Chlorpyrifos

Because of the clonal instability of the PC12 cell line (Fujita et al. 1989 (link)), the experiments were performed on cells that had undergone fewer than five passages, and all studies were repeated several times with different batches of cells. As described previously (Crumpton et al. 2000a (link); Qiao et al. 2003 (link); Song et al. 1998 (link)), PC12 cells (1721-CRL; American Type Culture Collection, Manassas, VA) were seeded onto 100-mm poly-d-lysine-coated plates in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% inactivated horse serum (Sigma Chemical Co., St. Louis, MO), 5% fetal bovine serum (Sigma Chemical Co.), and 50 μg/mL penicillin streptomycin (Invitrogen). Cells were incubated with 7.5% CO₂ at 37°C, and the medium was changed every 2 days. For studies in the undifferentiated state, cells were seeded at varying densities so that, regardless of the total time of incubation, the cells would reach a final confluence of 60–70%. Twenty-four hours after seeding, the medium was changed to include the various test substances: chlorpyrifos (Chem Service, West Chester, PA), diazinon (Chem Service), parathion (Chem Service), physostigmine (Sigma Chemical Co.), dieldrin (Chem Service), or NiCl₂ (Sigma Chemical Co.). Because of their poor water solubility, the pesticides were dissolved in dimethyl sulfoxide (Sigma Chemical Co.), achieving a final concentration of 0.1% in the culture medium; accordingly, all cultures included this vehicle, which had no effect on the PC12 cells (Qiao et al. 2001 (link), 2003 (link); Song et al. 1998 (link)).
For studies in differentiating cells, 3 × 106 cells were seeded; 24 hr later, the medium was changed to include 50 ng/mL 2.5 S murine NGF (Invitrogen), and each culture was examined under a microscope to verify the subsequent outgrowth of neurites. The test agents were added concurrently with the start of NGF treatment.

All experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources 1996 ) as adopted and promulgated by the National Institutes of Health. Timed-pregnant Sprague-Dawley rats (Charles River, Raleigh, NC) were housed in breeding cages, with a 12-hr light/dark cycle and free access to food and water. On the day of birth, all pups were randomized and redistributed to the dams with a litter size of 9–10 to maintain a standard nutritional status; for treatment groups with high pup mortality rates (not used for neuro-chemical analyses), litter sizes were maintained in this range by combining groups of survivors. Chlorpyrifos, diazinon, and parathion (all from Chem Service, West Chester, PA) were dissolved in DMSO to provide consistent absorption (Whitney et al. 1995 (link)) and were injected subcutaneously in a volume of 1 mL/kg once daily on postnatal days (PND) 1–4; control animals received equivalent injections of the DMSO vehicle. For chlorpyrifos, we used daily doses of 1 mg/kg and 5 mg/kg, straddling the threshold for growth retardation and systemic toxicity (Campbell et al. 1997 (link); Whitney et al. 1995 (link)). The lower dose produces neurotoxicity in developing rat brain with only 20% cholinesterase inhibition (Slotkin 1999 (link), 2004 (link); Song et al. 1997 (link); Whitney et al. 1995 (link)), well below the 70% threshold necessary for symptoms of cholinergic hyperstimulation (Clegg and van Gemert 1999 ). This treatment thus resembles the nonsymptomatic exposures reported in pregnant women (De Peyster et al. 1993 (link)) and is within the range of expected fetal and childhood exposures after routine home application or in agricultural communities (Gurunathan et al. 1998 (link); Ostrea et al. 2002 (link)). For diazinon and parathion, prior information on systemic toxicity using this vehicle and route was not available, so we evaluated a wider range of doses: 0.05–5 mg/kg for diazinon and 0.01–5 mg/kg for parathion. As shown in “Results,” just as for chlorpyrifos, the diazinon and parathion doses ranged from those with no discernible effect on growth or viability to those lying above the threshold for overt toxicity.
On PND5, one male and one female pup were selected from each of six litters in each treatment group and were used for neuro-chemical evaluations. Animals were decapitated, the cerebellum was removed, and the brainstem and forebrain were separated by a cut made rostral to the thalamus. Tissues were weighed, flash-frozen in liquid nitrogen, and maintained at −45°C until analysis.

Sorghum [Sorghum bicolor (L.) Moench] inbred line BTx623, which was a parent for several mapping populations in sorghum and the genotype for sequencing the sorghum genome, was used to generate the mutant populations [51 (link)-53 (link)]. The mutagenesis scheme is outlined in Figure 1. BTx623 seeds were obtained from the National Germplasm Resources of USDA-ARS. Initial observations found that the seedlings from the original seeds showed minor variations in height and panicle size, however, no genetic heterogeneity was detected using 10 publicly available SSR markers. To ensure the homogeneity of the seeds used for mutagenesis, the original line was self-fertilized for six generations by single seed descent (SSD). At every generation, one plant that displayed the most typical characteristics of the original BTx623 was selected for propagating to the subsequent generation. Batches of 100 g of dry seed (~3300 seeds) were soaked with agitation (16 hours at 50 rpm on shaker) in 200 ml of tap water containing EMS concentrations ranging from 0.1 to 0.6% (v/v). The treated seeds were thoroughly washed in about 400 ml of tap water for five hours at ambient temperature, changing the wash water every 30 min. The air-dried seeds were planted at 120,000 seeds per hectare. Before anthesis, each panicle was bagged with a 400 weight rain-proof paper pollination bag (Lawson Bags, Northfield, IL) to prevent cross pollination. After bagging, each bag was injected with 5 ml chlorpyrifos (Dow AgroSciences) at 0.5 ml/liter to control corn earworms that could hatch within the bag and destroy the seeds. Sorghum panicles were harvested manually and threshed individually. Each fertile panicle was planted as an M₂ head row. Three panicles were bagged for each row before anthesis and only one fertile plant was used to produce the M₃ seeds. Duplicate leaf samples were collected from the same fertile plant for extracting DNA, and both the leaf samples and the panicle were barcoded. To avoid cross-contamination of leaf samples with dead pollen that could fall onto the leaves during pollen shedding, leaves were thoroughly rinsed with de-ionized water before sampling. The seeds from the barcoded plants were harvested and used to propagate the M₃ generations. In some cases, because a substantial number of lines could not produce sufficient seeds even at the M₃ generation, 10 panicles were bagged for each M₃ head row and pooled as M₄ seeds. The M₄ seeds will be distributed to the sorghum research community for forward and/or reverse genetic studies.

It was mainly the end of 3rd instar to the beginning of 4th instar larvae that were chosen to monitor the insecticide resistance. And we adopted the impregnation technique recommended by WHO (refer to GB/T26347-2010) to determine LC50 and calculate RR (17 (link)). In the meantime, the WHO recommended contact tube method (refer to GB/T26347-2010) was used to monitor the resistance of adult mosquitoes to insecticides, and thereby the mortality of adult mosquitoes at diagnostic dose was determined. We selected 11 different types of pesticides, namely: 0.2% bendiocarb, 0.2% fenitrothion, 0.03% deltamethrin, 0.04% permethrin, 0.5% propoxur, 0.5% malathion, 0.08% beta-cypermethrin, 0.07% lambda-cyhalothrin, 2% chlorpyrifos, 0.4% beta-cypermethrin. See Supplementary Text S1, p. 3–6 for specific experimental methods.

In the present study, 60 samples of rice, including 15 samples of each Pakistani, Indian, Thai, and Iranian, were collected from March 2018 to March 2019 and sent to the reference laboratory. The samples selected among the food commodities were submitted to the other laboratories by the food importers, local store owners, or the farmers in Bushehr, Iran. Their ash, moisture, and mold content, mycotoxins (AFB1, OTA, ZEN, and DON), the concentration of PTEs (Pb, As, Cd, and Ni), and pesticides residues (chlorothalonil, diazinon, chlorpyrifos, trichlorfon, and fenitrothion) were examined and compared with the acceptable ranges of Institute of Standards and Industrial Research of Iran (ISIRI). According to the concluded data, a health risk assessment (carcinogenic and non-carcinogenic) was performed [11 (link),54 ].

GST inhibition analysis was performed using the CDNB/GSH system, in the presence or absence of 25 μM pesticide diluted in acetone (fenvalerate, atrazine, carbaryl, malathion, alachlor, carbofuran, permethrin, pirimicarb, endosulfan, zoxium zoxamide, metalaxyl, ksesoxim-methyl, boscalid, iprodione, carbedazim, thiachloprid, picoxystrobin, clothianidin, chlorpyrifos). During the course of the assay (30–60 s), no measurable pesticide/GSH conjugation was observed. The IC50 values were determined by fitting the concentration-response data to Equation (4): $$\% \mathrm{inhibition}=\frac{100}{1+\left(\frac{IC50}{\left[\mathrm{I}\right]}\right)}$$
where [I] is the pesticide concentration. The IC50 values were determined using the program GraphPad Prism version 7.00.