of mice increased significantly after exposure to PM 2.5 via inhalation.Exposure pathway on the other hand might have also played a role in relevant expressions of certain biomarkers.Here, the exposure is the direct inject of PM 2.5 into the blood circulation.Further work is needed to elucidate the possible mechanisms for the observed decrease of TNF-α level in the high-dose exposed groups.
As for 8-OHdG biomarker, we did not observe a significant change 1 hour after the PM 2.5 injection.The 8-OHdG biomarker is described to be produced as a result of DNA oxidative damage (Vattanasit et al., 2014).DNA molecules can be oxidized by ROS to form 8-OHdG, which is shown to be readily excreted in urine as well as other biofluids (Huang et al., 2012;Vattanasit et al., 2014).The increased release of 8-OHdG implied an oxidative damage, which was discussed to be induced by exposure to air particles (Møller et al., 2014).The oxidative damage in cells in turn caused inflammatory responses to take place, thus the higher expression of IL-6 was observed in the in vitro study by Vattanasit et al. (2014).In our study, we found a high level of serum IL-6 but a low level of 8-OHdG (changes of 8-OHdG in all groups were less than 11%) 1 hour after the injection of PM 2.5 .The oxidative DNA damage process is probably slower than the inflammation process.To further study the relevant biomarker levels and possible oxidative damages, we have continuously monitored their biomarker levels over a 9-day time period.
Fig. 4 shows the biomarker levels over the course of a 9day time period.The concentration levels of the biomarkers are shown with percentage changes compared with the first blood sample sampling right before the PM 2.5 injection.Each line represents the variations of corresponding biomarker from one particular rat.Several lines were absent for certain days before the 9th day after the injection due to the block of the catheter for certain rats.As observed in Fig. 4, the IL-6 levels for high-dose group reached its maximum at the fifth day after the injection and then started to decrease; while those for other groups remained in the normal range over the entire observation period.The results indicate that inflammatory responses lasted for a few days and then started to decrease due to possible immune responses and other self-defending mechanisms.The concentrations of serum CRP and endotoxin for the high-dose and middledose groups also increased on the fifth day after the exposure.For endotoxin, its increase over the 9-day time period might be largely due to the death of bacteria (endotoxin was released after the microbial death) from PM 2.5 samples injected into the blood circulation.Here, we have observed increases in both endotoxin and CRP over the course of the experiments as shown in Fig. 4. Thus, it is highly likely that the endotoxin from PM 2.5 played a major role in the observed increase in CRP in our work.
Interestingly, we have observed an increase in 8-OHdG up to more than 300% and TNF-α up to 80% on the 3rd day after the injection as shown in Fig. 5. TNF-α is produced mainly by activated macrophages, which are found in essentially all tissues patrolling for potential pathogens by amoeboid movement.Exposure to PM 2.5 via inhalation will increase the permeability of the alveolar capillary (Chang et al., 2005), thus letting the monocytes pass into the interstitial alveolar to phagocytose foreign agents and release cytokines.In the past, a number studies investigating PM 2.5 -induced Fig. 4. Concentration percentage changes of IL-6, CRP and endotoxin in sera of SD rats from different groups (Control-1,2,3 from control group; L-1,2,3 from low-dose group; M-1,2,3,4 from middle-dose group; H-1,2 from high-dose group) before exposure and 3 days, 5 days, 7 days, 9 days after the PM 2.5 extracts injection, respectively.Each line represents one particular rat over the 9-day time period.pulmonary injury using mice or rats via intratracheal instillation or inhalation detected a significant increase of TNF-α in BALF or lung tissue.Here, we have observed that the level of TNF-α started to decrease 3 days post-exposure, as seen in Fig. 5.In clear contrast, serum 8-OHdG in 2 rats, one from the high-dose group and another from the middledose group, increased sharply 5-7 days after the injection although we did not observe an increase 1 hour after the injection as shown in Fig. 3.However, it took only 1 hour to allow IL-6 to elevate with an increase of 102%.These results suggest that IL-6 is a sensitive biomarker for PM 2.5 exposure, and that PM 2.5 is able to induce oxidative DNA damage, though it will take a longer time than the inflammation.Møller et al. (2014) pointed out that DNA lesions take some time to accumulate to a level that can be distinguished from the background levels of DNA damage.Wang et al. (2016) studied the PM 2.5 oxidative DNA damage effect in the human vascular endothelial cells and found out exposure to coal-fired PM 2.5 for 24 hours resulted in high levels of 8-OHdG.Vattanasit et al. (2014) also found a 2.1-4.6 fold increase in 8-OHdG concentrations in human lymphoblastoid cells (RPMI 1788) after 24-hour treatment of diesel exhaust particles (DEP).Overall, in this work we have observed that the injection of the PM 2.5 into the blood circulation indeed Fig. 5. Concentration percentage changes of TNF-α and 8-OHdG in sera of SD rats from different groups (Control-1,2,3 from control group; L-1,2,3 from low-dose group; M-1,2,3,4 from middle-dose group; H-1,2 from high-dose group) before exposure and 3 days, 5 days, 7 days, 9 days after the PM 2.5 extracts injection, respectively.Each line represents one particular rat over the 9-day time period.induced oxidative DNA damage, although it took longer time to take place compared to the inflammatory responses.

Anatomy and Histopathological Analysis of Experimental Rats
In our work, hemorrhage as marked by arrows and shown in Fig. 6 was observed in lung alveoli of rats from the highdose groups (568 µg PM 2.5 injection).A recent study found the increase ratio of Th17/Tregs in rats with smoke-induced acute lung injury can play an potential role in the pathogenesis of hemorrhage in lung alveoli.In that study, serum IL-6 as a closely related cytokine with Th17 was observed to be significantly higher in exposed group compared with that in normal control group.Here, we also observed a significantly higher IL-6 levels following the injection of the PM 2.5 , which was also accompanied by a hemorrhage as shown in Fig. 6.This suggests that the same mechanism may exist in the process of pulmonary hemorrhage for the high-dose PM 2.5 exposed rats in our work.However, no histopathological changes were observed for other organs during the 9-day time period.Yan et al. (2014) found that a 16-week exposure of ambient PM 2.5 could induce the heart and kidney damages in diabetes mellitus rats through chronic hyperglycemia and systemic inflammation.Here, due to the limitation of cages and resources for breeding rats, we only kept the rats for 9 days.Thus, there is a possibility that damages to other organs could also occur if longer observation period could follow.Yet, caution should be taken regarding the observed lung damage.For example, He and Nan (2016) observed hemorrhage and edema in the interstitium and lung alveoli of Westar rats after an acute spinal cord injury.Nonetheless, we did not observe any destructive changes to the lung in the control group using the spinal cord euthanization.This might on the other hand imply that the observed hemorrhage could be in part associated with the PM 2.5 injection.The relevant damage could be attributed due to PM-borne agents coming out from the blood circulation.The results from this work suggest that the lung could be the most susceptible organs to the PM 2.5 exposure among the studied ones.

Elemental and Biological Composition of PM 2.5 Extracts
The concentrations of metals from PBS and PM 2.5 extracts for different groups are shown in Fig. 7.All the metal concentrations extracted from the PM 2.5 collected on the haze day were significantly higher than those from PBS and also those collected from the clear day.The metal Vanadium, which is commonly used in the production of steel, was only found in sample from the haze day.Previous studies pointed out that metals bound to PM 2.5 could be the important toxicants that are responsible for ROS generation  extracts for high-dose group injection from PM 2.5 sample collected on a haze day when daily PM 2.5 concentration was 140 µg m -3 , PM 2.5 in clear day: extracts for mi and the induction of oxidative stress (Ghio, 2004;Rhoden et al., 2004;Ramanathan et al., 2016).One report showed that oxidant radical generation and cytokine production (IL-6 and TNF-α) in airways were significantly increased after dosing metal-rich ambient PM 2.5 into contralateral lung segments of healthy volunteers (Schaumann et al., 2004).Studies in Utah Valley, where high levels particles came from a principal source being a steel mill, provided more evidences for the role of metals in the biological effects of ambient PM (Ghio, 2004).In another work, it was shown that inflammation in the lower respiratory tract with increased IL-8 and TNF-α was caused after human exposure to the aqueous PM extracts containing high metal content, including Fe, Cu, Zn, Pb, Ni and V (Ghio, 2004).The redox potential of metals was thought to play an important role in the inflammatory injury (Ghio, 2004).Agasanur et al. (2001) found that incubation of 2'-deoxyguanosine (dG) with oil-derived fly ash (OFA) and ROFA rich in watersoluble V, Ni and Fe resulted in a 16.5-fold and 625-fold increase, respectively, in dG hydroxylation to 8-OHdG.The metal Zn has been shown to induce pulmonary cell reactivity and epithelial damage in mice (Adamson et al., 2000).Rhoden et al. (2004) found a strong association between increased thiobarbituric reactive substances accumulation and the content of Al, Si and Fe in concentrated ambient particles.In another study, PM 2.5 samples containing higher amounts of metals such as Fe, Cu, Ni and Mn, e.g., collected from a traffic site, were shown to exhibit higher oxidative ability using dithiothreitol (DTT) assay (Fujitani et al., 2017).Most of the metals mentioned above can be found in extracts of PM 2.5 in our study (Fig. 7).The results here suggested that metal elements in the PM 2.5 extracts could have contributed to the oxidative damage and inflammatory responses as evidenced by the release of IL-6, CRP and 8-OHdG observed here.
In addition to the metal content analysis, we have also analyzed bacterial species in the PM 2.5 samples.The top five genera of bacteria detected in PM 2.5 extract from haze day were Methylobacterium, Brevundimonas, Streptococcus, Thermus and Escherichia/shigella.In contrast, the top five genera of bacteria detected in PM 2.5 extract from clear day were Brevundimonas, Streptococcus, Thermus, Methylobacterium and Pseudomonas.There was a slight difference observed in the abundance of the dominant bacterial genera between different PM 2.5 samples.The death of bacteria in the blood circulation due to the innate immune response could attribute to endotoxin increase, which promoted the generation of CRP as investigated in a previous work (Patterson and Higginbotham, 1965).In addition, some potential opportunistic pathogens could cause potential blood infection if they grow inside the circulation.Of course, there are also other biological components in the PM 2.5 samples, e.g., viruses, fungal spores, allergens.Here, we only analyzed bacterial structures and their abundances in the samples.In the literature, there are a large volume of health studies focusing on endotoxin exposure, however its synergetic roles with other chemical components remain to be relatively limited.The PM 2.5 components are very complex, which likely vary significantly from one place to another.It is rather challenging to differentiate between different materials for their individual health effects.In the future, more biological material related studies should be conducted to elucidate mechanisms of the PM 2.5 component-based health effects.In this work, we have injected all the extracted samples into the blood circulation.As indicated in the manuscript, this work was intended to see how toxic the PM 2.5 is when injected into the blood circulation.Of course, the results from this experiment might not represent the real scenarios, but gave us an idea about the highest toxicity that PM 2.5 can act upon people.In our forthcoming study, we have filtered the extracted samples using 0.4 µm-pore size filter, under which all particles could be inhaled and have the potential to enter the blood circulation.Thus, more results will be obtained with respect to the toxicity when particles enter the blood circulation.Nonetheless, a lot of materials from the extracted samples are dissolvable and can enter the blood circulation for the experiments conducted in this work.With respect to the rat's behavioral changes, numerical ranks could be sought in future studies for better characterization.

CONCLUSIONS
We developed a new PM 2.5 -toxicity study protocol using rats injected with catheters, which allowed us to observe health effects that have previously not been reported.Our study revealed that PM 2.5 in the rats' blood circulation induced acute health effects involving oxidative DNA damage and an inflammatory response, which were characterized by elevated serum biomarker (IL-6, CRP, and 8-OHdG) concentration levels, lung damage, and changes in behavior.The acute inflammation was observed 1 hour after the injection, while the oxidative DNA damage occurred five or more days later.Water-soluble metals, such as Fe, Cu, Al, Ni, Zn, and V, were detected in PM 2.5 extracts.According to our video recordings, it is highly possible that the injection of PM 2.5 could have negatively influenced the nervous system in rats that received the high dosage.Despite all these PM 2.5 -induced ailments, we observed that the exposed groups, including the high-dosage ones, were able to survive the challenge and behaviorally appeared to be as normal as the control group after 5 days of the 9-day period because of their immune defense.Our work provides a new perspective on the toxicity of PM 2.5 , including tolerance of injected PM 2.5 and the battle between PM 2.5 damage and the immune defense.After testing the worst-case scenario, the results suggest that the health effects of PM 2.5 might have been exaggerated in the literature.Nonetheless, we did not filter the extracted PM 2.5 samples, and the injections might not represent realistic exposure scenarios.In addition, we only assessed the acute effects, which were based on short-term observation, whereas the chronic toxicity of PM 2.5 in the blood warrants future in-depth investigation.The information obtained in this study may be helpful for implementing relevant air-pollution control measures and rethinking PM 2.5 toxicity in terms of human health.

Fig. 6 .
Fig. 6.The histology images of lung, heart, kidney, liver and spleen of SD rats from different groups by HE stain (10X) (PBS: control group, n = 3, L: low-dose group, n = 3, M: middle-dose group, n = 4, H: high-dose group, n = 2).Hemorrhage in lung alveoli was marked with black arrows in the figure.

Fig. 7 .
Fig. 7. Concentrations of metals from PBS(for control group) and PM 2.5 extracts for different groups (PM 2.5 in haze day: extracts for high-dose group injection from PM 2.5 sample collected on a haze day when daily PM 2.5 concentration was 140 µg m -3 , PM 2.5 in clear day: extracts for mi