Transgenerational Response of Germline Nuclear Hormone Receptor Genes to Nanoplastics at Predicted Environmental Doses in Caenorhabditis elegans

Transgenerational nanoplastic toxicity could be detected in Caenorhabditis elegans after exposure at the parental generation (P0-G); however, the underlying mechanisms remain largely unclear. We aimed to examine the role of germline nuclear hormone receptors (NHRs) in controlling the transgenerational toxicity of polystyrene nanoparticles (PS-NPs) based on gene expression screening and functional analysis. Among germline NHR genes, daf-12, nhr-14, and nhr-47 expressions were increased and nhr-12 expression was decreased by PS-NPs (1 and 10 μg/L). Transgenerational alterations in expressions of these four NHR genes were also induced by PS-NPs (1 and 10 μg/L). RNAi of daf-12, nhr-14, and nhr-47 caused resistance, whereas RNAi of nhr-12 conferred susceptibility to transgenerational PS-NP toxicity. After PS-NP exposure, expressions of ins-3, daf-28, and ins-39 encoding insulin ligands, efn-3 encoding Ephrin ligand, and lin-44 encoding Wnt ligand, as well as expressions of their receptor genes (daf-2, vab-1, and/or mig-1), were dysregulated by the RNAi of daf-12, nhr-14, nhr-47, and nhr-12. Therefore, alteration in certain germline NHRs could mediate the induction of transgenerational nanoplastic toxicity by affecting secreted ligands and their receptors in the offspring of exposed organisms.


Introduction
Due to improper handling, plastic pollution has been recognized as a global environmental hazard problem [1,2].Moreover, because of insufficient degradation, microplastics and even nanoplastics are generated from waste plastics [3,4].Nanoplastics have been widely distributed in different environments, such as water and soil [5,6], and can be transferred through environmental media [7,8].Predicted environmental doses (PEDs) of nanoplastics range from ng/L to µg/L [9].Nanoplastics were further detected and accumulated in the organs of several organisms, such as zebrafish [10,11].Considering the existence of nanoplastics in terrestrial or aquatic food webs, their exposure risk for human health has been further implied [12,13].Nanoplastics could even be discovered in human blood [14].Once bioavailable to organisms, nanoplastics could be further internalized in cells and transported to certain organelles, such as mitochondria [15].Accompanied with bioavailability and accumulation, some adverse effects on the development and functions of organs or tissues could be observed in different organisms after nanoplastic exposure [16][17][18][19].Nanoplastic toxicity could be induced at both exposed parental generations (P0-G) and their offspring [20][21][22].
Caenorhabditis elegans exhibits high susceptibility to pollutant toxicity [23][24][25][26], which makes it suitable for assessing the toxicity of pollutants at PEDs [27][28][29].Meanwhile, C. elegans has a relatively short life cycle [30], which makes it suitable to evaluate transgenerational toxicity after pollutant exposure [31,32].In nematodes, transgenerational damage in gonads and neurons could be detected after exposure to pristine and aged nanoplastics [33,34].For example, using locomotion behavior as the toxicity assessment endpoint, exposure to 10 µg/L of polystyrene nanoparticles (PS-NP) could cause the toxicity induction from P0-G to F4-G [35].As the classic model animal, C. elegans can provide profound insights into the toxicological mechanisms of pollutants [36,37].Some secreted germline ligands (such as insulin, FGF, and Wnt) and their receptors were shown to play an important function in governing transgenerational nanoplastic toxicity [38][39][40].
Some mechanisms (such as epigenetic regulation) exist to regulate the function of targets by affecting target expressions [41].microRNAs and histone methylation regulation were identified to control transgenerational PS-NP toxicity [42][43][44].As transcriptional factors, nuclear hormone receptors (NHRs) can respond to certain ligands to regulate some biological events by affecting target gene expression [45,46].Among members in C. elegans [47], some NHRs (such as DAF-12) were proven to regulate the response to pollutants [48,49].Environmental pollutants have been further implied to act as possible ligands to trigger or inhibit certain NHRs in nematodes after exposure [50,51].We assumed that nanoplastics may induce transgenerational toxicity by activating or suppressing certain germline NHRs.Thus, in the current study, we aimed to identify germline NHRs involved in the regulation of transgenerational nanoplastic toxicity in nematodes.The PS-NP was selected as an example of nanoplastics.Moreover, the underlying mechanism for candidate germline NHRs in controlling the transgenerational PS-NP toxicity was further determined.Our results suggested the crucial function of germline NHRs in regulating the induction of transgenerational nanoplastic toxicity.The identified germline NHRs provide an important molecular basis for further elucidation of the molecular mechanisms for transgenerational nanoplastic toxicity in organisms.

Nematode Maintenance
As described by Brenner [52], animals (wild type, N2) were allowed to grow on nematode growth medium (NGM).E. coli OP50 was fed C. elegans as food.Both E. coli and all the nematodes were obtained from Caenorhabditis Genetics Center (CGC).The solution (2% HOCl, 0.45 M NaOH) was applied for synchronizing adults to collected eggs [53], which were then transferred onto new NGMs to grow into L1 larvae.Chemical reagents were purchased from Aladdin Industrial Corporation (La Puente, CA, USA) and Sangon Biotech Co., Ltd.(Shanghai, China).

Nematode Maintenance
As described by Brenner [52], animals (wild type, N2) were allowed to grow on nematode growth medium (NGM).E. coli OP50 was fed C. elegans as food.Both E. coli and all the nematodes were obtained from Caenorhabditis Genetics Center (CGC).The solution (2% HOCl, 0.45 M NaOH) was applied for synchronizing adults to collected eggs [53], which were then transferred onto new NGMs to grow into L1 larvae.Chemical reagents were purchased from Aladdin Industrial Corporation (La Puente, CA, USA) and Sangon Biotech Co., Ltd.(Shanghai, China).

Exposure
Concentrations (0.1-10 µg/L) of PS-NPs were used as described [54], which are the PEDs of nanoplastics [9].The purchased PS-NPs were suspended in water.The working PS-NP suspensions were prepared by diluting the stocking solution with K buffer.To assess transgenerational PS-NP toxicity, C. elegans was exposed to PS-NPs from the L1 larval stage to adult day 3, referred to as P0-G.During the entire exposure period, PS-NP

Exposure
Concentrations (0.1-10 µg/L) of PS-NPs were used as described [54], which are the PEDs of nanoplastics [9].The purchased PS-NPs were suspended in water.The working PS-NP suspensions were prepared by diluting the stocking solution with K buffer.To assess transgenerational PS-NP toxicity, C. elegans was exposed to PS-NPs from the L1 larval stage to adult day 3, referred to as P0-G.During the entire exposure period, PS-NP solutions were replaced daily to maintain consistent conditions.Eggs of P0-G were transferred to an NGM plate with OP50 added, allowing them to develop to the adulthood stage, which was called F1-G.The following offspring (F2-G to Fn-G) were generated successfully in this way.All the experiments were repeated three times.After the treatment, the animals were used simultaneously and separately for different research purposes.During the assessment of the endpoints for toxicity, animals were selected randomly.

Endpoints
To analyze reproductive capacity, the total number of offspring was defined as the brood size during the process of egg-laying [55].To analyze locomotion, the frequency of body bend, as well as head thrash, was assessed [56].After recovery on NGM for 1 min, these behaviors were examined under a stereomicroscope Nikon C-DSS230 stereomicroscope (Nikon, Japan) [57].For each exposure, 50 C. elegans were examined.Three replicates were carried out.

Gene Expression
Trizol (Sangon Biotech Co., Ltd., Shanghai, China) was applied together with ceramic beads to grinder animals.Using M-MuLV reverse transcriptase (Sangon Biotech Co., Ltd., Shanghai, China), cDNAs were prepared.Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out on a SYBR Green qRT-PCR master mix (Vazyme Biotech Co., Ltd., Nanjing, China).tba-1 acted as the reference gene for the normalization of target genes [58].To analyze the change in gene expression in the germline, intact gonads were separated and collected by removing them from the body of nematodes using a glass knife.Information on the primers is provided in Table S1.Three biological replicates were carried out.

RNA Interference (RNAi)
RNAi was generated through feeding with dsRNA-generating bacterial cells [59].On an RNAi plate, L1 larval animals developed into adults.Their offspring were exposed to PS-NPs.L4440, an empty vector, functioned as the control [60].DCL569 is a tool strain for the germline RNAi of genes.qRT-PCR was performed to analyze gene RNAi efficiency (Figure S1).

Data Analysis
To ensure the quality of data and avoid random errors, 50-100 nematodes were chosen randomly to measure the relative evaluation indexes.Meanwhile, three parallel experiments were performed simultaneously.Data were all continuous and passed the normality test and homogeneity test of variance in SPSS 26.0.Significant difference among treatments was analyzed by one-way or two-way ANOVA (for multi-factor comparison) followed by a post hoc test.A p-value of <0.01 (**) was considered as significant statistically.

Identification of Germline NHRs in Response to PS-NP Exposure
In C. elegans, there are 33 NHR genes that can be expressed in the germline (Figure 1C).Among these 33 germline NHR genes, only 4 NHR genes could be dysregulated by exposure to PS-NPs (10 µg/L) (Figure 1D).The nhr-12 expression was significantly decreased by exposure to 10 µg/L of PS-NPs, and the expressions of nhr-14, nhr-47, and daf-12 were significantly increased by exposure to 10 µg/L of PS-NPs (Figure 1D).

RNAi of daf-12, nhr-12, nhr-14, and nhr-47 Affected Transgenerational PS-NP Toxicity
With locomotion behavior as the endpoint to reflect the function of motor neurons, the transgenerational PS-NP toxicity to decrease locomotion behavior was inhibited by germline RNAi of daf-12, nhr-14, and nhr-47 (Figure 3).Different from this, the transgenerational PS-NP toxicity to decrease locomotion behavior could be strengthened by germline RNAi of nhr-12 (Figure 3).
In addition to the response to PS-NP at P0-G, we further observed the response of germline NHR genes in the offspring.Increased germline daf-12, nhr-14, and nhr-47 expressions and decreased nhr-12 expression were detected from P0-G to F2-G of PS-NP (1 µg/L)-exposed nematodes and from P0-G to F4-G of PS-NP (10 µg/L)-exposed nematodes
In addition to the response to PS-NP at P0-G, we further observed the response of germline NHR genes in the offspring.Increased germline daf-12, nhr-14, and nhr-47 expressions and decreased nhr-12 expression were detected from P0-G to F2-G of PS-NP (1 µg/L)-exposed nematodes and from P0-G to F4-G of PS-NP (10 µg/L)-exposed nematodes (Figure 2).Thus, after nanoplastic exposure at PEDs at P0-G, these four germline Toxics 2024, 12, 420 9 of 13 NHR genes could exhibit a response across multiple generations.Additionally, the effect of these four germline NHR genes was not restricted at P0-G, and they would exert their effect on the offspring of PS-NP-exposed nematodes once activated or inhibited by PS-NPs at P0-G.
Moreover, using locomotion behavior and reproduction as endpoints, we detected resistance of daf-12(RNAi), nhr-14(RNAi), and nhr-47(RNAi) nematodes and susceptibility of nhr-12(RNAi) to transgenerational PS-NP toxicity (Figures 3 and 4).At P0-G, the resistance of daf-12(RNAi) and nhr-14(RNAi) nematodes to nanoplastic toxicity was observed previously [40,59].These findings demonstrated that, in the germline, the transgenerational activation of DAF-12, NHR-14, and NHR-47 and transgenerational suppression in NHR-12 mediated PS-NP toxicity formation across multiple generations.That is, the transgenerational response of these four germline NHRs functioned as a crucial contributor to the induction of transgenerational nanoplastic toxicity in nematodes.Nevertheless, after P0-G PS-NP (10 µg/L) exposure, activation of DAF-12, NHR-14, and NHR-47 and suppression in NHR-12 were recovered to control levels at F5-G (Figure 2).This implied that some unidentified signals exist to inhibit or block the further transgenerational germline NHR response caused by P0-G PS-NP exposure.The specificity of the RNAi effect to recover locomotive behavior and reproductive capacity using selected NHR genes in the germline (i.e., daf-12, nhr-12, nhr-14, and nhr-47) was not evaluated using other NHR genes whose expression was unaffected by exposure to PS-NP (Figure 1).Therefore, we cannot exclude the possibility that RNAi of some of them may also affect transgenerational PS-NP toxicity.
In C. elegans, insulin receptor DAF-2, Ephrin receptor VAB-1, and Wnt receptor MIG-1 have been proven to regulate nanoplastic toxicity [39,40,62].RNAi of daf-2, vab-1, and mig-1 further induced resistance to transgenerational PS-NP toxicity [39,40,62].Additionally, these four germline NHRs control transgenerational PS-NP toxicity by differentially targeting insulin, Wnt, and/or Ephrin ligands to a certain degree, which further affects the function of their receptors in their offspring.Nevertheless, the expression of germline egl-17 and lag-2 was not affected by RNAi of these four NHR genes under PS-NP exposure conditions (Figure 5).The RNAi of egl-17 and lag-2 also caused resistance to transgenerational PS-NP toxicity [38,61].This implies that certain unidentified upstream regulators exist to activate or inhibit FGF and Notch ligands that mediate transgenerational nanoplastic toxicity.

Figure 1 .
Figure 1.Effect of PS-NP exposure on expression of germline NHR genes.(A) TEM image of PS-NPs before sonication.(B) Raman spectrum of PS-NP.(C) NHR genes expressed in the germline.(D) Effect of PS-NP (10 µg/L) exposure on expressions of germline NHR genes.In total, 30 intact gonads were used for the qRT-PCR assay for each treatment.Data are presented as mean ± standard deviation (SD).** p < 0.01 vs. control.

Figure 1 .
Figure 1.Effect of PS-NP exposure on expression of germline NHR genes.(A) TEM image of PS-NPs before sonication.(B) Raman spectrum of PS-NP.(C) NHR genes expressed in the germline.(D) Effect of PS-NP (10 µg/L) exposure on expressions of germline NHR genes.In total, 30 intact gonads were used for the qRT-PCR assay for each treatment.Data are presented as mean ± standard deviation (SD).** p < 0.01 vs. control.

Author Contributions:
Investigation, Z.L. and Y.W.; conceptualization, methodology and supervision, Q.B. and D.W.All authors have read and agreed to the published version of the manuscript.