Background

Human exposure to micro- and nanoplastic particles (MNPs) has gained significant attention in the past few years due to their increased production, widespread distribution in the environment, and recent detection in human tissues and fluids [1,2,3,4,5,6]. MNPs are generated through mechanical, thermal, and photochemical degradation of the millions of tons of plastic produced and discarded each year [7]. The resulting sizes of the particles generated by these processes fall in the micro- (< 5 mm and > 100 nm) or nano- (< 100 nm) ranges allowing the fraction of particles that are 10 μm or less to be readily aerosolized and inhaled. The potential for ubiquitous exposure, combined with the current knowledge of the toxicity of aerosolized particles, raises questions about the health effects of MNP inhalation.

Recent studies have detected airborne MNPs in both indoor and outdoor environments [8,9,10] and unequivocally identified microplastic particles in lung samples from patients and human cadavers [3, 5]. Occupational inhalation of plastic nylon fibers has been shown to cause pulmonary inflammation known as Flock disease or Flock worker’s lung [11]. Earlier studies have demonstrated that aerosolized particles like particulate matter (PM) or engineered nanomaterials (ENM) cause aberrant pulmonary function [12,13,14], pulmonary inflammation [14,15,16,17,18,19,20], systemic inflammation [17, 21,22,23,24,25], and impaired cardiovascular activity [15, 17, 23,24,25,26,27,28,29,30].

A variety of adverse cardiovascular outcomes can occur after aerosolized particle exposure. Affected portions of the cardiovascular system include the heart [31,32,33], the macrocirculation (conduit vessels > 150 microns in diameter) [34, 35], and the microcirculation (vessels < 150 microns) [36,37,38,39]. Exposure to particulate mixtures such as PM or diesel exhaust often lead to increases in blood pressure [40, 41], whereas ENM exposure do not generate these changes [42, 43]. Vascular dysfunction has also been described after particulate exposure [42, 44, 45]. This is characterized by decreased vasomotor function of the vessels in response to endothelial or vascular smooth muscle stimuli. Effects on the macrocirculation and microcirculation, however, are often different, and may vary between vascular beds [43, 46,47,48]. Changes in cardiovascular function after particle inhalation are thought to occur as a consequence of systemic inflammation, direct particle interaction with vessels, and/or decreased neurological control of the heart and vasculature [49]. The effect of MNP inhalation on the cardiovascular system are unknown.

The inflammatory and cardiovascular effects of particle inhalation vary depending on physicochemical properties including particle chemistry, size, and surface area. Among MNPs, different plastic polymers exhibit unique physicochemical properties that influence their interactions with biological tissues. Plastics are a toxicologically unique aerosol because polymer materials are frequently manipulated with chemical additives, known as plasticizing compounds, often to promote flexibility or hardness characteristics in the final products. These chemical additives, including bisphenols and phthalates, have been identified as endocrine disruptors due to their ability to mimic endogenous reproductive hormones [50]. Disruption of reproductive hormones, including estradiol and progesterone, may also play an important role in vascular responsivity and systemic inflammation [51, 52]. While many studies have theorized that MNPs may act as a vector for endocrine disruption chemicals, these outcomes remain unclear.

The purpose of this study was to characterize MNP aerosol toxicity in a whole-body rodent inhalation exposure model utilizing a novel MNP. Polyamide served as a representative plastic test material that is not produced with bisphenol analogs or phthalates [53,54,55] and has no known endocrine disrupting properties. We observed robust cardiovascular alterations and systemic inflammatory and endocrine responses following polyamide exposure of female rats during estrus. These results are important as they are the first to demonstrate whole-body polyamide MNP inhalation in a laboratory model for the assessment of systemic toxicities. Furthermore, we identified toxicities pertaining to inflammatory, cardiovascular, and endocrine activity.

Results

Particle characterization

The measured Brunauer–Emmett–Teller (BET) specific surface area (SSA) of the polyamide powder was 9.89 ± 0.55 m2/g and the calculated equivalent BET diameter of the particles was 0.53 ± 0.03 μm. A visual representation of the whole-body inhalation platform used for polyamide powder aerosolization is shown in Fig. 1. The total aerosol number concentration measured by the Scanning Mobility Particle Sizer (SMPS) (8–300 nm) averaged 220 ± 78 particles/cm3 (range: 110–356 particles/cm3) with a count median mobility diameter of 17.2 ± 1.7 nm (Fig. 2A). The average particle number concentration from the Aerodynamic Particle Sizer (APS) (0.5–20 μm) was 90 ± 34 particles/cm3 (range: 42–334 particles/cm3) with a narrow unimodal size distribution having a median aerodynamic diameter of 3.0 ± 0.1 μm (Fig. 2B). The bulk of the PM mass (~ 95%) was contained within the PM2.5−10 aerodynamic size fraction and the remaining mass (~ 5%) resided in the PM0.1−2.5 range as measured by the Harvard Compact Cascade Impactor (CCI) (Fig. 2C). This is in agreement with APS data and was confirmed using a High-Resolution Electrical Low Pressure Impactor (HR-ELPI) (data not shown). These data identify the polyamide aerosols as MNPs with a median diameter of 2.81 μm (GSD = 1.3). As the aerosolization platform produced respirable particles throughout the exposure, the time-weighted average for which rats were exposed is lower than OSHA’s permissible exposure limit for particles of this size. Characterization of the filtered air exposure revealed negligible levels of particles produced by hair, dander, and excrement (data not shown).

Fig. 1
figure 1

General schematic of the rodent whole-body inhalation platform used for experimentation

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Fig. 2
figure 2

Real-time size characterization of the whole-body inhalation facility shows the presence of nanoscale particles (A), and microparticles (B). Time-integrated characterization of the overall particle size fractionation during exposure was quantified using a Harvard Compact Cascade Impactor (C). Data are presented as mean ± SEM.

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Animal characteristics and pulmonary deposition modeling

Animal age and heart weight were not significantly different between the control and exposure groups. Inhalation of polyamide MNP resulted in a 14% increase in mean arterial pressure (MAP) (Table 1).

Table 1 Animal age and weight, heart weight, and mean arterial pressure. Data are presented as mean ± SEM, n = 9–17, *Significantly different (p < 0.05) from control animals as determined by a two-tailed t-test assuming equal variance between groups
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Without clearance, the estimated mass fraction of the inhaled particles that was deposited across the different respiratory regions was 76.7%, with the most deposition predicted in the head (extra-thoracic) region (49.9%), followed by the tracheobronchial (23.4%), and alveolar (3.4%) regions. The predicted deposited particle doses in these regions over the 4 h exposure period were 21.8, 3.25, and 0.035 µg/cm2, respectively. Assuming particle clearance for 20 h after the end of the exposure period, the estimated retained doses in the tracheobronchial and alveolar regions were 0.003 and 0.033 µg/cm2, respectively, indicating a high rate (> 99.9%) of clearance from the tracheobronchial area and negligible clearance from the alveolar region. No clearance calculation for the head region was available in the Multiple-Path Particle Dosimetry (MPPD) model.

Pulmonary histopathology and inflammation

Histopathological analysis of pulmonary tissue did not reveal any significant differences in infiltrating immune cell populations, morphological damage to pulmonary structures, or damage to the pulmonary vasculature (Fig. 3). Pulmonary tissue showed a continuous layer of ciliated epithelia in the higher-level conducting airways in both groups (Fig. 3A-B and D-E). There were no changes in the structure of the vasculature, or thickness of parenchymal cells lining the bronchi and terminal bronchioles (Fig. 3B and E). A comparable number of macrophages and neutrophils in the alveolar sacs and vasculature was observed in both groups (Fig. 3C F). Exposure did not disrupt the continuity and thickness of the alveolar barrier or proximity to pulmonary capillaries (Fig. 3C F). Overall, the epithelial barrier remained intact and pulmonary inflammation was not identified 24 h after a single exposure to MNP aerosols in virgin female rats.

Fig. 3
figure 3

Representative images of H&E-stained sections at 20x, 100x, and 200x magnification from naïve female rat controls (n = 9) or polyamide exposed rats (n = 8) are above. Low magnification morphological appearance of the conducting airways can be seen at 20x magnification (A and D). The proximal bronchioles (red arrows) and the distal bronchioles (black arrows) are shown at a 100x magnification (B and E). Representative images of the alveolar region are shown at 200x magnification (C and F)

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Analysis of bronchoalveolar lavage (BAL) fluid showed a significant yet physiologically negligible increase in the percentage of neutrophils in exposed animals (Table 2). No differences in protein levels or cell numbers were detected (Table 2).

Table 2 BAL fluid immune cell and protein levels. Significance was assessed by comparing outcomes with a two-tailed t-test assuming equal variance between groups. If data were not normally distributed and variance was unequal between groups, the Wilcoxon Rank Sum test was used for comparison of data sets. Data are presented as mean ± SEM, n = 9, *Significantly different (p < 0.05) from naive controls as determined by visual identification and quantification of Giemsa-stained cells
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Macrocirculation vascular reactivity

Aortic and uterine artery segments were assessed for their vascular reactivity to investigate alterations within the macrocirculation (conduit vessels > 150 microns) induced by polyamide inhalation. Uterine vessels were selected for investigation to represent a distal vascular bed for which vascular reactivity varies based on estrous stage [37]. Four parameter logistic regression modeling of the concentration response curves identified no significant differences in the reactivity of the aortas in rats exposed to polyamide relative to filtered air control rats (Fig. 4A-C). However, the left uterine artery exhibited both endothelium-dependent and -independent impairment in the rate of relaxation after methacholine (MCh) and sodium nitroprusside (SNP) stimulation, respectively (Fig. 4D and E). There were no alterations in vascular smooth muscle responses after stimulation with phenylephrine (PE) (Fig. 4F). These data suggest vasodilation pathways within the macrocirculation are selectively impaired 24 h after a single exposure to MNP aerosols, while vasoconstriction pathways are spared.

Fig. 4
figure 4

Vascular reactivity in the abdominal aorta and uterine artery was assessed by establishing concentration response curves with the endothelial-dependent vasodilator methacholine (A and D), the endothelial-independent vasodilator sodium nitroprusside (B and E), and the vasoconstrictor phenylephrine (C and F). Significance was assessed by comparing overall reactivity via a four-parameter nonlinear regression analysis. Data are presented as mean ± SEM, n = 9–11, *Significantly different (p < 0.05) from filtered air controls

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Microcirculation vascular reactivity

Resistance vessels are the major regulators of tissue perfusion. We assessed the vascular reactivity of the premyometrial radial artery, as a representative vessel of the microcirculation. Polyamide MNP inhalation led to a significantly decreased dilation response to MCh, an endothelium-dependent dilator (Fig. 5A). Radial artery segments from filtered air controls and MNP exposed rats exhibited comparable vascular reactivity for the endothelium-independent vasodilation and smooth muscle constriction pathways in response to SNP and PE, respectively. (Fig. 5B and C). These data indicate that MNP inhalation may impair dilation of the microcirculation primarily by affecting the endothelium.

Fig. 5
figure 5

Vascular reactivity in the radial artery was assessed by establishing drug response curves with the endothelial-dependent vasodilator, methacholine (A), the endothelial independent-vasodilator, sodium nitroprusside (B), and the vasoconstrictor, phenylephrine (C). Significance was assessed by comparing overall reactivity via a four-parameter nonlinear regression analysis. Data are presented as mean ± SEM, n = 7–9, *Significantly different (p < 0.05) from filtered air controls

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Systemic inflammation

Biomarkers of systemic inflammation were measured in plasma from control rats and MNP exposed rats (Fig. 6). Circulating levels of IL-6 were significantly elevated in 24 h post-MNP inhalation. CRP and MCP-1 levels were also elevated, but these values did not reach significance (p = 0.06 and 0.10, respectively).

Fig. 6
figure 6

Enzyme-linked immunosorbent assays for IL-6 (A), CRP (B), and MCP-1 (C) were carried out for assessment of systemic inflammation. Samples with analyte levels below the level of quantification and above the level of detection were assigned the value LOQ/2. Significance was assessed by comparing outcomes with a two-tailed t-test assuming equal variance between groups. Data are presented as mean ± SEM, n = 7–9, *Significantly different (p < 0.05) from filtered air controls; TTrending difference (p ≤ 0.10) from filtered air controls

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Hormonal mediators of vascular reactivity

In further studies, female rats were exposed during estrus to control for hormonal differences that may affect vascular reactivity. As potential endocrine disruption after MNP inhalation has not been investigated, we measured circulating levels of 17β-estradiol and progesterone in rats exposed to MNPs. Levels of circulating 17β-estradiol were significantly decreased in MNP exposed rats when compared to controls while progesterone was unaffected (Fig. 7A and B).

Fig. 7
figure 7

Enzyme-linked immunosorbent assays for 17β-estradiol (A) and progesterone (B). Samples with analyte levels below the level of quantification and above the level of detection were assigned the value LOQ/2. Significance was assessed by comparing outcomes with a two-tailed t-test assuming equal variance between groups. Data are presented as mean ± SEM, n = 7–9, *Significantly different (p < 0.05) from filtered air controls

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Discussion

Herein, we present novel data on the pathophysiological effects of inhalation exposure of estrus-staged virgin Sprague Dawley rats to a single dose of polyamide MNPs. Our findings identify multiple adverse cardiovascular outcomes including elevated blood pressure and impaired uterine vascular dilation. MNP inhalation also induced systemic inflammation, as evidenced by increased levels of proinflammatory cytokines in plasma. Decreases in circulating levels of 17β-estradiol after MNP inhalation suggest that polyamide may be endocrine disrupting, a property that would further impact cardiovascular function.

In these studies, we characterized the inhalation exposure platform and demonstrated that polyamide MNPs can readily be aerosolized for inhalation delivery to rodents. Using time-integrated sampling, we showed that particles in the PM2.5−10 aerodynamic size fraction contributed most to the mass of aerosolized particles. However, the PM< 0.1 size fraction had the highest detectable particle number/cm3. In our MPPD model, the percentage of deposited mass in the alveolar region was low in comparison to the tracheobronchial region. Nevertheless, the retained dose in the alveolar region was an order of magnitude greater than the tracheobronchial region. Measurements of other solid aerosols yield comparable results in terms of particle contribution to mass [56] and pulmonary deposition [57], but differed in terms the percentage of particle mass in each size fraction [58]. It should be noted that the MNPs in this study are a pristine, bulk polyamide material, as they have not undergone significant weathering since initial production. The exposure concentration used in this study cannot yet be compared to real-world exposure assessments due to limitations in detection of nano-sized particles. The spheroidal shape of the particles used in this study differs from the fragments and fibers detected environmentally and in tissue. Physicochemical properties of secondary particles resulting from mechanical degradation or incineration may modulate the toxicological outcomes compared to what we observed in this model. Due to the lack of available research in this area, it is unclear whether the overall mobility of particles and pulmonary deposition that we observed are consistent with environmental sources of MNPs.

A single exposure to polyamide aerosol did not generate notable pulmonary inflammation in our experimental model. Thus, 24 h post-exposure, there was no apparent histopathology or inflammatory changes in lung tissue. Negligible changes in the inflammatory cell population percentages and protein levels were noted in BAL fluid. While inflammation is often associated with pulmonary particle exposure, the absence of this outcome has been described in other studies of solid aerosols, despite higher airborne particle numbers than observed in our platform [39, 59]. Whether repeated inhalation exposure to MNPs would impact pulmonary inflammation remains to be determined.

Both systemic and local cardiovascular impairments were identified in our experimental rats. Increases in MAP after MNP inhalation indicates that this exposure generates systemic vascular impairments. This observation is distinct from other classes of homogenous particles such as metallic or carbonaceous materials [42, 43]. However, exposure to 3D printing aerosols, a conglomerate of particles and carcinogenic gases [60], has been shown to elevate MAP within 24 h of inhalation exposure [38]. In our study, large conduit vessels in exposed rats showed varied changes after polyamide MNP inhalation with the aorta being unaffected while the uterine artery exhibited blunted endothelium-dependent and -independent dysfunction. Polyamide exposure generated differential outcomes in the uterine microcirculation with only endothelium-dependent dilation being impaired. Other solid aerosols impair systemic dilation after pulmonary exposure, and the impact can vary between the macrocirculation and microcirculation even when the test material is the same [44]. The uterine vasculature has been identified as a vascular bed affected by particle inhalation through varying mechanisms [37, 45, 47, 61,62,63]. In sexually mature female animals, the uterine vasculature must have the appropriate response to stimuli in order to support reproductive functions of uterine tissue [51, 64, 65]. Findings presented here indicate that polyamide MNPs may impact vascular beds, conduit vessels, and resistance vessels through different, as of yet unidentified mechanisms.

Our data suggest that MNP inhalation increases circulating levels of proinflammatory cytokines. Other markers of acute inflammation such as IL-4, IL-5, and IFNγ were included in our initial assessment, but these cytokines were below the limit of detection in our assays. Selection of these cytokines for measurement of systemic inflammation was based on frequency of a detectable change in these biomarkers at a 24 h post-exposure timepoint in previous studies [66,67,68,69,70]. IL-1β and TNFα were not included due to their earlier peak following exposure to compounds known to cause inflammation [66, 67, 69, 71]. We found that circulating IL-6 was significantly higher in MNP exposed rats 24 h after exposure; moreover, biomarkers induced by IL-6, such as CRP and MCP-1, were also elevated, however, these data were not significant. These findings are consistent with epidemiological and animal studies that suggest pulmonary particle exposure generates systemic inflammation [21,22,23, 69, 72,73,74]. Further analysis of systemic inflammation caused by MNPs is required to assess the temporal relationship between this exposure and inflammatory cytokines.

To our knowledge, this report is the first to demonstrate endocrine disruption after MNP inhalation. We selectively focused on reproductive hormones that influence vascular reactivity. We controlled for hormonal cycling by exposing female rats only in estrus, the most fertile stage of their reproductive cycle. Consequently, the observed decrease in 17β-estradiol can be attributed to the inhalation of polyamide. Recently, Dusza et al. reported that hsd17b1, a gene which regulates 17β-estradiol biosynthesis, was downregulated after MNP exposure in a placental cell culture model [75]. This outcome occurred independently of systemic inflammation. To our knowledge, there is no evidence to suggest that systemic inflammation modulates reproductive hormone production unless the inflammation is severe enough to affect hormone producing tissues. However there is evidence indicating that estrogen and progesterone may act as anti-inflammatory compounds or in the resolution of inflammation [76,77,78,79]; therefore, it is more likely that the reduction in 17β-estradiol reported in these studies influences systemic inflammation.

As indicated above, the polyamide powder used in this study served as a representative MNP that is not known to be generated with bisphenol analogs or phthalates [53,54,55] and has no reported endocrine disrupting properties. However, polyamide has a high affinity of estrogenic compounds [80,81,82] which may act as endocrine disruptors in biological settings and explain the reduction in 17β-estradiol reported in these studies. The consequences of endocrine disruption induced by polyamide inhalation are unknown. Further studies are ongoing to examine how repeated exposure to MNP may affect circulating hormones in females and males, as well as their impact on pregnancy and fetal growth.

The toxicokinetics of MNPs are of equal importance as the toxicodynamics we have addressed in this study. Future studies will aim to visualize MNP in the rat lung as well as translocation to distal tissues after inhalation exposure. Direct interactions of MNPs with biological barriers and other tissues may drive their mobility and toxicity in living systems. Research of other solid particles suggests that their mobility in biological tissues is due, in part, to cellular uptake [83,84,85], and that they can cause oxidative stress [86, 87], as well as DNA damage [87,88,89]. Some studies suggest MNPs may have similar outcomes [90], but these parameters have not been investigated.

Conclusions

In these studies, we present a well characterized rodent whole-body inhalation exposure system that can be used for the toxicological evaluation of MNP inhalation. MNPs are a particle type that has been frequently overlooked despite the likelihood of human exposure and health concerns. To our knowledge, no research group has aerosolized MNP for use in a whole-body exposure chamber. Taken together, our results reveal the immune, cardiovascular, and endocrine systems as potential targets of MNP inhalation after a single exposure. It is imperative that MNPs be investigated for potential toxicity given their ubiquitous nature in the ambient and indoor environment. Furthermore, plastics may act as a toxicological vector due to their ability to adsorb and/or absorb biological, organic, and chemical agents. The pervasive nature of MNPs and their adverse effects as presented in this study highlight the need for better understanding of MNP toxicities to support consumer choice, regulatory policy, and environmental remediation.

Methods

Animal model

Female Sprague Dawley rats were purchased from Charles River Laboratories (Kingston, NY) at 8–10 weeks of age. Rats were delivered to an AAALAC accredited vivarium at Rutgers University where they were provided at least 72 h to acclimate. In the vivarium, food and water were available to the rats ad libitum. Females were randomly assigned to each group (n = 12 /group) and their estrous cycle status monitored. Estrous cycle stage was confirmed by vaginal smear cytology. Vaginal smears were obtained using cotton swabs moistened with deionized water as previously described [37, 91]. The vaginal mucus and cell populations were transferred to glass slides by rolling the swab on the slides. Using a microscope, the cellular populations in the vaginal orifice of the rat were identified and the stage of the estrous cycle was confirmed. To limit vascular and endocrine variability, all rats were exposed in the estrus phase of their reproductive cycle. All experiments were performed with Rutgers IACUC approval.

Polyamide powder and bulk characterization

Orgasol® 2001 UD NAT 2, a polyamide ultrafine powder, was obtained from Arkema (King of Prussia, Pennsylvania). Commercial characterization identifies a powder composed of polyamide-12 particles with a nearly round shape and a diameter of 5 ± 1 μm [92]. Physicochemical properties of these particles were confirmed in-house via Brunauer-Emmett-Teller specific surface area analysis. The polyamide powder was characterized as is for SSA using the multi-point BET nitrogen adsorption method in the NOVATouch® LX4 surface area and pore size analyzer (Quantachrome Instruments, Boynton Beach, FL). Using the measured SSA (m2/g), an equivalent BET diameter (µm) of the polyamide particles was also computed, assuming a spherical shape and equal size of all particles, using the following equation:

BET Diameter (µm) = 6 / [ρ (g/cm3) ´ SSA (m2/g)]

where the value for density was 1.14 g/cm3 as derived from previous characterization of polyamide [93].

Inhalation exposure

Rats were exposed to polyamide particles (9.53 ± 1.03 mg/m3) or filtered air over an average of 4.35 ± 0.39 or 4.5 h, respectively, using a custom rodent inhalation facility designed for whole-body aerosolized particle inhalation (IEStechno, Morgantown, WV). In brief, particles were aerosolized by an acoustic generator and rats exposed in an 84 L exposure chamber as previously described [43, 94]. The animal inhalation exposure chamber was connected to state-of-the-art real-time and time-integrated instrumentation in order to enable a detailed physicochemical, morphological, and toxicological characterization of the generated and sampled polyamide aerosol. An exposure concentration of near 10 mg/m3 was selected based on the American Conference of Governmental Industrial Hygienists (ACGIH) guidelines for poorly soluble and non-cytotoxic inhalable aerosols, as described in the Safety Data Sheet provided by the manufacturer [95]. This concentration is more conservative that the OSHA guidelines of 15 mg/m3. Rats were exposed to a concentration comparable to the current average daily concentration assessments of plastic particles in occupational settings [11, 96].

Real-time and time-integrated particle characterization of polyamide aerosol

The particle number concentrations as a function of size of the generated polyamide aerosol were monitored in real-time using a SMPS (TSI Model 3080, Shoreview, MN) for aerosols in the nano size regime (8–300 nm), and an APS (TSI Model 3321, Shoreview, MN) for submicron- and micron-sized aerosols (0.5–20 μm). The instruments were connected to two separate ports on the exposure chamber via flexible and conductive silicone rubber tubing to minimize particle losses. Measurements were initiated approximately 1 h after the aerosol generation was started to ensure sufficient time for the particle concentrations in the chamber to stabilize. Real-time measurements were collected for at least 1 h. The SMPS recorded a measurement every 2.25 min and the APS recorded every 20 s. The total aerosol number concentrations and the number-size distributions were averaged across the individual measurements over the 1 h monitoring period, and the average aerosol size statistics (i.e. median, mode, mean, geometric mean, and geometric standard deviation) were reported.

The polyamide aerosols were also measured in a size-fractionated manner using a High-Resolution Electrical Low Pressure Impactor (Dekati HR-ELPI+, Kangasala, Finland) or the Harvard Compact Cascade Impactor (CCI) [97]. Four aerodynamic size fractions of the aerosol, i.e., PM< 0.1, PM0.1−2.5, PM2.5−10 and PM> 10, were collected in the CCI. The PM< 0.1 was collected on Teflon filters (PTFE membrane disc filter: 2 μm pore size, 47 mm diameter, Pall Corporation, Port Washington, NY) while the larger PM were collected on polyurethane foam substrates. The aerosol sampling flow from the exposure chamber was 10 L/min and supplemented with HEPA-filtered ambient air to make up the 30 L/min total flow required by the CCI. Aerosol sampling was continued for 2 h. Post sampling, gravimetric analysis of the PM substrates was performed to calculate the time-averaged aerosol mass concentrations as a function of the different aerodynamic size fractions. Size fractionation measure by the CCI was confirmed with HR-ELPI+.

Pulmonary deposition modeling of polyamide aerosol

The deposition of the inhaled polyamide aerosol in the different lung regions (i.e. head, tracheobronchial, alveolar) of the exposed virgin female rats was modeled using the Multiple-Path Particle Dosimetry model (MPPD v. 3.04) for a single exposure period of 4 h to 9.53 mg/m3 polyamide MNPs, assuming no particle clearance as well as a clearance period of 20 h post exposure [98,99,100]. The concentration of 9.53 mg/m3 was chosen for the model to reflect the average exposure concentration in the experimental animal chamber. The aerosol exposure parameters input into the model were based on real-time monitoring and time-integrated sampling data: Count median diameter = 2.81 μm, GSD = 1.3 and Density of 1.14 g/cm3 which has been reported in the literature for polyamide [93]. The following rat lung and breathing parameters were used in the model: Symmetric Sprague Dawley Rat; Body weight = 233 g; Breathing Frequency: 124.6 #/min; TLC = 11.59 mL; FRC = 3.03 mL; Lung (distal) volume = 3.78 mL; URT = 0.348 mL; Tidal volume = 1.6 mL; Inspiratory fraction = 0.5; Pause fraction = 0.0; Body orientation: on stomach; Breathing scenario: Whole-body exposure; Default clearance parameters. The estimated deposited dose of the polyamide particles in the different lung regions was reported in units of particle mass per unit surface area (µg/cm2).

Mean arterial pressure and plasma collection

Twenty-four hr after the exposure, rats were anesthetized using isoflurane (5% induction and 3% maintenance). While anesthetized, animals were weighed, and placed in a nose cone on a surgical board in a supine position where the anesthetic was maintained. Once the animal was secured to the board, the carotid artery was isolated and cannulated with polyethylene tubing with an inner diameter of 0.58 mm and outer diameter of 0.965 mm (BD Intramedic, Franklin Lakes, NJ). MAP was obtained using a BLPR2 pressure transducer as well as a blood pressure monitor (World Precision Instruments, Sarasota, FL). After a stable MAP reading was recorded, approximately 6 mL of blood was collected directly from the cannula into BD Vacutainer® Plus whole blood tubes lined with 100% Dipotassium EDTA Dihydrate. Whole blood was centrifuged (Sorvall™ ST 8, Thermofisher Scientific, Waltham, MA) at 1100 RCF for 10 min (Thermofisher Scientific, Waltham, MA). The plasma was then removed using a pipette, aliquoted for future study, and snap frozen in liquid nitrogen.

Tissue harvest

The right and left uterine horns were carefully excised with the ovary intact and placed in 4° C physiological salt solution (PSS). The thoracic aorta was also excised and placed in 4° C PSS.

Pulmonary histology

A subset of animals (18 total) was used for histology and BAL collection. The control rats used for these studies were naïve. Animals housed in the aformentioned AAALAC accredited vivarium breathe filtered air by means of a HEPA filter overlaying the air inlet on their cages. Therefore, the authors cite no reason to assume pulmonary inflammation would differ between naïve and filtered air controls. For all other studies, the stress of the exposure may influence outcome and therefore filtered air controls were used. To our knowledge, cycling reproductive hormones do not affect pulmonary inflammation so estrous cycle was not considered as a factor for histology or BAL analysis.

Lungs were excised and examined for any gross morphological changes. Lungs were perfused with 3% paraformaldehyde in PBS, fixed in 3% paraformaldehyde for 12 h, and then transferred to 50% ethanol. Tissues were embedded in paraffin wax and sectioned. Paraffin-embedded tissues were sectioned at 5 μm, stained with hematoxylin and eosin, and examined by a board-certified veterinary pathologist via light microscopy. Pulmonary histomorphological endpoints for gas exchange and air conduction parenchyma included edema, white blood cell numbers, as well as changes to alveolar walls, alveolar epithelium, and vasculature. Incidence and severity scoring utilized appropriate controls for semiquantitative grades from 0 to 5 with grade 0 indicating none or background changes; grade 1, minimal changes; grade 2, mild changes; grade 3, moderate changes; grade 4, marked changes, and grade 5, severe changes.

Bronchoalveolar lavage fluid collection and analysis

A 16-gauge blunted needle was inserted in the trachea and secured. Using a syringe, 10 mL of PBS was instilled into the lung and subsequently removed. The collected fluid was centrifuged at 300 RCF for 8 min. The fluid supernatant was collected and stored at -80° until further analysis. Cell pellets were resuspended in 1 mL PBS. BAL cell differentials and albumin content were analyzed as previously described [101, 102].

Wire myography

Wire myography (DMT, Ann Arbor, MI) was used to assess vessel reactivity of the aorta and the uterine artery. Vessels were excised and placed in cold Wire Myography Physiological Salt Solution (WM-PSS: 130 mM NaCl, 4.7 mM KCl, 1.18 mM KH2PO4, 1.17 mM MgSO4 7H2O, 14.9 mM NaHCO3, 5.5 mM glucose, 0.03 mM EGTA, and 1.6 mM CaCl2). Excess connective tissue was removed to isolate the vessel. Segments of the vessel (2 mm) were cut, mounted on two wires within 1 h of tissue harvest, and used for experimentation as previously described [43]. Endothelium-dependent relaxation, endothelium-independent relaxation, and smooth muscle contraction were separately evaluated via cumulative additions of 60 µL of MCh (MP Biomedicals, Solon, OH CAT 190,231), SNP (Thermofisher Scientific, Waltham, MA CAT 211,640,250), and PE (Thermofisher Scientific, Waltham, MA CAT 207,240,100), respectively (10− 9 to 10− 4 M for each pharmacological application). Responses to chemical agents were randomized.

Wire myography data are presented as a percentage of maximum tension after incubation with High Potassium Physiological Salt Solution (KPSS: 74.7 mM NaCl, 60 mM KCl, 1.18 mM KH2PO4, 1.17 mM MgSO4 7H2O, 1.6 mM CaCl2, 14.9 mM NaHCO3, 0.03 mM EDTA, and 5.5 mM glucose) for 5 min, which reflects the maximum tension placed on the wire in the myograph chamber by the vessel.

Pressure myography

The right uterine horn was stabilized in a bath of 4° C Microvessel Physiological Salt Solution (MV-PSS: 119 mM NaCl, 4.7 mM KCl, 1.17 mM MgSO4 7H2O, 1.6 mM CaCl2 2 H2O, 1.18 mM NaH2PO4, 24 mM NaHCO3, 5.5 mM glucose, and 0.03 mM EGTA) to visualize the premyometrial radial artery, which is representative of the microcirculation in the uterus [103]. A single radial artery was excised, mounted, and pressurized to 60 mmHg in a bath of MV-PSS prior to experimentation and within 1 h of tissue harvest as described [36, 48, 103]. The equilibrated artery was incubated with various pharmacological agents to measure concentration dependent physiological responses. MCh was used to test endothelium dependent vascular smooth muscle relaxation. SNP was used to test endothelium independent vascular smooth muscle relaxation. PE was used to test smooth muscle contraction. The vessel was exposed stepwise to increasing concentrations (10− 9 to 10− 4 M) of each given pharmacological agent. The order of execution for each concentration response curve was completely randomized.

Pressure myography data are presented as a percentage of maximum relaxation after incubation with Ca2+ Free Microvessel Physiological Salt Solution (Ca2+ Free PSS: 120.6 mM NaCl, 4.7 mM KCl, 1.17 mM MgSO4 7H2O, 1.18 mM NaH2PO4, 24 mM NaHCO3, 5.5 mM glucose, and 0.03 mM EGTA) for 20 min, which reflects the maximum dilation of the vessel measured by the caliper when maximum dilation was elicited by Ca2+ Free PSS.

Systemic inflammation

Proinflammatory cytokines including IL-6, MCP-1, IL-4, CRP, IFNγ, and IL-5 were measured in plasma using Enzyme-Linked Immunosorbent Assays (ELISAs) (Sigma Aldrich, St. Louis, MO CAT RAB0311, RAB0057 RAB0301, RAB0097 and RAB0227 and Abcam, Cambridge, UK CAT ab267811). For samples with analyte concentrations above the limit of detection but below the level of quantification, a value of half the limit of quantification was assigned as described [104,105,106,107].

Measurement of hormonal mediators of vascular function

ELISA bioassays were used to measure 17β-estradiol (Abcam Cambridge, UK CAT ab108677) and progesterone (Novus Biologicals, Littleton CO CAT NBP2-60127), which are hormonal mediators of vascular function, in plasma. For samples with analyte concentrations above the limit of detection but below the level of quantification, a value of half the limit of quantification was assigned as described [104,105,106,107].

Statistics

Student’s t-test assuming equal variance between groups was used to compare animal characteristics, pulmonary inflammation, and ELISA data. If the data set did not follow a Gaussian distribution and variance was unequal between groups, the Wilcoxon Rank Sum Test was used for comparison. Overall vascular reactivity using pressure myography and wire myography drug response curves were compared using a four-parameter nonlinear regression analysis. GainData (ELISA) analysis tool was used to convert absorbance values to analyte concentrations for each ELISA (Arigo Biolaboratories, Hsinchu City, Republic of China). Statistical analyses were completed with GraphPad Prism 9.0 (San Diego, CA, USA). Data are presented as mean ± SEM and significance is set at p ≤ 0.05.