Organophosphates

The 8 × 15 K Agilent microarray design chip (A-MEXP-2196) (Mitchell et al., 2012 (link)) was used to detect the set of genes differentially expressed between the resistant population of Vallée du Kou and a susceptible laboratory colony Ngoussou. Each array contains 60 mer probes designed from all 13,000 transcripts of the Ensembl P3.5 A. gambiae genome annotation, plus additional probes for the detoxification genes from a previous microarray design, the ‘detox chip’, used previously to explore metabolic resistance in A. gambiae (David et al., 2005 (link)).
RNA was extracted from three batches of ten females 3 day old A. gambiae s.s. from a F₁ sample from the VK6 population (nonexposed to insecticide but known to be resistant to multiple insecticides from bioassays results of VK) and from the Ngoussou strain which is fully susceptible to pyrethroids, DDT, carbamates and organophosphate with 100% mortality observed 24 h after 1 h exposure. RNA was isolated using the Picopure RNA isolation kit (Arcturus). The quantity and quality of extracted RNA were assessed using NanoDrop ND1000 spectrophotometer (Thermo Fisher) and Bioanalyzer (Agilent, Santa Clara, CA, USA) respectively. Complementary RNA (cRNA) of each sample was amplified using the Agilent Quick Amp labeling Kit (two-color) following the manufacturer's protocol. cRNA from the VK6 samples were labeled with cy3 dye while the cRNA from the susceptible strain Ngoussou was labeled with the cy5 dye. cRNA quantity and quality were checked before labeling using the NanoDrop and Bioanalyzer. Labeled cRNAs were hybridized to the arrays for 17 h at 65 °C according to the manufacturer's protocol. Five hybridizations between cRNA from VK and Ngoussou were carried out by swapping the biological replicates (Fig. S6).
Microarray data were analyzed using Genespring GX 11.0 software. In order to identify differentially expressed genes, a cut-off of 2-fold-change and a statistical significance of P < 0.05 and P < 0.01 were applied. The P values were generated from a t-test against zero using the data from the five hybridizations (Fig. S6) after a multiple testing correction using the Benjamin–Hochberg test. Enrichment analysis was carried out using the Blast2Go software (Conesa et al., 2005; Gotz et al., 2008 ) to detect the major Gene Ontology (GO) terms over-represented among the set of probes up or under-transcribed in the VK population in comparison to the entire microarray chip using a Fisher's test for statistical significance. The microarray data from this study were submitted to Array Express, accession number: E-MTAB-1083.

Occupational exposure to pesticides in the month prior to the interview was characterized in number of days. Several forms of exposure were investigated in relation to the most recent contact with pesticides, such as applying herbicides or insecticides/fungicides, mixing/preparing mixture, cleaning equipment, washing contaminated clothing, having contact in transportation and storage, through wet clothing, reentering into the plantation without protection after application, and spilling on the body or on clothing. We also characterized the sum of the forms of pesticide exposure.
In order to gather information on what types of chemicals were used on the farms in the last 30 days, plastic cards were prepared with pictures of the main pesticides used in tobacco farming in the region, totaling 56 commercial formulations and more than 22 products (referred to as others). The date of most recent contact was also collected for each product reported. The chemical groups analyzed were organophosphorus compounds (organophosphates), neonicotinoids, pyrethroids, carbamates, benzoylurea, fipronil, clomazone, glyphosate, sulfentrazone, triazines, dithiocarbamates, metalaxyl, iprodione and copper compounds. The sum of the types of chemicals used was taken as an indicator of multi-chemical exposure.
The personal protective equipment (PPE) investigated were boots, masks and gloves to protect against chemicals, protective clothing, hat/headgear, characterizing the number of days they were used during work in the last 30 days. We also examined the sum of the PPE that each worker reported always using.

The effects of the compounds 1–5 and resveratrol used as the reference on mitochondria were evaluated as for their capacity to restore mitochondrial function after organophosphate pesticide (glyphosate and chlorpyrifos) treatment. Briefly, BEAS-2B cells (3 × 10⁴ cells/well in a 96-well) were treated with either glyphosate or chlorpyrifos and at two concentration levels (10 and 100 µM) in the presence or absence of the test compounds (50 µM). After 24 h of incubation, the changes in mitochondrial potential and mitochondrial reducing activity (MRA) were evaluated. The changes in the mitochondrial potential were detected by 5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolylcarbocyanine iodide/chloride (JC-1), a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by fluorescence emission shift from red (∼590 nm) to green (∼525 nm). MRA was assessed by the resazurin assay. To this aim, cells were incubated with resazurin (6 µM) in the presence and absence of the compounds (50 µM) and the fluorescence intensity evaluated over time (0–240 min), in a plate reader (Infinite F200 PRO, Sunrise, Tecan, Männedorf, Swiss), and the results were normalized to the total protein using the Bradford assay (Sigma) [39 (link)]. Each experimental step was carried out in six replicates.

Cortical organoids were treated on alternating days between days 61 to 70 with quaternary ammonium and phosphonium compounds or organophosphate flame retardants at their approximate IC₉₀ and IC₇₅ concentrations respectively. Organoids were harvested on day 70, washed in PBS, and fixed overnight with ice-cold 4% Paraformaldehyde (Electron Microscopy Sciences, HP1–100Kit). On the following day organoids were washed with PBS and cryoprotected using a 30% sucrose solution. Organoids were then embedded in OCT and sectioned at 15 μM. Slides were washed with PBS and incubated overnight with anti-SOX10 (1:200, R&D, AF2864) and anti-APC CC1 (1:200, Millipore, MABC200), followed by labeling with Alexa Fluor-conjugated secondary antibodies (2 μg/mL, Thermo Fisher). Slides were imaged at 10x magnification using a Hamamatsu Nanozoomer S60. Quantification of positive cells was performed using QuPath software (https://qupath.github.io/)^{52 (link)}.