For cell culture, Gibco (Carlsbad, CA) supplied Dulbecco's modified Eagle minimum essential/Ham's F-12 (DMEM/F12) plus Glutamax media fetal bovine serum (FBS), penicillin G/streptomycin mix, and enzyme-free PBS-based cell dissociation buffer. Sigma-Aldrich (St. Louis, MO) provided the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reagent and the trypan blue stain. We used commercial wettable powder (WP) mancozeb 80% (Manzate® 80, UPL Ltd., Mumbai, India).

The human neuroblastoma cell line SH-SY5Y was a generous donation of Dr. Javier Saez-Castresana (University of Navarra, Spain). Undifferentiated SH-SY5Y cells (immature state) were cultured to confluence (> 80 %) in growth medium [DMEM/F12 (1:1) medium GlutaMAX, 10% v/v fetal bovine serum (FBS), 100 U/mL penicillin G, and 100 µg/mL streptomycin, and kept at 37 °C in a humidified atmosphere of 95 % oxygen and 5 % carbon dioxide. The medium was replaced every 2 to 3 days. After the culture had grown, the cells were harvested using an enzyme-free phosphate-buffered saline (PBS)-based cell dissociation solution.

We chose undifferentiated cells since retinoic acid (RA) differentiation (Cheung et al., 2009[8]) significantly changes SH-SY5Y cells frequently making them more resilient and resistant to Parkinsonian specific neurotoxins like 6-OHDA (6-hydroxydopamine) or overall oxidative stress because of improved defensive systems (Schneider et al., 2011[62]). Although not exactly resembling mature neurons, undifferentiated SH-SY5Y cells have a star-like shape with short neurites that provide a useful expandable model for general in vitro neurotoxicity research. The use of RA differentiated cells is more relevant when examining the neurobiology of Alzheimer's diseases (de Medeiros et al., 2019[15]).

The concentrations of sheer fungicide were set up according to the percentage of the active ingredient specified by the manufacturer (80 % w/v). Mancozeb or MCZ (zinc;manganese(2+);N-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate; chemical formula: C₄₀H₆₀Mn₉N₂₀S₄₀Zn (M.W.= 2664 g/mol); CAS Ref.#: 8018-01-7) were freshly prepared in a stock solution of sterile water just before treating the cells and then serial dilutions were directly made in the growth medium (covered concentration range: 0.1 to 16 µM). Because of the poor solubility of MCZ in water, Manzate® contains the active ingredient in a wettable powder formulation. Thanks to the combination of wetting excipients, the fungicide quickly and evenly disperses when added to water. As a result, the serial dilutions of MCZ were entirely clear, even though the stock solution was slightly cloudy.

The first measure of cell viability was the reduction of the thiazolyl blue tetrazolium bromide, or MTT dye, to formazan (Mosmann, 1983[50]). In 96-well microplates, cells were seeded at a density of 6 x 10⁴ cells per well (200 µL) for MTT assays. After a 48-hour incubation period at 37 ºC, a stock of MTT salt (5 mg/mL in PBS) was diluted tenfold in the culture medium. The most active mitochondrial reductase enzymes of living cells converted the yellow MTT salt to purple formazan after a 2-hour incubation at 37 °C, enough time for formazan crystals to form in SH-SY5Y cells (Ramirez-Cando et al., 2024[57]). After aspirating the medium, the formazan crystals were solved in 100 µL of pure DMSO. Using a microplate reader (Rayto RT-2100C Microplate Reader, Rayto Life and Analytical Sciences Co. Ltd., China), the optical density (O.D.) was measured at 560 nm with the appropriate DMSO serving as a blank. The absorbance of the control (non-treated) wells was set to 100 to standardize the absorbance values of the treated wells. A well was treated with 10 % DMSO to serve as a positive cell death control. Eight duplicates of each treatment condition were conducted for every independent experiment.

Further information on cell viability was obtained by measuring the percentage of viable cells that remained intact after applying the vital-dye trypan blue (TB) exclusion test (Strober, 2015[65]). Cells were seeded at a density of 5 x 10³ cells per well (400 µL) in 24-well plates and MCZ treatments (0, 0.1, 0.25, 0.5, 1, and 2 µM) were applied 48 hours later. Analysis of cell viability with exclusion dye was done 24 and 72 hours after treatment. Under the light of an inverted microscope (Motic AE31E, Motic China Group Co., Hong Kong, China), TB was dissolved directly into the culture medium (0.02 % v/v) to assess the fraction of dead (TB-tangible) cells versus total cell counts at 200-fold magnification. Pictures were taken with a digital camera connected to MotiConnect software (Motic China Group Co., Hong Kong, China). Cell counts were performed using the free ImageJ software (http://imagej.nih.gov/ij) on a minimum of 500 cells in several fields (ten replicates per treatment condition).

Endpoints for screening neurotoxic effects also included the impairment of important developmental processes such as neurite outgrowth inhibition (Lee et al., 2022[37]). Up to 400 µL of culture medium was used to seed cells at a density of 5 x 10³ cells/well in 24-well plates (4 wells per concentration). After 48 hours, MCZ (0, 0.1, 0.2, 0.5, 1 and 2 µM) was administered and the 24 and 72-hour treatment´s visual appearance of the cells was examined in phase-contrast images on ImageJ. Each well was the subject of digital micrographs that were saved on a PC. Neurite-like structures or neurites were measured and examined in ten replicates, with at least 500 cells in each treatment condition. The number of cells displaying neurites that were twice as long as the width of the cell body was used to measure neurite outgrowth. To rule out artifacts from varying cell counts, the averaged neurite length per cell was assessed in relation to control (untreated or 0µM) (Lee et al., 2022[37]).

Reactive oxygen species (ROS) were measured in treated cells using the probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA), which is an indirect and non-specific biomarker of oxidative stress (Wang et al., 1999[69]), as our laboratory previously described (Ramirez-Cando et al., 2023[56]; 2024[57]). In brief, cells were seeded onto coverslips placed in Petri dishes at a density of 6 x 10⁶ cells/ Petri dish with 8 mL of media. These cells were then allowed to proliferate for 48 hours, and finally the fungicide was applied for a 24-hour period. Following treatment, the cells were incubated in a growth medium with 10µM of DCF-DA for 30 minutes. They were then fixed for 10 minutes in ice-cold PBS containing 3.7 % formaldehyde, rinsed three times with PBS, allowed to dry on air, and mounted on glass slides using Entellan^TM rapid mounting medium (Merck-Millipore, Germany). Cells were photographed in six fields per experimental condition at a magnification of 200x with the assistance of a Leica DM4000 B imaging fluorescence microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany). Utilizing excitation and emission filters set at 485 ± 10 nm and 530 ± 12.5 nm, respectively, fluorescence intensity was calculated with the use of a blue filter (450-490 nm) and the Leica Application Suite X software for research. Finally, ImageJ was used to evaluate the microphotographs (four images per treatment condition).

The corresponding calculations were performed following the cell counting to achieve an initial cell density of 2.5 x 10⁴ cells/mL. Cells were cultured on coverslips (22 x 22 mm with a thickness of 0.13-0.17 mm) placed in four glass Petri dishes (ϕ = 30 mm). The Petri dishes were allocated into one control Petri dish and three Petri dishes for different MCZ concentrations. After placing the coverslips inside the Petri dishes, 25 mL of the DMEM solution with cells was poured into each Petri dish. The Petri dishes were incubated at 37 °C in a humidified O₂ (95 %) and CO₂ (5 %) atmosphere for 24 hours. Cells were then treated with MCZ at 0.1, 0.25, and 1.0 µM for 24 and 48 hours, since pilot studies demonstrated that this concentration range worked out better for morphological analysis. Samples were incubated in 3.7 % formaldehyde/PBS solution at room temperature for 20 minutes to fix cells and subsequently washed in distilled water for 3 seconds.

The Atomic Force Microscope (AFM, NX7 model, Park System) was equipped with an integrated On-axis Optic featuring a Vision Camera and an objective lens with 10x magnification for biomechanical analysis. An NSC36 Type A cantilever (Park System, Suwon, Korea) was utilized for contact analysis according to manufacturer's recommendation for contact force exceeding 0.2N/m. The AFM setting was calibrated following the manufacturer's specifications to ensure accurate analysis of the samples. Data from the AFM study of the cell's mechanical properties were collected using the XEI software (TEST Park Systems Corp, Suwon, Korea). Among the several AFM measurements, the sample analysis focused primarily on Young's modulus.

The Rstudio free software (https://posit.co/download/rstudio-desktop/) was used for statistical analysis (Supplementary material includes R codes). To measure neurite outgrowth and % of dead cells (vita dye exclusion test) replicates were conducted in four wells per MCZ concentration, results were expressed as median ± IQR to provide a clearer view of data distribution and variability capturing more information (based on boxplot), and later analyzed through the one-way ANOVA combined with the Dunnet´s test as the post-hoc analysis.

In the case of MTT data, data were normalized with respect to control, and a scatter graph was plotted from which a concentration-response curve was adjusted by a four-parameter log-logistic function utilizing the 'drm()' function within the RStudio software package 'drc()' for concentration-response analysis. The logistic function applied for this analysis is given by the expression:

where b represents the relative slope around e, c the lower limit, d the upper limit, and e the EC₅₀.

The relative intensity of ROS was plotted on a scatter graph. Data underwent normalization against control values. A 4^th order polynomial concentration-response curve was selected as the best fit as follows:

Lastly, using Monte Carlo simulations, an ANOVA was performed on the biomechanical characteristics of the cells acquired from a single AFM experiment and subsequent repeated results (up to 500 per treatment condition). Dunnett's test was performed as the post-hoc test. P < 0.05 was regarded as statistically significant. for all data analysis.

Ethical approval was not required to use SH-SY5Y cells because they are a commercially available, established cell line, not primary human cells taken directly from a patient. In other words, no human subjects were involved, since SH-SY5Y cells are considered a pre-existing source for research.

The interest was to know whether acute (24 hours) to sub-chronic (72 hours) exposure to increasing concentrations (0.5 to 16 µM) of MCZ fungicide altered cell viability. Figure 2(Fig. 2) represents the concentration-response curves, plotted as the percentage of the untreated control, of the MTT viability test at different treatment periods. The ANOVAs of the MTT test results revealed a significant treatment effect at 24 hours (F(5,42) = 53.90, P < 0.001) and 72 hours (F(5,42) = 100.30, P < 0.001)). The adjusted R-squared values were set above 0.7 in both cases, which indicated a good correlation between the model predictions and real values.

Because the MTT assay is no longer acceptable as a single measure of cell viability, other parameters were required to determine whether MCZ was truly causing cell death. To corroborate MTT data, living cell cultures were examined for the presence of TB-tangible (dead) cells (Figures 3(Fig. 3) and 4(Fig. 4)). The ratio of dead cells caused by the fungicide concentration-dependently increased after 24 (F(5,60) = 117.2, P < 0.001) and 72 hours (F(5,60) = 357.1, P < 0.001) of treatment. AMCZ concentrations over 1µM and 0.5µM resulted in cell death rates of almost 100% following 24- and 72-hour exposure.

Figure 5(Fig. 5) shows the concentration-dependent evolution of neurite outgrowth after 24-hour exposure to MCZ. As shown in Figure 5(Fig. 5), there was a significant treatment effect in the average neurite length per cell (F(5,60) = 120.4, P < 0 .001); as well as in the number of neurites per cell (F(5,60) = 81.9, P < 0.001). The characteristic polygonal-shaped soma of SH-SY5Y cells gradually transformed into a more circular shape, while concentration-dependently decreasing in the number and length of neurites per cell.

The DCHF-DA reagent exhibited a noticeable increase in fluorescence in ROS generating cells (Figure 6(Fig. 6)). The results obtained after 24-hour treatment with MCZ suggested that the fungicide prompted a significant increase in ROS levels compared to control (as no treatment). ROS level reached a maximum at 4 µM of MCZ to posteriorly decrease due to the massive cell death at higher concentrations. The concentration-response curve of ROS generation by MCZ treatment in SH-SY5Y cells moderately followed a biphasic model given by a 4^th-grade polynomial curve in a range from 1 to 10 µM (Figure 7(Fig. 7)). The adjusted R-squared value was set to 0.754 (F(5,24) = 9.89, P = 0.3272).

Young's modulus was used to evaluate the biomechanical response of cells, which depends on a combination of intrinsic cellular factors (like cytoskeleton, cellular shape, and molecular motors) and extrinsic factors (such as external forces, matrix stiffness, and biochemical cues), to different concentrations of MCZ. Statistical analysis via ANOVA of 500 Monte Carlo-outcomes mimicking one real-life AFM experiment revealed a significant treatment effect on the modulus of elasticity of both cell nuclei (24-hour MCZ: F(3,1996) = 175.15; P < 0.001; 48-hour MCZ: F(3,1996) = 876.06, P< 0.001) and axons (24-hour MCZ: F(3,1966) = 477.18; P < 0.001; 48-hour MCZ: F(3,1996) = 1643.21, P < 0.001) (Figure 8(Fig. 8)). Subsequent Dunnett's test pinpointed robust differences across treatments, with statistically significant deviations from untreated (control) cells. As shown in Figure 8(Fig. 8), the increase of Young's modulus was the largest after exposure to 0.25 µM of MCZ in both the axon and the nucleus, regardless of treatment time. This phenomenon suggests a possible activation of cellular response mechanisms that increased stiffness. This concentration-dependent behavior suggests that, whereas low MCZ concentrations fortify the cellular structure, higher ones undermine it (see Figure 9(Fig. 9)). More interestingly, the pattern of elastic modulus changes in the nucleus after 24-hour MCZ treatment (Figure 8A(Fig. 8)) was like the axonal response to 48-hour MCZ treatment (Figure 8C(Fig. 8)), thus highlighting nuanced, concentration-dependent effects on cell mechanics shifting from the nucleus to cell body throughout time. After 48-hour treatment, all tested concentrations of MCZ significantly influenced the nucleus's mechanical properties, underscoring the pronounced impact on cellular rigidity (Figure 8C(Fig. 8)), while in axons, only the treatment with 0.25 µM of MCZ produced a robust stiffness increase (Figure 8D(Fig. 8)).

See also Table 1(Tab. 1) and 2(Tab. 2).

We are grateful to the Programa de las Naciones Unidas para el Desarrollo (PENUD) of the Ecuadorian Government. The authors specially thank Prof. Lola de Lima and Prof. Carlos Reinoso for their technical support with the chemical characterization of MCZ and with the AFM respectively.

This work was supported by the Secretaria Nacional, Ciencia, Tecnología e Innovación (SENESCYT) grant of the Ecuadorian Government (Ref. PIC-18-INE-YACHAY-002) to Santiago J. Ballaz and Lenin J. Ramirez-Cando.

None.

LJR-C and SJB: Conceived and design the study, analyzed and interpreted the data, and contributed with reagents, materials, analysis tools or data; EGC-B: Performed the in vitro experiments, and wrote the original draft; JC and RAO: Performed AFM experiments and analysis. All the authors wrote and approved the final version of the manuscript.

The datasets generated analyzed during the current study are available from the corresponding author on reasonable request.

The authors declare that generative AI was not used during the writing and the research process. The authors are solely responsible for the finished manuscript.