Advances of In Vitro Inhalation Bioaccessibility for the Contaminants in Atmospheric Particulate Matters

Laijin Zhong; Zhijie Tang; Xin Hu; Hongzhen Lian

doi:10.7536/PC200853

Progress in Chemistry ›› 2021, Vol. 33 ›› Issue (10) : 1766-1779 DOI: 10.7536/PC200853

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Advances of In Vitro Inhalation Bioaccessibility for the Contaminants in Atmospheric Particulate Matters

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Abstract

Exposure to toxic elements or organic contaminants associated with atmospheric particulate matters(APM) via inhalation may result in potential health risks to human. Up to day, various inhalation bioaccessibility procedures(IBAcP) have been advocated to investigate the bioaccessible concentrations of these contaminants in APM for the easy and fast risk-based assessment. In this review, the inhalation bioaccessibility of the toxic elements and organic contaminants in APM and the current IBAcP for the hazards assessment are reviewed and evaluated. In addition, the defects and challenges existed in current IBAcP are disclosed and the possible solutions are proposed.

Contents

1 Introduction

2 Main procedures for inhalation bioaccessibility

3 Inhalation bioaccessibility of inorganic and organic contaminants

3.1 Inhalation bioaccessibility of inorganic toxic elements

3.2 Inhalation bioaccessibility of organic contaminants

4 Validation of inhalation bioaccessibility procedures via in-vivo correlation

5 Challenge and prospective

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Keywords

inhalation bioaccessibility / inhalation bioavailability / atmospheric particulate matters / toxic elements / organic contaminants

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Laijin Zhong, Zhijie Tang, Xin Hu, Hongzhen Lian. Advances of In Vitro Inhalation Bioaccessibility for the Contaminants in Atmospheric Particulate Matters. Progress in Chemistry, 2021, 33(10): 1766-1779 DOI:10.7536/PC200853

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1 Introduction

Atmospheric particulate matter (APM) is a kind of solid and liquid suspended particulate matter which is relatively stable and uniformly dispersed in the atmosphere.It includes primary particulate matter directly discharged into the atmosphere from natural and anthropogenic sources and solid or liquid particulate matter (secondary particulate matter) generated by a series of chemical or physical processes of gaseous pollutants such as sulfur dioxide, nitrogen oxides and hydrocarbons in the atmosphere^[1⇓⇓~4]. According to the aerodynamic size (Aerodynamic diameter,D_ae),APM, it is usually divided into total suspended particles (Total suspended particulate,TSP,D_ae≤100μm), inhalable particles (Inhalable particles,PM₁₀,D_ae≤10μm) and fine particles (Fine particulate matters,PM_2.5,D_ae≤10μm), and the particles with a diameter less than 0.1 μm are called ultrafine particles^[3,5⇓~7]. The composition of APM is complex, including elemental carbon, inorganic salts, heavy metals, organic carbon and microorganisms^[1⇓⇓~4]. Harmful components such as toxic elements and organic pollutants in APM mainly come from anthropogenic sources such as biomass/fossil fuel combustion, mining, smelting and vehicle exhaust emissions, or from natural sources such as volcanic eruptions and sandstorms^[2]. The inhalation of these harmful ingredients will bring potential harm to the physical and mental health of residents.

When APM is inhaled into the lungs, some particles interact with pulmonary surfactants and extraalveolar fluid, and enter the liquid phase from the gas phase^[8]. Dissolved components and ultrafine insoluble particles or insoluble components in APM enter the circulatory system through the air-blood barrier and accumulate in the body, some of which are transported to the kidney and gastrointestinal system and excreted in the form of urine and feces, or accumulated in hair and nails^[9,10]. Some particles or insoluble components are cleared by the lung's own clearance function, but when the accumulation rate of insoluble components exceeds the lung clearance rate, particles will deposit in the lung, leading to alveolar fibrosis and so on^[11]. Inhaled APM particles deposited in the lungs may cause a range of adverse health effects^[12,13]. Studies have shown that the heavy metals and organic pollutants loaded by APM particles are important reasons for the health damage caused by APM particles. For example, harmful components in particulate matter are dissolved out, causing local pulmonary toxic reactions, and these dissolved harmful components can cross the air-blood barrier and enter the circulatory system^[14,15]^[14]. Some epidemiological studies have shown that long-term human exposure to fine particulate matter in the atmosphere can cause a sharp increase in the concentration of heavy metals in blood and urine^[16,17]. For example, Zn and Cd can weaken lung and kidney function, As inhalation may cause lung cancer, and Pb and Cd inhalation can damage DNA fragments^[18,19]^[20]^[21]. In addition, studies have shown that the inhalation of heavy metals may damage the cardiovascular system, reproductive system and endocrine system^{[22⇓⇓~25]}^[26]^[27].

At present, the assessment methods of APM hazards include epidemiological investigation, animal exposure experiments, in vitro cell exposure experiments and inhalation bioavailability studies. These assessment methods include biological assessment methods from cell level, animal individual level to population survey, and non-biological in vitro chemical analysis assessment methods. Epidemiological investigation method refers to the relationship between the status and distribution of APM in the investigation area and the disease and health status of the designated population in the area.Animal exposure experiment refers to the exposure of a certain amount of APM to the respiratory system of experimental animals through inhalation under laboratory conditions, which can be used to study the cytotoxicity, inflammatory proteins, specific diseases or genetic mutations caused by exposure^[28,29]^{[30⇓⇓~33]}. In vitro cell exposure experiments refer to experiments in which cells are stimulated directly by APM or by water extracts or organic solvent extracts of soluble components in APM, which can be used to study the cytotoxicity, lung inflammatory factors, gene expression upregulation, and specific behaviors related to cardiovascular and cerebrovascular diseases induced by APM^[34⇓~36]. Due to the time-consuming, laborious, high economic cost and high experimental uncertainty, biological assessment methods are difficult to carry out on a large scale, and can not meet the needs of health risk assessment in different regions with large spatial and temporal variations. Therefore, non-biological in vitro chemical analysis and evaluation methods have attracted wide attention. At present, the determination of the total amount of some pollutants in APM is a routine requirement in the ambient air quality standards of various countries. For example, China's ambient air quality standards (GB 3095-2012) stipulate that pollutants such as lead and benzopyrene are compulsory items, while cadmium, mercury, arsenic and chromium are optional items^[37]. Environmental science research shows that the biological toxicity of pollutants in the environmental matrix mainly depends on the amount of soluble pollutants (bioavailable), rather than the total amount of pollutants^[6]. Therefore, the bioavailability or bioavailability of pollutants has been widely concerned, and a variety of chemical extraction methods for bioavailable forms of pollutants in various environmental matrices and exposure pathways have emerged^[38]^[38]. Inhalation is the direct route of human exposure to APM, so researchers have proposed a variety of chemical formulations simulating lung fluid to study the dissolution effect of certain harmful components in APM in vitro, inhalation bioavailability, and to quickly assess the potential risk of harmful components in APM^[39,40]. In this study, the inhalation bioavailability of harmful elements in TSP and PM_2.5 in Nanjing was studied to assess the potential carcinogenic and non-carcinogenic risks^[41,42]. The results show that the potential risk of harmful elements based on the bioavailability of inhalation is less than that based on the total amount of harmful elements. Therefore, the potential risk assessment of harmful elements based on inhalation bioavailability can overcome the deficiency of overestimating the potential risk of harmful elements in APM and reduce the excessive management of APM. At present, the inhalation bioavailability of harmful components in APM has attracted wide attention.

2 Inhalation bioavailability of harmful components in APM

The health risk assessment model proposed by the United States Environmental Protection Agency (United State Environmental Protection Agency, U. S. EPA) is the most widely used risk assessment model for inhalation exposure of harmful components in APM. In this model, the dimensionless Hazard quotient (HQ) and Carcinogenic risk (CR) are used to evaluate the non-Carcinogenic and carcinogenic potential risks of harmful components in APM, and the expression formula is as follows:^[43]

(1)

EC = C × ET × EF × ED / AT

(2)

HQ = EC / (Rf C i × 1000 μg · m g - 1)

(3)

CR = IUR × EC

Where EC (Exposure concentration) is the Exposure concentration of harmful components (μg·m^-3);C is the concentration of harmful components bound by AMP in the air (μg·m^-3);ET is exposure time (h·day^-1);EF is exposure frequency (day·year^-1);ED is exposure period (year); AT is the average exposure time (H) (carcinogenic risk :AT=70 year×365 day·year^-1×24 h·day^-1); The non-carcinogenic risk :AT=ED×365 day·year^-1×24 h·day^-1);RfC_i is the inhalation reference concentration (Inhalation reference concentrations,mg·m^-3) and the IUR is the inhalation unit carcinogenic factor (Inhalation unit risk,(mg·m^-3)^-1).

The assessment model was initially based on the total amount of harmful components in the APM. However, studies have shown that the absorption rate of harmful components in APM in the lungs is hardly 100%, which is affected by the composition and distribution of harmful components in APM and their interaction with lung fluid^[38,44]. Therefore, the concepts of Bioavailability (BA,%) and relative bioavailability of harmful components in APM were introduced into the risk assessment of U. S. EPA^[10,43,44]. Obtaining the bioavailability of inhalation requires a large number of animal experiments, which not only takes a long time, costs a lot, and has differences in animal species, but also involves animal ethics and human differences. Researchers hope to find a simple and efficient in vitro experimental method that can reflect the bioavailability, and the formulation of in vitro simulated lung fluid based on lung body fluid has been proposed one after another. Bioaccessible concentration (BC) is defined as the extractable fraction of the extractable fraction extracted from the simulated lung fluid in vitro, and Bioaccessibility (BAc,%) is defined as the percentage of the Bioaccessible fraction of a component in the APM relative to the total amount of that component^[6]. BAc can be obtained by simulating the extraction process of physiological environment through the method of preparing simulated body fluid in vitro, and the extraction process is not only simple and efficient, but also low in cost. The risk assessment model based on bioavailability or bioavailable concentration overcomes the shortcoming that the potential risk may be overestimated based on total concentration.

At present, in vitro extraction methods of available concentrations of harmful components in APM can be divided into three categories: chemical extraction method, continuous extraction method and simulated lung physiological environment extraction method. The chemical extraction method comprises pure water and aqueous solutions with different pH values (such as an ammonium citrate solution with a pH value of 4.4, an ammonium acetate solution with a pH value of 7.0, and a citric acid solution with the pH value of 2.1), the matrix of the method is simple, and the solutions with different pH values can simulate different lung exposure areas^[3,6,7]; Continuous extraction method refers to the extraction of different chemical forms (such as water-soluble, exchangeable, carbonate, sulfide and oxide) of harmful components in APM with different solvents, such as BCR continuous extraction method^[45,46]. The extraction method of simulating lung physiological environment is a method of simulating lung physiological solution through the composition of lung physiological solution prepared in vitro. Its extraction conditions are very close to the respiratory system, which can more truly reflect the available concentration of harmful components in APM exposed to human body^[47⇓~49]. This type of formulation, which simulates the physiological solution of the lung to study the Inhalation bioaccessibility of harmful components in APM, is collectively referred to as the Inhalation bioaccessibility procedure (IBAcP).

IBAcP originated from the composition of extracellular fluid of skeletal muscle proposed by Gamble in 1967. It was later modified by Moss in 1979 to replace the organic acid and protein components in Gamble formula with citric acid and acetic acid for the dissolution experiment of uranium in body fluids, and was named Gamble solution^[50]^[48]. The initial simulated epithelial interstitial fluid of the lung consists primarily of inorganic salts (Na⁺, K⁺, Ca²⁺,Mg²⁺, Cl^-,

S O 4 2 -

HP O 4 2 -

and

HC O 3 -

) and organic acid (citric acid and acetic acid),Later, a variety of formulations simulating lung physiological solutions were developed by adding or adjusting relevant components.These components include various organic acids (vitamin C, uric acid, lactic acid, pyruvic acid), amino acids (glycine, l-cysteine), polypeptides (glutathione), proteins (mucin, albumin, transferrin), chelating agents (dimethyltriaminepentaacetic acid,DTPA), surfactant (dipalmitoylphosphatidylcholine, DPPC), bacteriostat (benzalkonium chloride), antioxidant (vitamin E) and cation (

NH 4 +

)^{[6,39,40,51,52]}.

After entering the lung, APM not only contacts with the fluid on the surface of the lung, but also is phagocytosed by pulmonary macrophages and degraded in lysosomes (Figure 1). Therefore, on the basis of Gamble solution, Artificial lysosomal fluid has been developed, whose pH value is between 4.5 and 4.55. Among them, Artificial lysosomal fluid (ALF) proposed by Midander et al. In 2007 is the most widely used one, which is mainly composed of inorganic salts and organic acids^[7,53,54]^[7]. Table 1 lists the specific components and contents of several common simulated lung fluids. The synthetic lysosomal fluid can be used to reflect the state of the inflamed lung, while the simulated epithelial interstitial fluid of the lung is used to reflect the state of the normal lung^[52]. Figure 2 depicts the basic experimental steps of the current IBAcP. The experimental parameters of IBAcP, such as solid-liquid ratio (g·mL^-1), extraction temperature, extraction time, oscillation mode and rate, have not yet been standardized, and need to be optimized and standardized^{[6,39,40,52,55⇓⇓~58]}.

3 Inhalation bioavailability of harmful components in APM

3.1 Inhalation bioavailability of toxic elements in APM

At present, studies on the inhalation bioavailability of toxic elements in APM mainly focus on cadmium, lead, zinc, copper, manganese and arsenic^{[39,49,51,52]}. Table 2 summarizes the inhalation bioavailability of toxic elements in the recent literature. As can be seen from Table 2, in general, the inhalation bioaccessibility of arsenic is high, and the inhalation bioaccessibility of cadmium, zinc and lead is low. Table 2 also shows that there are differences in the inhalation bioavailability of harmful components in APM samples from different sources. First, different emission sources affect the inhalation bioavailability of the hazardous constituents. For example, when Julien et al. used J-GS to compare the inhalation bioaccessibility of cadmium, lead and zinc in vehicle exhaust and fly ash, they found that the bioaccessibility of cadmium, lead and zinc in vehicle exhaust (NIST-SRM: NIES 8) ( (74.3 ± 4.6)%, (45.2 ± 3.5)% and (92.5 ± 2.5)%, respectively) was significantly higher than that in fly ash (NIST-SRM: BCR 038) ( (11.2 ±^[6]. They believe that fly ash is mainly composed of aluminum silicate salts, quartz and iron oxides, with low solubility, resulting in low bioavailability of cadmium, lead and zinc. Secondly, there are differences in the inhalation bioavailability of harmful components in different space APM samples. For example, when our research group studied the inhalation bioaccessibility of heavy metals in indoor and outdoor particulate matter (less than 3.3 μm) in spring and winter in Nanjing from 2015 to 2016, it was found that there was a significant difference between the inhalation bioaccessibility of manganese in indoor and outdoor particulate matter extracted with SLF (a simulated lung epithelial interstitial fluid) in winter (5.7% ± 1.2% and 22.6% ± 6.5%, respectively)^[41]. Furthermore, changes in sampling time affect the bioavailability of harmful components in APM. For example, we found that the inhaled bioavailability of indoor lead, zinc, and manganese varied significantly with the seasons, with values of 0.9% ± 0.2%, 1.4% ± 0.5%, and 5.7% ± 1.2% in winter, respectively, but increased significantly in the following spring, with values of 4.3% ± 1.2%, 4.5% ± 0.6%, and 19.0% ± 15.1%, respectively^[41]. Li et al. studied the inhalation bioavailability of lead in Nanjing PM_2.5 during and after the 2014 Youth Olympic Games (YOG), and found that the inhalation bioavailability of lead in ALF extracted PM_2.5 decreased during YOG (59% -79% during YOG, 55% -87% during non-YOG), but the J-GS results were opposite (11% -29% during YOY, 5.3% -21% during non-YOG)^[61]. In addition, the difference of particle size of APM is also one of the factors that cause the difference of inhalation bioavailability of harmful components in APM. For example, Wiseman et al. Found that the inhalation bioaccessibility of arsenic in PM₁₀ was significantly lower than that in PM₁ (57% and 80%, respectively)^[51]; We also found that the inhalation bioaccessibility of lead in ALF-extracted PM_2.5 was significantly higher than that in total suspended particulate matter (45.1% ± 15.8% and 17.8% ± 5.2%, respectively)^[42]. In conclusion, the source, particle size, and temporal and spatial variability of APM have different effects on the inhalation bioavailability of toxic elements.

Table 2 shows that the formula, solid-liquid ratio, extraction time and oscillation mode selected by different laboratories will affect the inhalation bioavailability of heavy metal pollutants. For example, the results of our research group showed that the bioavailability of nickel and strontium in TSP extracted in SLF for 48 H was significantly lower than that extracted in SLF for 72 H^[42]. Kastury et al. Studied the inhalation bioaccessibility of lead and arsenic in four kinds of PM₁₀, and the results showed that the inhalation bioaccessibility of lead and arsenic obtained at different extraction stages at the solid-liquid ratio of 1:5000 was significantly higher than that at 1:100^[62]; Our study also confirmed that the inhalation bioavailability of lead in the PM_2.5 was affected by the solid-liquid ratio^[63]. Kastury et al. Also studied the effects of four oscillation modes (magnetic stirring, tumbling stirring, random stirring and directional rotation) on the inhalation bioavailability results, and the results showed that the effect of extracting lead and arsenic by magnetic stirring was significantly better than that by the other three modes^[62].

In addition, different sampling membranes also affect the inhalation bioavailability of harmful components in APM^[42,64]. For example, when we compared the inhalation bioaccessibility of heavy metal contaminants in APM collected with different sampling membranes, 24 H extraction results for both SLF and ALF showed significant differences between the inhalation bioaccessibility of copper and cobalt in PM_2.5 collected with polytetrafluoroethylene (PTFE) membranes and quartz membranes (SLF: 14.9% ± 6.0%, 1.64% ± 0.71% and 25.8% ± 5.0%, 19.9%)^[42]. The influence of sampling membrane may be due to the different hydrophilicity of different sampling membrane materials.

3.2 Inhalation bioavailability of organic pollutants in APM

In recent years, more and more attention has been paid to the inhalation bioavailability of organic pollutants in APM. The organic pollutants in APM mainly include some Semi-volatile organic compounds (SVOCs), such as Polycyclic aromatic hydrocarbons (PAHs), Organophosphorus flame retardants (OPFRs), Halogenated flame retardants (HFRs) and some compounds with lower volatility, such as Phthalate esters (PE. These organic pollutants can cause a series of health risks, such as neurotoxicity, asthma, carcinogenesis and so on^[65,66].

Table 3 summarizes the results of recent studies on the inhalation bioaccessibility of organic pollutants in APM. The method for extract that inhalation bioavailability of organic pollutants in APM is the same as that for extract heavy metals and other pollutants. For example, Gao et al. Used ALF and GS methods to study the total inhalation bioavailability and carcinogenic risk of 12 PAHs in the PM_2.5 of four types of cities (provincial capital, energy-mining area, agriculture, forest) in northern China^[67]. In another study, they found that the bioavailability of PAHs in PM_2.5 was higher in neutral GS than in acidic ALF^[68]. Liu et al. found that the inhalation bioavailability of PAHs in the biochar-based APM-PAHs was very low (GS: 0.43% ~ 1.49%, ALF: 0.34% ~ 1.31%); Li et al. found that the inhalation bioavailability of PAHs in the PM_2.5-PAHs was significantly higher (SELF: 3.21% ~ 44.2%), which may be due to the strong hydrophobicity of PAHs and the strong adsorption effect of biochar on PAHs^[69]^[70]. They also found that the toxicity of PM_2.5-PAHs to A549 cells was more related to the inhaled bioavailability of PAHs (compared to total PAHs concentration)^[70]; Zeng et al. Found that the bioavailability of PAHs in PM_2.5 extracted in J-GS and ALF for 15 days was 0.7% ~ 24.5%^[71].

It can be seen from Table 3 that the inhalation bioavailability of PAHs extracted by traditional IBAcP is very low, which may be due to the strong hydrophobicity of PAHs and their easy readsorption by APM during extraction. PAHs released from APM into lung fluid may be absorbed by lung tissue, which may lead to underestimation of inhalation bioavailability of PAHs by traditional extraction methods. In the study of oral bioavailability of PAHs in soil, Tenax (a porous resin) is often added to simulated gastrointestinal fluid to increase the extraction efficiency^[72]. Accordingly, Xie et al. improved the inhalation bioavailability method of PAHs in APM by adding Tenax (wrapped with a mesh and made into a rod) as an adsorbent for PAHs when extracting PAHs in APM with X-GS and ALF^[60]. The results showed that the inhalation bioavailability of PAHs was greatly improved by the addition of Tenax. It was also found that the inhalation bioaccessibility of PAHs in large particle size APM was 8.7 ~ 9.1 times higher than that in small particle size APM. When Kademoglou et al. studied the inhalation bioavailability of PEs in indoor dust with GS and ALF, they found similar results, with the inhalation bioavailability of PEs with strong hydrophobicity being less than 5% and that of PEs with weak hydrophobicity being more than 75%^[55].

In summary, the inhalation bioavailability of organic contaminants in APM is related to the partitioning of the organic phase (organic contaminants) between the solid phase (APM) and the aqueous phase (simulated lung fluid). Wei et al. Proposed a mathematical model for the inhalation bioavailability of SVOCs in APM based on the gas-solid and liquid-solid partition models and the tissue absorption model of SVOCs in APM. The model consists of three parts: (1) the spontaneous release of loaded SVOCs into the lung gas after inhalation of particles into the lung^[73]; (2) The particles deposited in the lung enter the lung fluid, and the SVOCs in the APM are dissolved in the lung fluid; (3) SVOCs in gas phase and liquid phase were absorbed by lung tissue. The biggest difference between this method and the inhalation bioavailability study based on simulated lung fluid is that this method takes into account the spontaneous release process of SVOCs in the gas phase of APM. They used the first two parts of the model to study the inhalation bioaccessibility of 72 SVOCs loaded by APM in French residential areas. The results showed that the inhalation bioaccessibility of PAHs ranged from 48% to 90%, and that of Polychlorinated biphenyls (PCBs) ranged from 48% to 56%^[74].

4 In vitro- in vivo experimental validation of IBAcP

At present, although the application of IBAcP is very extensive, the establishment and development of IBAcP lacks the support of in vivo experimental data. At the same time, there is no corresponding standard for the selection of the components of the inhalation bioavailable simulated lung fluid formula and the experimental parameters of the extraction process. Therefore, it is urgent to establish the corresponding biological experimental verification of IBAcP.

From the establishment and development of Oral bioaccessibility procedures (OBAcP), it can be seen that the application of an Oral bioaccessibility method needs to be verified by in vivo experimental data. bioavailability data and Bioaccessibility data are not directly used in the validation of oral bioaccessibility methods. Bioaccessibility Research Group Europe (BARGE) proposed the Relative bioavailability (RBA) validation criteria for oral bioaccessibility methods^[38]. Relative bioavailability refers to the ratio between the proportion of the amount of harmful components in soil entering the corresponding organs or tissues of experimental animals (Organ T_soil,mg·kg^-1) to the total amount of exposure (T dose_soil,mg·kg^-1) after exposure for a certain period of time and the proportion of the amount of reference substances (water-soluble compounds of harmful components) entering the organs or tissues after exposure (Organ T_reference,mg·kg^-1) to the total amount of exposure (T dose_reference,mg·kg^-1), which is defined as follows:

(4)

RBA = Organ T soil / T dos e soil Organ T reference / T dos e reference × 100 %

BARGE believes that the following criteria need to be met to establish an RBA method:

(1) The dose-response curves of soil and reference material should be linear in a certain range of exposure dose (ΔT = K · T dose + t);

(2) The intercept of the dose response curve of the soil is the same as that of the reference substance;

(3) The time T at blank exposure is less than t.

This method requires a dose response experiment for each soil sample, which is very demanding and complicated to practice, and researchers tend to obtain only the dose response curve of the reference substance.When the dose-response curve has good linearity and the slope is close to 1 and the intercept is close to 0, it is considered that the bioavailability method can be verified by using the obtained RBA data in the corresponding exposure dose range^[75⇓~77].

There are many challenges in the validation of IBAcP, such as: (1) The choice of exposure mode: The advantage of inhalation exposure mode is to use the active respiration of experimental animals to carry out APM exposure, which largely restores the inhalation exposure process, but it is difficult to control the exposure dose during the experiment, so it is difficult to obtain stable RBA data. On the one hand, it is difficult to obtain uniform APM dispersion gas for a long time during inhalation exposure; On the other hand, the amount of inhalation between different experimental animals during exposure is not controllable. Tracheal/nasal instillation is different from real breathing, but the exposure dose can be controlled more accurately. (2) Selection of exposure time: Inhalation exposure is different from oral exposure. The cleaning cycle of oral exposure is short, and the end point of single exposure is easy to determine. Generally, the exposed samples are basically emptied after eating 6 to 8 hours, while the deposition time of particulate matter in the lungs is long.There are great individual differences in the cleaning cycle, and it is difficult to determine the end point of a single exposure (for example, after several days of inhalation exposure, there may still be samples in the lungs, the harmful components in these samples can continue to be released, and the exposure is still not over)^[78]. For example, our research group took Balb/C mice as the exposure object (Figure 3), and chose the nasal drip exposure mode to study the inhalation exposure metabolic characteristics of simulated PM_2.5 particles contaminated by lead compounds, and found that the lead content in the lung PM_2.5 decreased sharply within 24 hours of exposure (y=-59.5x+2037.3,R²=0.99;y refers to the lead content in the lung, ng; X refers to the time after exposure, H); After exposure for 24 ~ 72 H, the content of lead in lung PM_2.5 decreased slowly (y=-8.9x+859.3,R²=0.99). After 72 hours of exposure, the change of lead content in lung PM_2.5 tended to be flat (y=-1.6x+283.3,R²=0.92)^[63]. This phenomenon of rapid metabolism in a short period of time was also found in the relevant reports on the metabolism of particulate matter in the lungs of mice. The reason for this phenomenon is considered to be that the response of lead in the organs of mice after inhalation exposure is closely related to the rapid metabolic process in the lungs within 24 hours, and the intensity of lead release from the lungs after 24 hours is low, which has little effect on the metabolic process of lead in the organs^[79⇓~81]. Therefore, we controlled the end point of inhalation exposure at 24 H, and obtained a good dose-response curve (R²=0.999) of inhalation exposure with the mouse kidney as the target. Based on the obtained relative bioavailability data of inhalation exposure, we selected W-GS for the inhalation bioavailability study of lead in in vitro correlation, optimized the solid-liquid ratio to 1∶1000 g·mL^-1, and its In vivo-in vitro correlation curve (IVIVC) was :IBAc=1.07RBA-3.86,R²=0.73. This study shows that there is a gap between the inhalation bioavailability of lead in some PM_2.5 analyzed by IBAcP and the absorption of lead in experimental animals. Of course, the species differences of experimental animals may also affect the IBAcP validation. The animal experimental validation of the bioavailability of other harmful elements and organic pollutants for inhalation of IBAcP has not been reported in the literature, and further research is needed.

5 Conclusion and prospect

Risk assessment based on the total amount of harmful ingredients may overestimate the potential risk, leading to unnecessary governance by decision makers. The establishment of risk assessment based on the total amount of bioavailable forms can overcome the shortcomings of overestimating the potential risk and scientifically assess the potential risk. At present, this method has been widely recognized and has a good application prospect. However, there are still many challenges in the study of bioavailability and the verification of IBAcP.

(1) At present, there is a lack of systematic comparison of the selectivity and extraction performance of IBAcP formula components used to study the bioavailability of harmful components in APM, and there is no uniform standard for the corresponding parameters of extraction experiments.Some research reports do not even publish the corresponding bioavailability method parameters, which leads to the lack of scientific and reliable comparison of inhalation bioavailability data between different laboratories^[2,64]. Therefore, it is necessary to optimize and standardize the IBAcP and experimental parameters for the inhalation bioavailability of harmful components in APM.

(2) Inhalation-clearance pathway of particulate matter exposure. Kastury et al. proposed that the particulate matter discharged from the lungs would be ingested through the mouth and contribute to the bioavailability during the Inhalation bioaccessibility study, so they believed that the inhalation bioaccessibility of particulate matter should be studied in combination with the oral exposure experiment (Inhalation-ingestion bioaccessibility model, IIBA)^[62]. They conducted tracheal instillation experiments with lead-loaded fly ash in mice, combined with X-ray fluorescence (XRF) microscopy, and found that lead signals were produced in the intestines and stomach of mice after exposure for a period of time, which was used as a basis for proving that particulate matter discharged from the lungs could enter the digestive system.From the results of in vitro extraction experiments, it can be seen that the bioavailability of lead in particulate matter in simulated gastrointestinal solution is significantly higher than that in inhalation, so they claim that the bioavailability of this part of particulate matter from the lungs through oral intake is greater than that from the lungs^[81]. However, their animal experiments ignored the possibility that lead absorbed by the lungs was excreted through the gastrointestinal tract, and the results could not determine the proportion of particulate matter excreted by the lungs that was ingested orally.More experimental data are needed to support the contribution of oral ingestion of particles from the lungs to bioavailability and the need to use IIBA mode for inhalation bioavailability studies^[10].

(3) The research on the application of IBAcP to organic pollutants in APM is still in the initial stage, and many problems are still worthy of further study. Does the composition of the simulated lung fluid affect the extraction of organic pollutants? Does the extraction process require the addition of Tenax, or is there a better adsorbent than Tenax? Is the traditional inhalation bioavailability method applicable to the study of organic pollutants in various types of particulate matter? Is there a strong relationship between the inhalation bioaccessibility of organic pollutants and their hydrophobicity and can Log K_ow be used to predict the inhalation bioaccessibility of organic pollutants? The accuracy of the mathematical model proposed by Wei to predict the inhalation bioavailability of PAHs in APM and the possibility of expanding the application of the model (for example, can the model used by Wei to study PAHs and PCBs be applied to other SVOCs)?

(4) Validation of the inhalation bioavailability method. At present, the study of inhalation bioaccessibility of harmful components in APM still needs a lot of bioaccessibility validation of animal inhalation exposure for the components and extraction parameters of IBAcP. Although we have proposed a method to verify the relative bioavailability of IBAcP by inhalation, this method is only used to verify the IBAcP of lead inhalation bioavailability study in PM_2.5, and more in vivo experimental data need to be established to verify the IBAcP of other harmful components in inhalation bioavailability study^[63]. Moreover, only mice were used as exposure subjects in the study, and the method of relative bioavailability study still needs to be further improved. Furthermore, even for the more developed oral exposure studies, there is still no clear in vivo validation for the bioaccessibility of organic pollutants. Therefore, the validation of inhalation bioavailability of organic contaminants in APM is more challenging.

And (5) establish an inhalation bioavailability database. By conducting a large number of applied studies with validated and effective inhalation bioavailability methods, the accumulated data and experience can be further established into a database, which can greatly save time and resource costs for assessing the health risks of APM. For example, the main physical and chemical parameters of APM in the case to be assessed, including the size of APM, the form and distribution characteristics of harmful components in APM, can be obtained through research, and the corresponding inhalation bioavailability results can be output through big data analysis, which is more conducive to decision makers to scientifically judge the pollution level and formulate reasonable control measures.

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