Combined Effects of Micro- and Nanoplastics at the Predicted Environmental Concentration on Functional State of Intestinal Barrier in Caenorhabditis elegans

The possible toxicity caused by nanoplastics or microplastics on organisms has been extensively studied. However, the unavoidably combined effects of nanoplastics and microplastics on organisms, particularly intestinal toxicity, are rarely clear. Here, we employed Caenorhabditis elegans to investigate the combined effects of PS-50 (50 nm nanopolystyrene) and PS-500 (500 nm micropolystyrene) at environmentally relevant concentrations on the functional state of the intestinal barrier. Environmentally, after long-term treatment (4.5 days), coexposure to PS-50 (10 and 15 μg/L) and PS-500 (1 μg/L) resulted in more severe formation of toxicity in decreasing locomotion behavior, in inhibiting brood size, in inducing intestinal ROS production, and in inducting intestinal autofluorescence production, compared with single-exposure to PS-50 (10 and 15 μg/L) or PS-500 (1 μg/L). Additionally, coexposure to PS-50 (15 μg/L) and PS-500 (1 μg/L) remarkably caused an enhancement in intestinal permeability, but no detectable abnormality of intestinal morphology was observed in wild-type nematodes. Lastly, the downregulation of acs-22 or erm-1 expression and the upregulation expressions of genes required for controlling oxidative stress (sod-2, sod-3, isp-1, clk-1, gas-1, and ctl-3) served as a molecular basis to strongly explain the formation of intestinal toxicity caused by coexposure to PS-50 (15 μg/L) and PS-500 (1 μg/L). Our results suggested that combined exposure to microplastics and nanoplastics at the predicted environmental concentration causes intestinal toxicity by affecting the functional state of the intestinal barrier in organisms.


Introduction
Due to the insufficient recycling and reusing system, large quantities of plastics have been randomly released into ecosystems, including the terrestrial ecosystem, marine ecosystem, and freshwater ecosystem [1]. Generally, plastics refer to various morphological debris that originates from multiple plastic product degradation [2]. Such plastic debris can be further degraded into microplastic particles (<5 mm) or nanoplastic particles (<100 nm) [3,4]. As is well known, particle size plays a dominant role in determining particle biodistribution and biotoxicity [5]. Long-term exposure to small sizes of nanopolystyrene particles (20 nm) could cause more severe exposure risk in causing transgenerational toxicity than larger sizes of nanopolystyrene particles (100 nm) in nematodes [6]. The particle size is inversely proportional to micro-and nanoplastics absorption in organisms [7][8][9]. In vertebrates, polystyrene microplastic exposure induced testis developmental disorder and affected male fertility in mice [10]. In addition, alteration in functional group modification of nanopolystyrene particles could also cause different toxicity formations. For example, sulfonate-or amino-modified nanopolystyrene could cause more serve neurotoxicity or reproductive toxicity in Caenorhabditis elegans [11,12]. UV-aged microplastics induced neurotoxicity via regulating the neurotransmission in larval zebrafishes [13]. However, the working suspensions monodisperse. The FTIR spectrum of PS-50 and PS-500 is shown in Supplementary Figure S1.
Toxics 2023, 11, x FOR PEER REVIEW 3 of 13 working suspensions were made by diluting the stock solution with liquid K-medium. Before exposure, a 30 min sonication at 40 kHz (100 W) was performed to make the working suspensions monodisperse. The FTIR spectrum of PS-50 and PS-500 is shown in Supplementary Figure S1.

Animal Maintenance
C. elegans (wild type, Bristol strain N2) were acquired from School of Medicine, Southeast University (Nanjing, China, accessed on 16 May 2022) and cultured on nematode growth medium plates containing Escherichia coli OP50, the food source, at 20 °C without light [31]. To extract worms at the same developmental stage, we prepared a bleaching solution (2% HOCl, 0.45 M NaOH) to synchronize pregnant worms over a 5 min period to discharge enough eggs [32]. These worms were then rinsed thrice with Kmedium (0.032 M KCl, 0.051 M NaCl) [33], before transfer to fresh NGM plates with OP50 feeding, and incubated at 20 °C for 24 h without light to acquire L1 stage worms.

Exposure and Assessment Endpoints
The estimated environmental PS-50 concentrations were chosen as 5, 10, and 15 μg/L [20,21]. The selected working concentrations of PS-500 were 0.1 and 1 μg/L [20]. Longterm treatment (L1-larvae to adult day-1) was used to treat the examined worms and the exposure suspensions were introduced to E. coli OP50 (~4 × 106 colony-forming units (CFUs)). During the exposure period, the suspensions were replenished daily. Herein, some endpoints were performed to detect the potential combined toxicity between PS-50 and PS-500 at the predicted environmental concentration on nematodes.
Locomotor behavior, including head thrashing and body bending, was employed to assess the motor neuronal operative status as described [6]. Head thrashes were quantified according to the posterior bulb direction (y-axis) alterations, with an assumption that the x-axis was the traveling direction [24,32]. Body bends were quantified as the midbody bending directional alterations [24,32]. For individual contacts, 40 worms were analyzed.
Brood size was used to assess the reproductive capacity [34]. The total number of the progeny during the development beyond the eggs was counted [35]. For each exposure treatment, 20 worms were analyzed.
Intestinal ROS synthesis was assessed as reported earlier [31]. C. elegans were exposed to 1 μM 5′,6′-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate (CM-H2DCFDA), prior to a 3 h incubation without light. The tested organisms were rinsed thrice in K-medium (0.032 M KCl, 0.051 M NaCl), and then mounted on a 2% agar pad to evaluate intestinal fluorescent ROS production, using a fluorescence microscope with an excitation wavelength at 488 nm and an emission filter of 510 nm. The relative fluorescence intensity representing intestinal ROS production was semiquantified in relation to the intestinal autofluorescence. Overall, 40 animals were assessed per group, and each group was tested three times.

Animal Maintenance
C. elegans (wild type, Bristol strain N2) were acquired from School of Medicine, Southeast University (Nanjing, China, accessed on 16 May 2022) and cultured on nematode growth medium plates containing Escherichia coli OP50, the food source, at 20 • C without light [31]. To extract worms at the same developmental stage, we prepared a bleaching solution (2% HOCl, 0.45 M NaOH) to synchronize pregnant worms over a 5 min period to discharge enough eggs [32]. These worms were then rinsed thrice with K-medium (0.032 M KCl, 0.051 M NaCl) [33], before transfer to fresh NGM plates with OP50 feeding, and incubated at 20 • C for 24 h without light to acquire L1 stage worms.

Exposure and Assessment Endpoints
The estimated environmental PS-50 concentrations were chosen as 5, 10, and 15 µg/L [20,21]. The selected working concentrations of PS-500 were 0.1 and 1 µg/L [20]. Long-term treatment (L1-larvae to adult day-1) was used to treat the examined worms and the exposure suspensions were introduced to E. coli OP50 (~4 × 106 colony-forming units (CFUs)). During the exposure period, the suspensions were replenished daily. Herein, some endpoints were performed to detect the potential combined toxicity between PS-50 and PS-500 at the predicted environmental concentration on nematodes.
Locomotor behavior, including head thrashing and body bending, was employed to assess the motor neuronal operative status as described [6]. Head thrashes were quantified according to the posterior bulb direction (y-axis) alterations, with an assumption that the x-axis was the traveling direction [24,32]. Body bends were quantified as the midbody bending directional alterations [24,32]. For individual contacts, 40 worms were analyzed.
Brood size was used to assess the reproductive capacity [34]. The total number of the progeny during the development beyond the eggs was counted [35]. For each exposure treatment, 20 worms were analyzed.
Intestinal ROS synthesis was assessed as reported earlier [31]. C. elegans were exposed to 1 µM 5 ,6 -chloromethyl-2 ,7 -dichlorodihydro-fluorescein diacetate (CM-H2DCFDA), prior to a 3 h incubation without light. The tested organisms were rinsed thrice in Kmedium (0.032 M KCl, 0.051 M NaCl), and then mounted on a 2% agar pad to evaluate intestinal fluorescent ROS production, using a fluorescence microscope with an excitation wavelength at 488 nm and an emission filter of 510 nm. The relative fluorescence intensity representing intestinal ROS production was semiquantified in relation to the intestinal autofluorescence. Overall, 40 animals were assessed per group, and each group was tested three times.
Intestinal autofluorescence brought on by the lipofuscin-mediated lysosomal deposition refers to the aging process in worms [35]. After contact, we mounted nematodes Toxics 2023, 11, 653 4 of 12 on a 2% agar plates and measured them under the DAPI channel of a microscope. The fluorescence intensity was quantified using Image J.
Intestinal permeability reflecting the functional state of the intestine barrier was routinely performed to exhibit the intestine damage induced by environmental pollutants [29]. Erioglaucine disodium (5.0%) was diluted using K-medium with OP50 addition as a food source [23]. Then, the exposed worms were soaked in the prepared staining liquid for 3 h in the dark. After staining, we used K-medium to wash the nematodes six times. Image capture was performed under the bright field. Overall, 30 animals were employed for individual treatments, with three biological replicates per treatment.

Statistical Analyses
SPSS 12.0 was employed for all data analyses, and data was provided as mean ± standard derivation (SD). Intergroups assessment utilized the one-way or two-way ANOVA with multiple-factor comparison followed by a post hoc test. A probability level of 0.01 (**) was set as the significance threshold.  S2). Interestingly, combined exposure of PS-500 (1 µg/L) and PS-50 (5 µg/L) noticeably reduced locomotion behavior rather than brood size in wild-type nematodes (Supplementary Figure S2).

Combined Effects of PS-50 and PS-500 on Intestinal Morphology
After prolonged exposure, we first examined intestinal morphology to detect the potential toxicity on intestinal structure. Whether coexposure or single-exposure to PS-50 and PS-500, it did not all cause remarkable changes in the intestinal lumen ( Figure 3). Furthermore, no aberration was confirmed for the intestinal cells in nematodes co-or single-exposed to PS-50 and PS-500 ( Figure 3). Therefore, the interplay of PS-50 and PS-500 at the predicted environmental concentration did not influence the intestinal morphology in worms.

Combined Effects of PS-50 and PS-500 on Intestinal Morphology
After prolonged exposure, we first examined intestinal morphology to detect the potential toxicity on intestinal structure. Whether coexposure or single-exposure to PS-50 and PS-500, it did not all cause remarkable changes in the intestinal lumen ( Figure 3). Furthermore, no aberration was confirmed for the intestinal cells in nematodes co-or single-exposed to PS-50 and PS-500 ( Figure 3). Therefore, the interplay of PS-50 and PS-500 at the predicted environmental concentration did not influence the intestinal morphology in worms.

Discussion
To date, sufficient evidences provided by previous studies have raised that prolonged exposure to micro-or nanoplastics results in severe multiorgan toxicity in environmental organisms, including neurotoxicity, reproductive toxicity, and immunotoxicity in environmental organisms [11,26,[40][41][42]. However, the combined effects of microplastics and nanoplastics, especially at predicted environmental relevant concentration, on or- and oxidative-stress-related gene expression (B). Long-term exposure was provided from L1-larvae to adult day-1. "+", with; "−", lacking. Control, without polystyrene particle treatment. Bars denote means ± SD. ** p < 0.01 vs. control (if not specially indicated); NS, no significant difference.

Discussion
To date, sufficient evidences provided by previous studies have raised that prolonged exposure to micro-or nanoplastics results in severe multiorgan toxicity in environmental organisms, including neurotoxicity, reproductive toxicity, and immunotoxicity in environmental organisms [11,26,[40][41][42]. However, the combined effects of microplastics and nanoplastics, especially at predicted environmental relevant concentration, on organisms remains unclear. Herein, locomotion behavior and brood size, as sensitive ecological indicators, could more effectively reflect the potential biotoxicity of environmental pollutants [43,44]. Firstly, we used sensitive endpoints (brood size and locomotion behavior) to examine the combinational effects between PS-50 and PS-500 in nematodes. We revealed that long-term PS-500 (1 µg/L) exposure could prominently enhance PS-50 (15 µg/L) toxicity in diminishing locomotion behavior and brood size (Figure 2). That is, although PS-500 (1 µg/L) alone would not cause toxicity, a synergistic effect between PS-500 (1 µg/L) and PS-50 (15 µg/L) on nematodes can be formed. That is, coexposure to micro-and nanoplastics at estimated environmentally significant concentrations showed more serve toxicity on organisms in the environment. As reported, prolonged exposure to nanopolystyrene particles (100 nm) at estimated environmentally significant concentrations (1 µg/L) did not promote toxicity in nematodes [27]. The above observation suggests that the interplay of PS-50 and PS-500 might cause more severe toxicity on motor neuronal activity in wild-type nematodes compared to that in separate exposure groups. Besides this, no detectable alteration in brood size was detected in worms cotreated with PS-500 (1 µg/L) and PS-50 (5 µg/L) (S2B), which implies that brood size may not be so sensitive in examining the improvement in combined toxicity. Differently, brood size showed a more susceptible property than locomotion behavior in controlling MC-LR toxicity [45], likely because of differences in sensitivity to different toxicants or exposure durations.
The broadened intestinal lumen is frequently used as an indicator to reflect the abnormality in intestinal morphology [23,35]. Recently, some environmental stress, such as simulated microgravity, can alter the intestinal morphology [23]. In contrast, neither single-exposure or coexposure to PS-500 (0.1, 1 µg/L) and PS-50 (15 µg/L) resulted in the noticeable structural changes in the intestinal lumen or intestinal cells in nematodes ( Figure 3). That is, the interplay of PS-50 and PS-500 at environmental relevant concentration was not sufficient to damage the intestinal morphology. Similarly, exposure to graphene oxide or 6-PPDQ in the range of µg/L did not also induce the abnormality of intestinal morphology [35,46].
Intestinal permeability is usually performed to assess the functional state of the intestinal barrier [35,47]. Different from the observation of intestinal morphology, we observed remarkable improvement in intestinal permeability only after coexposure in the PS-50 (15 µg/L) and PS-500 (1 µg/L) group rather than that in other groups (Figures 4A and S3). Subacute PS-MPs exposure also caused a hyperpermeable state of the intestinal barrier, indicating that PS-MPs exposure resulted in intestinal damage to nematodes [30]. Interestingly, the blue dye was translocated from the intestinal lumen to intestinal cells, but the blue dye was not further translocated into the body cavity in nematodes coexposed to PS-50 (15 µg/L) and PS-500 (1 µg/L) ( Figure 4A). The results imply that combined exposure to PS-50 and PS-500 at environmental relevant concentration generated severe intestine toxicity in enhancing intestinal permeability and damaging the functional state of the intestinal barrier rather than single-exposure to PS-50 or PS-500. More importantly, Liang et al. also showed that polystyrene micro-and nanoplastics synergistically induced intestinal barrier dysfunction via an ROS-mediated epithelial cell apoptotic pathway in mice [19]. Additionally, nanopolystyrene at the estimated environmental concentration can enhance microcystin-LR toxicity via intestinal destruction in Caenorhabditis elegans [48].
Some crucial regulators are involved in controlling for intestinal permeability. Similar to the observation of intestinal permeability, coexposure to PS-50 (15 µg/L) and PS-500 (1 µg/L) remarkably decreased the intestinal acs-22 and erm-1 expressions ( Figure 4B), which may be intricately linked to the detectable rise in intestinal permeability. In C. elegans, ACS-22 encodes a protein homologous to mammalian fatty acid transport protein and is necessary for controlling intestinal permeability [23]. ERM-1 encodes an Ezrin-radixinmoesin protein needed for maintaining the intestinal barrier functional state [23]. Hence, the underlying mechanism of intestinal barrier function is potentially changed by coexposure to PS-50 and PS-500 at the predicted environmental concentration. Furthermore, no significant changes in intestinal act-5, pkc-3, and hmp-2 contents were confirmed ( Figure 4B), which were also important contributors to maintain the normally functional state of intestinal barrier in nematodes.
To explore the underlying intracellular mechanism required for the PS-50 and PS-500 coexposure-mediated modulation of intestine toxicity, we proposed oxidative stress and intestinal autofluorescence in this study. Intestinal autofluorescence is generated via lysosomal deposition of lipofuscin, which accumulates over time in aging nematodes [49]. Oxidative stress is the major contributor to the toxicity formation of toxicants [50]. Prolonged exposure to PS-500 (1 µg/L) enhanced PS-50 (10 and 15 µg/L) toxicity in inducing intestinal autofluorescence and intestinal ROS synthesis ( Figures 5, 6A, S4 and S5); this observation further supports the more severe intestine toxicity caused by the combined exposure to PS-50 and PS-500 at environmentally relevant concentration. Similarly, exposure to polystyrene microplastics (1 µm) at 72 h also induced oxidative stress and intestinal injury in nematode Caenorhabditis elegans [30], which implies the close association between oxidative stress and intestine toxicity caused by polystyrene microplastic exposure. Intriguingly, coexposure to PS-500 (1 µg/L) and PS-50 (5 µg/L) produced a noticeable rise in intestinal ROS synthesis rather than intestinal autofluorescence synthesis (Supplementary Figures S4 and S5). The reason may be that intestinal ROS synthesis is more sensitive than intestinal autofluorescence. Furthermore, nanopolystyrene particles at the estimated environmentally significant concentration enhanced the environmental ENMs (TiO 2 -NPs) toxicity on nematodes by diminishing locomotion behavior and enhancing intestinal ROS synthesis [47].
In C.elegans, SODs, catalases, and mitochondrial complex components act as crucial regulators to maintain the oxidative stress balance [51,52]. Compared to the control group, PS-500 (1 µg/L) did not produce any alteration among the examined genes required for controlling ROS production ( Figure 6B). Differently, obvious rises in sod-3, clk-1, and gas-1 expressions occurred in worms exposed to PS-50 (15 µg/L). More interestingly, coexposure to PS-500 (1 µg/L) and PS-50 (15 µg/L) further dramatically enhanced the sod-2, sod-3, isp-1, clk-1, gas-1, and clt-3 expressions. SOD-2 and SOD-3 cooperate with CTL-3 to modulate the oxidation-antioxidation system in nematodes [23,35]. ISP-1, CLK-1, and GAS-1 localize to the mitochondria electron transport chain to sever as important components to control intestinal ROS production [47]. Environmentally, these observations provided the indirect evidence for the more severe toxicity formation in nematodes coexposed to PS-50 and PS-500 at environmentally relevant concentration compared with single-exposure to PS-50 or PS-500. It is implied that nanoplastic collaboration with microplastics synergistically contributes to the remarkable enhancement of toxicity in organisms.
Noteworthily, based on the above observations, the combined effects to PS-50 and PS-500 were only observed at the upper concentration range for both plastic particles. However, the estimated environmental nanoplastic concentration for 50 nm plastic particles is speculated to be 1 pg L -1 -15 µg/L and ≤1 µg/L for 500 nm plastic particles [20,21]. In a real environment, the environmentally relevant concentrations for the particle sizes used in the present study are most likely lower than the upper concentration. That is, from an environmental perspective, the observed combined toxicity of PS-50 and PS-500 on the functional state of the intestinal barrier may be overrated. Nevertheless, the potentially intergenerational effects of coexposure to PS-50 and PS-500 at the predicted environmental concentration cannot be neglected.

Conclusions
Taken together, coexposure to PS-50 and PS-500 was conducted to confirm our hypothesis, with the following conclusion: coexposure to micro-and nanoplastics at estimated environmentally significant concentrations could induced more severe deterioration of the functional state of the intestinal barrier than single-exposure to micro-or nanoplastics. In wild-type nematodes, cotreatment with PS-50 (15 µg/L) and PS-500 (1 µg/L) did not damage the intestinal morphology but enhanced intestinal permeability. Induction of intestinal ROS synthesis and intestinal autofluorescence production act as a cellular mechanism to powerfully explain the formation of intestine toxicity caused by coexposure to PS-50 (15 µg/L) and PS-500 (1 µg/L). Meanwhile, the downregulation of acs-22 or erm-1 expression and the upregulated expressions of oxidative stress-related genes serve as a molecular basis to strongly explain the formation of intestine toxicity caused by coexposure to PS-50 (15 µg/L) and PS-500 (1 µg/L). Our results suggested that combined exposure to microplastics and nanoplastics at the predicted environmental concentration notably causes intestinal toxicity by affecting the functional state of the intestinal barrier in organisms.