Perfluoroalkylated Substance Effects in <i>Xenopus laevis</i> A6 Kidney Epithelial Cells Determined by ATR-FTIR Spectroscopy and Chemometric Analysis

The effects of four perfluoroalkylated substances (PFASs), namely, perfluorobutanesulfonate (PFBS), perfluorooctanoic acid (PFOA), perfluorooctanesulfonate (PFOS), and perfluorononanoic acid (PFNA) were assessed in <i>Xenopus laevis</i> A6 kidney epithelial cells by attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy and chemometric analysis. Principal component analysis–linear discriminant analysis (PCA-LDA) was used to visualize wavenumber-related alterations and ANOVA-simultaneous component analysis (ASCA) allowed data processing considering the underlying experimental design. Both analyses evidenced a higher impact of low-dose PFAS-treatments (10^–9 M) on A6 cells forming monolayers, while there was a larger influence of high-dose PFAS-treatments (10^–5 M) on A6 cells differentiated into dome structures. The observed dose–response PFAS-induced effects were to some extent related to their cytotoxicity: the EC₅₀-values of most influential PFAS-treatments increased (PFOS < PFNA < PFOA ≪ PFBS), and higher-doses of these chemicals induced a larger impact. Major spectral alterations were mainly attributed to DNA/RNA, secondary protein structure, lipids, and fatty acids. Finally, PFOS and PFOA caused a decrease in A6 cell numbers compared to controls, whereas PFBS and PFNA did not significantly change cell population levels. Overall, this work highlights the ability of PFASs to alter A6 cells, whether forming monolayers or differentiated into dome structures, and the potential of PFOS and PFOA to induce cell death

Light micrograph of endothelium.

<p>Endothelial cell treated with 1/10 silver-intensified gold nanoparticles (<b>A</b>). Scale bar  = 10 µm. SERS-enhanced spectral map of the same region taken at a wavenumber of 611 cm⁻¹ (<b>B</b>).</p

Mean spectra (light blue) of the 1/10 silver-intensified gold samples.

<p>Showing the spectral peaks which demonstrate the highest levels of SERS-enhancement. The pack with the highest level of enhancement is at 611 cm⁻¹.</p

Mean spectra from the five different classes of samples; the spectra have been baseline corrected.

<p>The different treatments consist of: 1/10 silver-intensified gold (1/10 Au with Ag); 1/100 silver-intensified gold (1/100 Au with Ag); 1/10 gold without silver-intensification (1/10 Au only); silver-intensification solution without gold (Ag solution only) and PBS solution (PBS treatment only).</p

Three-D cluster plot of four different sample classes with 95% confidence ellipsoids.

<p>The classes consist of: 1/10 silver-intensified gold (1/10 Au with Ag); 1/100 silver-intensified gold (1/100 Au with Ag); 1/10 gold without silver-intensification (1/10 Au only) and PBS solution (PBS treatment only).</p

Histogram showing the range of diameters of silver-intensified cationic gold nanoparticles on the corneal endothelial cell surface.

<p>Histogram showing the range of diameters of silver-intensified cationic gold nanoparticles on the corneal endothelial cell surface.</p

The PCA cluster vectors with the PBS-treated control samples set as the origin.

<p>The green plot consists of 1/10 gold only samples deviates only slightly from the origin, but both plots from the 1/100 silver-intensified gold (red) and the 1/10 silver-intensified gold (light blue) samples are surprisingly similar over the 1000–600 cm⁻¹ spectral regions. Both plots show maximum deflection from the origin around the 611 cm⁻¹ spectral region.</p

Tentative peak assignations for the most enhanced peaks of the spectra.

<p>Tentative peak assignations for the most enhanced peaks of the spectra.</p

Scanning electron micrographs of the apical surface of endothelial cells.

<p>Corneal endothelium (<b>A, B</b>). Endothelium with 1/10 silver-intensified cationic gold (<b>C</b>). Endothelium with 1/10 silver-intensified cationic gold at a high magnification (<b>D</b>). (<b>A</b>) Scale bar  = 5 µm; (<b>B</b>) Scale bar  = 5 µm; (<b>C</b>) Scale bar  = 10 µm; and (<b>D</b>) Scale bar  = 5 µm. Insert ×5.</p

A schematic diagram of the silver-intensified cationic gold nanoparticle on the surface of the cell.

<p>The 15 nm cationic gold particle is shown interacting with the cell glycocalyx molecules.</p