A Novel Determination of the Relationship Between Exposure to Industrial Toxicant HFPO-DA and pqm-1-related Aging in C. elegans

Hexauoropropylene oxide dimer acid (HFPO-DA, ammonium salt with trade name “GenX”) is an industrial toxicant that has recently been detected in the environment [1]. However, HFPO-DA’s potential aging-related effects on organisms of higher trophic levels, including worms and humans, have not been extensively explored. The purpose of this study is to quantify inuences on C. elegans (free-living nematode) lifespan by HFPO-DA exposure, specically via ingestion of food to simulate the mechanisms of toxicant exposure, through lower trophic-level organisms, commonly found in nature. C. elegans N2 (wild-type) samples were prepared with a uracil-based medium and E. coli OP50 (food source) at room temperature; C. elegans in the experimental, treated sample was fed E. coli OP50 incubated with 280 ng/L HFPO-DA. The target gene pqm-1 was selected due to its role in an evolutionarily conserved insulin signaling pathway and in promoting development. Molecular biology laboratory techniques (RNA extraction, qRT-PCR, uorescence tagging, etc.) were used to quantify pqm-1 expression to yield four technical replicates for each sample. The data was analyzed through null hypothesis t-tests, heatmaps, protein interactions, and gene homology tools. HFPO-DA exposure through E. coli caused a statistically insignicant (0.811-fold) change in pqm-1-related aging in C. elegans. Future work includes investigating the effects of different levels of HFPO-DA exposure on C. elegans aging. HFPO-DA C. elegans Longitudinal studies conducted to explore the effects of direct HFPO-DA exposure on overall C. elegans development across the lifespan. Another possibility be to repeat the experiment with different means of HFPO-DA exposure or in a higher order organism. This study’s results may prove useful in future research on aging with other similar toxicants.


Introduction
Through this study, we sought to quantify the potential effects of HFPO-DA exposure on pqm-1 gene expression in C. elegans (a free-living, transparent nematode) in order to determine whether such chemical stress could in uence aging processes in C. elegans. HFPO-DA is an ammonium salt with the trade name "GenX" and is a potent toxicant used in manufacturing industries that has recently been detected in global river water ecosystems [1].
C. elegans was used as a model organism for various reasons. Its full genome has been sequenced, and C. elegans shares many of the same genes and biological pathways with humans [2]. C. elegans is relatively easy to grow in a short amount of time, producing more than 1,000 eggs daily and having a life cycle of only two weeks [2]. C. elegans is also inexpensive to grow and only requires cultures of bacteria and a nutrient source.
The target gene pqm-1 was selected for several reasons. In C. elegans, the PQM-1 protein (encoded by pqm-1) is an important nuclear transcription activator that binds to the DAF-16 Associated Element (DAE) upstream of development (Class II) genes [3,4]. In nature, reduced insulin/IGF-1-like signaling (IIS) leads to PQM-1 remaining in the cytoplasm and thus downregulation of Class II genes, increasing lifespan [3].
IIS is also conserved across many species, including mammals; therefore, the implications of IIS in C. elegans may re ect aging outcomes in humans.
Regarding the selection of the reference gene, previous studies have shown that tba-1 (alpha tubulin subunit) expression is predominantly localized to developing germ cells and nervous system to maintain embryonic viability [5]. Therefore, experimental factors are unlikely to signi cantly affect tba-1 expression in mature adult C. elegans, in which tba-1 is only expressed at the minimum level for survival. A previous toxicology study also showed that tba-1 expression in C. elegans was stable with exposure to nano-copper oxide (industrial toxicant similar to HFPO-DA), suggesting its reliability as a reference gene when measuring gene expression in C. elegans in response to HFPO-DA exposure [6].
It was hypothesized that the difference in relative pqm-1 gene expression between the experimental and control groups will not be statistically signi cant and that HFPO-DA will induce a statistically signi cant difference in relative pqm-1 gene expression.

Materials And Methods
Animal Care and Maintenance C. elegans N2 (wild type) was fed E. coli OP50, maintained at a temperature of 23℃, and kept in a uracil-based growth medium. The experimental group of C. elegans was fed E. coli incubated with 280 ng/L HFPO-DA, simulating how C. elegans and humans may be exposed to HFPO-DA in nature via lower trophic-level organisms.

RNA Extraction and Quanti cation
RNA extraction was commenced by using the M9 buffer to wash C. elegans off of the NGM plates, transferring the worms into conical tubes with a pipette, centrifuging C. elegans to the bottom of the tubes, and removing the liquid supernatant. C. elegans cells were then disrupted with Buffer RLT Plus. Four beads were added to each sample tube, and all the tubes were loaded to the Bead Mill for homogenization. Lysis and puri cation were conducted through the RNeasy Plus Mini Kit. The RNeasy Plus Mini Kit handbook by Qiagen was followed to ensure adherence to recommended procedural guidelines. NanoDrop 2000 was used to assess the quality and quantity of the extracted RNA; if needed, the RNA samples were exposed to DNase I and protease for further RNA puri cation.
qRT-PCR was used to produce quanti able gene expression results. For pqm-1, reverse transcriptase and the DNA forward and reverse primers CACCGCCGACTACTATGCC and TCGGCTGCATTAGGTTTACTGTG, respectively, were added to the RNA solutions. Taq polymerase, a heatstable DNA polymerase found in T. aquaticus, was also added Solutions the solutions. The solutions were then subjected to multiple heat cycles to separate DNA strands, allowing Taq polymerase to bind to primers and synthesize complementary DNA strands. The qRT-PCR was designed such that the positive control would be the samples with the reference tba-1 gene, the negative control would be samples missing a key cellular component, and the repeated measurements for each sample would serve as technical replicates.
The iTaq™ Universal SYBR® Green One-Step Kit was used for quanti cation of RNA products, as it contains SYBR Green I (a chemical that binds to the minor grooves in double stranded DNA and emits green light under blue light). ROX (emits red light) was used to provide a baseline re ectance level and normalize the results.

Data Analysis
The Google Sheets software was used to calculate data based on the threshold number of cycles (Ct) (i.e., the number of cycles needed to achieve a statistically signi cant signal increase relative to the background level). The ΔCt (difference between Ct values of target and reference genes within a sample), average ΔCt's for each condition, ΔΔCt (difference between average ΔCt's of experimental and control conditions), and 2 −ΔΔCt (fold relationship between target RNA transcripts of experimental and control conditions) values were calculated. A two-tailed t-test was conducted for the ΔCt values to assess the signi cance of the results (with a p-value threshold of 0.05).
The JMP Pro software was used to create heatmaps, a technique used to visualize gene expression data. The STRING Database was used to construct gene-to-gene and protein networks based on the pqm-1 gene and was also used to identify interactions and other pathways of interest. HomoloGene was used for gene homology analysis.

Results
The heatmap (refer to Fig. 1) shows a slight decrease in relative pqm-1 expression in the experimental condition compared to in the control condition. After using Google Sheets for data analysis (refer to Appendix A), it was also revealed that the average ΔCt values for the experimental (HFPO-DA exposure) and control conditions were 5.694 and 5.392, respectively. The ΔΔCt value was 0.302, and the fold relationship (2 −ΔΔCt ) value from the averaged ΔCt values was 0.811. The p-value from the aforementioned two-tailed null-hypothesis t-test was 0.770.
Pqm-1 was found to be only exclusive to C. elegans; thus, the gene homology graph compares pqm-1 with F10B5.3, another gene in C. elegans. Their query coverage and percent identity were 59% and 74.26%, respectively (refer to Fig. 3).

Discussion And Conclusion
As shown in the heatmap (Fig. 1), relative pqm-1 expression was slightly lower in C. elegans exposed to HFPO-DA than in the control group by a fold change of 0.811. However, we fail to reject the null hypothesis that the difference in relative pqm-1 gene expression between the experimental and control groups was not statistically signi cant (p = 0.770 > 0.05, two-tailed). It can thus be concluded that the results do not indicate a signi cant in uence of HFPO-DA exposure, via E. coli OP50, on aging in C. elegans speci cally in relation to pqm-1.
The STRING analysis suggests that PQM-1 is associated with not only members of the IIS pathway, but also other proteins that are involved in development, growth, and maturation of C. elegans. This underscores the integral role of pqm-1 expression in contributing to Class II (development) gene expression and thus aging in C. elegans. Although humans and mammals share IIS pathways as C. elegans, no direct homologues for pqm-1 were found outside of the C. elegans species. The pqm-1 expression patterns analyzed in this study may not directly apply to humans; however, the STRING analysis strongly suggests that relationships between HFPO-DA exposure and aging in C. elegans and in humans share commonalities on an IIS pathway level. Thus, future research may aim to con rm the existence of such a similar relationship between HFPO-DA exposure and human aging on a developmental pathway level.
This study's limitations include the number of technical replicates that were used for each condition. No biological replicates were analyzed; thus aging variability across the C. elegans species may not have been accurately re ected in the results. Due to time constraints, RNeasy was used for RNA extraction from the samples; however, the TRIzol protocol is more accurate and often results in higher yields.
Other future directions include exposing E. coli to different amounts of HFPO-DA and determining a potential threshold for HFPO-DA concentration and abnormal C. elegans aging. Longitudinal studies could also be conducted to explore the effects of direct HFPO-DA exposure on overall C. elegans development across the lifespan. Another possibility may be to repeat the experiment with different means of HFPO-DA exposure or in a higher order organism. This study's results may prove useful in future research on aging with other similar toxicants.

Declarations
Ethics Approval and Consent to Participate All experimental subjects were used in adherence to approved ethical standards.

Consent for Publication
Not applicable.
Availability of data and material Not applicable. All relevant data is included in the body of the manuscript. Data analysis of qRT-PCR results generated through Google Sheets from four and three technical replicates of experimental and control groups, respectively. Blue boxes, yellow boxes, and white boxes correspond to data pertaining to solely the experimental group, solely the control group, and both the experimental and control groups, respectively. Figure 1 Heatmap generated through JMP Pro showing relative pqm-1 gene expression (circled in yellow), as normalized to tba-1 gene expression, in C.

Figures
elegans. As shown in the legend on the right, box color is indicative of the magnitude of relative gene expression, ranging from red (higher expression) to green (lower expression). STRING protein interaction network centered on PQM-1 (10 network edges in total).