Human survey

A survey was conducted among the pesticide shop workers, pesticide applicators, and land owners in Vadapalanji Panchayat Union, Madurai district, Tamil Nadu, India (latitude 9.9272, longitude 78.0092; Additional file 1: Figure S1). Details on name, frequency, and volume of insecticides commonly being used were collected. The fungicides, herbicides, bactericides, and mineral supplements were not taken into consideration. In a subsequent study, the people (n = 3080) from villages in Vadapalanji Panchayat Union that incorporates Vadapalanji, Manapatti, Thenpalanji, Sundarajapuram, Nagamalai puthur and Palkalainagar, Vellaparaipatti, and Meenatchipatti villages of Madurai district, Tamil Nadu, India were surveyed for their diabetic status and exposure history to OPs using a questionnaire (Additional file 3). Participants under the age of 35 years and pregnant women were excluded from the study. In addition, sex, age, and familial diabetic history were also collected. The diabetic status of the participants was self-reported by answering “Yes” or “No.” Participants involved in OP spraying, mixing, and field workers in OP-based agriculture were defined as having direct exposure to OPs and those who were not associated with OP-based agricultural works were defined as having indirect exposure to OPs.

Human blood collection

Blood samples of 5 mL were collected in EDTA-coated tubes from a random subset of the population (n = 802) involved in an earlier survey. The details of age, sex, height, weight, occupation, dietary habits, smoking, alcohol and tobacco consumption, exposure history to pesticides, prevalence of diabetes or any other diseases, and familial diabetic history were collected using a standard questionnaire (Additional file 5). Obesity was defined as body mass index (BMI) > 30 kg/m2. The collection protocol was approved by the internal research and review board, ethical clearance, biosafety, and animal welfare committee of Madurai Kamaraj University. Laboratory personnel performing analyses of blood parameters were all blinded and provided with only the sample ID and no participant details.

HbA 1c analysis

Diabetic status was confirmed by HbA 1c analysis in whole blood by HPLC (D10, Biorad Inc., USA). Diabetes was defined as either having a history of diabetes on medications or glycated hemoglobin (HbA 1c ) of ≥ 6.5% based on International Expert Committee (IEC) recommendations.

Acetylcholine esterase assay

AChE assay was performed in blood plasma/serum using the Amplex Red acetylcholinesterase kit (Invitrogen Inc., USA; A12217) as per the manufacturer’s instructions. In this assay, AChE activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), a sensitive fluorogenic probe for hydrogen peroxide. First, AChE converts the acetylcholine substrate to choline, which is oxidized by choline oxidase to betaine and H 2 O 2 . The latter, in the presence of horseradish peroxidase, reacts with Amplex Red in a 1:1 stoichiometry and generates a highly fluorescent product, resorufin [37]. The fluorescence was measured using excitation in the range of 545 nm and emission at 590 nm. The reactions were performed with technical triplicates. The reference values are 3334–7031 mU/L for males and 2504–6297 mU/L for females.

OP residues analysis by GC/MS

Blood plasma samples were extracted by a dispersive liquid–liquid microextraction technique [38] by a modified method. Briefly, 200 μL of plasma sample was spiked with 1 mg/mL of azobenzene as internal standard followed by the addition of 20 μL of 5 N HCl and made up to 1 mL with deionized water. Subsequently, the sample is incubated at 70 °C for 30 min to avoid the interaction of OPs with proteins. After cooling down to room temperature, 150 μL of acetonitrile (dispersive solvent) and 50 μL of chloroform (extraction solvent) mixture was forcibly added to the sample using syringe and sonicated for 3 min followed by centrifugation at 10,000 rpm for 5 min. The organic phase at the bottom of the tube is carefully collected and dried under a gentle stream of nitrogen gas and dissolved in 20 μL of hexane. Sample preparation and analysis were done in a blinded fashion.

Primary stock solutions of each insecticide (1 mg/mL) were prepared in methanol. Working standard solutions of the compounds were prepared by combining the aliquots of each primary solution and diluting with hexane. The stock solutions were stored at −20 °C in the dark when not in use. The standards were run at different concentrations and peak area was observed and subsequently linearity was established. Limit of detection (LOD) and limit of quantification (LOQ) were determined by standard methods. The recovery efficiencies for each individual insecticide was determined by spiking known concentrations of insecticide and measurement by GC/MS (Clarus 680/ 600C; Perkin Elmer Inc., USA).

The GC injector temperature was set at 200 °C. The oven temperature program was optimized to hold at 120 °C for 1 min and then to increase by 10 °C min−1 up to 220 °C [39]. Helium gas was used as carrier gas. The transfer line temperature was adjusted to 280 °C. Mass spectrometry conditions were as follows: electron ionization source set to 70 eV, emission current 500 lA, MS Quad 150 C, MS Source 200 °C. The mass spectrometer was run in full-scan mode and in single ion monitoring mode. The m/z fragments monitored for each insecticide is provided in Additional file 1: Figure S2C.

Maintenance and breeding of animals

Mice of BALB/c strain (RRID:IMSR_HAR:1255) were obtained from Madras University, Chennai and maintained and bred in an animal house at 25–28 °C with 12 h day/night cycles. The animals were fed with deionized water and standard rat chow (Hindustan Lever Limited, India) ab libitum. All the experiments in this study were performed with female mice aged eight weeks weighing 20–28 g and maintained in a constant environment at 25–28 °C with 45–60% humidity. The health status of the mice was confirmed by continuously monitoring their activities, behavior, body weight, and feces. The animal protocols used in this study were approved by the internal research and review board, ethical clearance, biosafety, and animal welfare committee of Madurai Kamaraj University.

Administration of monocrotophos to animals

The mice were provided MCP (Sigma-Aldrich Inc., USA; 361173) at 10× TMDI dose [6] (28 μg/kg bodyweight/day) directly in drinking water for 180 days. The food and liquid intake and animal activity were monitored daily. The body weight of the animals was documented every 30 days.

Measurement of fasting blood glucose

The animals were allowed for overnight fasting prior to blood glucose measurement. The fasting glucose was measured using a biosensor based glucometer [40] (Johnson & Johnson Inc., USA; OneTouch) with a drop of blood from the tail vein and expressed as mg/dL.

Oral glucose tolerance test

OGTT was performed to analyze how quickly the glucose is cleared from the blood [41]. The animals were fasted overnight prior to execution of OGTT, blood was sampled by the tail vein, and glucose was measured using the glucometer (0 min). Subsequently, the animals were gavaged with glucose solution (1.5 g/kg body weight) and blood glucose was monitored at 15, 30, 60, 90, and 120 min. The data were plotted in blood glucose versus time curve and glycemic response was expressed as area under the curve (AUC, × 103).

Harvest of organs

The animals were anesthetized by subcutaneous injection of ketamine (100 mg/kg body weight). The blood was collected by cardiac puncture and the organs including the brain, heart, liver, kidneys, and large intestine were harvested and perfused in sterile PBS (10× g/ l: 25.6 Na 2 HPO 4, 80 NaCl, 2.0 KCl, 2.0 KH 2 PO 4 . pH 7.2) and stored at −80 °C.

Serum insulin

Insulin level in the serum was determined by immunoenzymometric assay based kit (Monobind Inc., USA; 5825–300) as per the manufacturer’s instructions.

Preparation of tissue homogenate

A total of 100 mg of the tissue was homogenized in 1 mL RIPA buffer (Sigma-Aldrich Inc., USA; R0278), supplemented with 100 μL of cocktail protease inhibitors (Sigma-Aldrich Inc., USA; P8340), and incubated in ice for 20 min. The homogenate was centrifuged at 12,000 rpm for 20 min at 4 °C and the supernatant was collected, aliquoted, and stored at −80 °C.

Protein estimation

The amount of protein in serum/tissue homogenate was estimated by Bradford assay [42] (Sigma-Aldrich Inc., USA; B6926) as per the manufacturer’s instructions. Bovine serum albumin was used as a standard and the reactions were performed with technical triplicates.

Protein carbonylation assay

The carbonyls produced by oxidation of protein measured spectrophotometrically by the dinitrophenyl hydrazine (DNPH) method [43]. Briefly, 100 μL of the serum/tissue homogenate were mixed with 400 μL of 10 mM DNPH dissolved in 2.5 M HCl and incubated for 60 min and the protein was precipitated with an equal volume of trichloroacetic acid (TCA) (10%). The resultant pellet was washed with 1:1 ethanol:ethyl acetate mixture and resuspended in 250 μL of 6 M guanidine HCl. The protein hydrozones were measured spectrophotometrically at 370 nm. The corrected absorbance (CA) for each sample was calculated by the difference between corresponding control. The concentration of protein carbonyls (nM) was determined as follows: ((CA)/0.011) (250/100)). The reactions were performed with technical triplicates.

Lipid peroxidation assay

The lipid peroxidation was determined by estimating malondialdehyde (MDA) [44] with minor modifications. Briefly, 100 μL of tissue homogenate was added with 200 μL of ice cold 10% TCA to precipitate the protein and kept in ice for 15 min. After incubation, the samples were centrifuged at 2200 rpm for 15 min at 4 °C. A total of 200 μL of supernatant was added with equal volume of 0.67% thiobarbituric acid (TBA) and then incubated in a boiling water bath for 10 min. 1,1,3,3’-tetramethoxypropane was used as the standard. The color developed was read at 532 nm and the amount of MDA was expressed as nM/mg protein. The reactions were performed with technical triplicates.

Total antioxidant assay

The total antioxidant assay in the serum was executed using the total antioxidant kit (Sigma-Aldrich Inc., USA; CS0790) as per the manufacturer’s instructions. The principle of this assay is the formation of a ferryl myogloblin radical from metmyoglobin and hydrogen peroxide, which oxidizes the ABTS (2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) to produce a radical cation, ABTS•+, a soluble green chromogen that can be determined spectrophotometrically at 405 nm [45]. Trolox, a water-soluble vitamin-E analog, serves as the standard. The reactions were performed with technical triplicates. The antioxidant concentration was expressed in mM relative to the concentration of the Trolox standard.

Histopathology

The perfused hepatic tissue was fixed with 10% formaldehyde and paraffin embedded by standard methods. The embedded tissues were sliced into 5-μm thin sections using rotatory microtome. The sections were stained with hematoxylin and eosin and mounted on slides. Finally, the tissue morphology was examined by light microscopy, recorded, and analyzed by a qualified clinical pathologist in blinded fashion.

Fecal transplantation

After 180 days of experiment, 200 mg of the fecal material from animals drinking pure water or MCP mixed water was collected and suspended in 5 mL of PBS, mixed and incubated for 5 min at room temperature for separation by gravity, and the upper phase was collected. The mice were randomly selected for the study and allowed for 4 h fasting before fecal transplantation. Mice were gavaged with 200 μL of suspension per day for seven consecutive days [27]. The mice gavaged with fecal suspension were maintained in a separate, adjacent glass chambers to avoid cross-contamination. Similar conditions of temperature, humidity, water, and feed were maintained between the control and fecal transplanted group. At the end of the week, an OGTT was conducted as described above. Twenty-four hours after OGTT, the mice were sacrificed and the intestine and liver were collected for other assays.

Ex vivo culture and feeding

A total of 200 mg of fecal content from randomly selected mice from different cages were collected and suspended in 5 mL of sterile PBS and vortexed. The mixture was allowed to stand at room temperature for 5 min for separation by gravity and the supernatant was collected. One milliliter of the supernatant was inoculated in 9 mL of Robertson cooked meat medium (composition g/L: beef heart solids 98; proteose peptone 20; dextrose 2; sodium chloride 5. pH 7.2) supplemented with different OPs (MCP, CHL, MAL, and M.PAR) (Sigma-Aldrich Inc., USA; 36173, 45395, 36143, 36187) at 0.2 mg/mL concentration and incubated at anaerobic condition at 37 °C for nine days [22, 27]. To maintain logarithmic growth, the culture was subcultured every three days. After nine days of growth, part of the culture was centrifuged at 3000 rpm for 5 min and the supernatant was collected while the remaining pellet was dissolved in same volume of PBS. As mentioned above in the fecal transplantation protocol, the mice were administered with 200 μL of whole culture/suspended cells/supernatant continuously for seven days and finally OGTT was performed. As described above, the mice fed with cultures were maintained in separate, adjacent glass chambers with the same environment of temperature, humidity, water, and feed. Twenty-four hours after OGTT, the mice were sacrificed and the intestine and liver were collected for other assays.

Metagenomic RNA isolation

Total RNA was extracted from the ceacal tissue along with its contents using TRI reagent (Sigma-Aldrich Inc., USA; T9424) as per the manufacturer’s instructions. The integrity was checked in the agarose gel and quality and quantity was determined spectrophotometrically.

Enrichment of bacterial RNA

Bacterial RNA was enriched from the total RNA by using MICROBEnrich kit (Ambion Inc., USA; AM1901) as per the manufacturer’s protocol. Here, hybridization capture technology was used to remove human, mouse, and rat RNA (both mRNA and rRNA) from complex host-bacterial RNA populations, leaving behind enriched microbial total RNA. In the first step of the procedure, host-bacterial total RNA is incubated with a mixture of capture oligonucleotides that bind the mammalian 18S and 28S rRNAs and polyadenylated RNAs. Next, the rRNA/oligo nucleotide hybrids and all polyadenylated mRNAs are removed from the mixture with oligonucleotide-derivatized magnetic beads. To ensure complete removal of eukaryotic mRNAs, complementary DNA was constructed with oligo-d(T) primers and polymerase chain reaction for the mouse GAPDH gene was executed and checked.

Enrichment of bacterial mRNA

Bacterial mRNA was enriched in the purified RNA by removing the 16S and 23S rRNAs using a MICROBExpress kit (Ambion Inc., USA; AM1905) as per the manufacturer’s instructions. The method employs a modification to sandwich capture hybridization protocols that were developed for the capture and detection of specific nucleic acid molecules with probes conjugated to magnetic beads. The bound RNA was separated by using magnetic field and the unbound RNA was dissolved in RNase free water. The enrichment of bacterial mRNAs and removal of rRNAs was confirmed by bioanalyzer (Agilent Inc., USA) analysis.

RNA sequencing and analysis

RNA library was constructed using TruSeq kit (Illumina Inc., USA) as per the manufacturer’s instructions. RNA-seq was done at Centre for Cellular & Molecular Platforms (Government of India), Bangalore with paired-end reads in Illumina HiSeq 1000 machine. The sequencing was performed in a blinded way. Raw data were processed using the Solexa software. Low-quality reads were filtered according to the base quality value. The reads were mapped with mouse genome, murine mRNAs, transfer RNAs, and rRNAs by Bowtie 2 [46] and the annotated sequences were removed.

We used a reference database of human microbiome to perform functional analysis of the RNA-seq data. This reference included 538 draft and finished bacterial genomes from the human microbiome consortium. High-quality reads were mapped using Bowtie 2 to our reference bacterial database. Subsequently, using the KEGG database, all predicted proteins from the reference genome database were annotated with KEGG orthologous groups (KOs). For query genes with multiple matches, the annotated reference gene with the lowest e value was used. When multiple annotated genes with an identical e value were encountered after a BLAST query, we included all KOs assigned to those genes. The number of transcripts assigned to each gene was then tallied and normalized to RPKM. To account for genes that were not detected owing to limited sequencing depth, a pseudocount of 0.01 was added to all samples. Genes were grouped by taxa, genomes, and KO by calculating the cumulative RPKM for each sample. HUMAnN [47] was used for metabolic reconstruction from metagenomic data followed by LefSe [48] analysis with bootstraping to identify significant biomarkers. The reads were annotated to metacyc enzyme database from the human microbiome consortium using BLASTN. The number of transcripts assigned to each enzyme were then tallied and normalized to RPKM. The enzymes of the same class were summed and expressed as single enzyme.

Esterase assay

A total of 200 μL of the culture was centrifuged at 12,000 × rpm for 10 min and the pellet was suspended in 200 μL of sterile PBS. Eighty microliters of suspension were used for esterase assay with ethyl butyrate as substrate as per Lisboa et al. [49]. The formation of carboxylic acid due to hydrolysis of substrates mediated by esterase causes a reduction in the pH, which changes the color of the medium from blue to yellow. This reaction can be observed or monitored spectrophotometrically at 616 nm. We used ethyl butyrate (Sigma Aldrich Inc., USA; 109959) as the substrate and bromothymol blue (Himedia labs, India; GRM120) as the pH indictor.

Metabolomics

The tissue processing for metabolomics was carried out in NIH Centre for Metabolomics, University of California, USA as per standard operating procedure [50]. The analysts were blinded of the sample information. A total of 50 mg of caecum tissue cleared of fecal matter was taken in a 25-mL polypropylene centrifuge tube and 2.5 mL of extraction solvent (acetonitrile:isopropanol:water 3:3:2) was added and homogenized for 45 s. In between every homogenization, the homogenizer was cleaned with solutions of methanol, acetone, water, and the extraction solvent. The homogenate was centrifuged at 2500 rpm for 5 min. The supernatant was aliquoted 2 × 500 μL and one of them stored at −20 °C for back up. The other aliquot of 500 μL was evaporated to complete dryness in a centrivap cold trap concentrator. The dried aliquot was resuspended in 500 μL of degassed 50% acetonitrile and centrifuged for 2 min at 14,000 rcf. The supernatant was collected in a fresh tube and evaporated to dryness in a centrivap cold trap concentrator and finally submitted to derivatization.

Primary metabolism by ALEX-CIS GCTOF MS

Data were acquired using the following chromatographic parameters as described by Fiehn et al. [51]. A Rtx-5Sil MS column (Restek Corporation) was used with helium as a mobile phase. A total of 0.5 μL of samples were injected at 25 splitless time into a multi-baffled glass liner with injection temperature of 50 °C ramped to 250 °C by 12 °C s−1. Oven temperature was programmed at 50 °C for 1 min, ramp at 20 °C per minute to 330 °C which was held constant for 5 min. Data processing and data reporting were done by NIH Centre for Metabolomics.

Raw results data were normalized to reduce the impact between-series drifts of instrument sensitivity, caused by machine maintenance, aging, and tuning parameters. We used a variant of vector normalization in which the sum of all peak heights for all identified metabolites excluding the unknown for each sample was calculated and termed as mTIC. mTIC was used to avoid the potential non-biological artifacts for the biological normalizations, such as column bleed, plasticizers, or other contaminants. mTIC averages were determined between different treatment groups and following equation was used for normalization of metabolite i of sample j:

$$ {\mathrm{Metabolite}}_{\mathrm{ij},\ \mathrm{normalized}} = \left({\mathrm{metabolite}}_{\mathrm{ij},\ \mathrm{raw}}/\ {\mathrm{mTIC}}_{\mathrm{j}}\right) \times {\mathrm{mTIC}}_{\mathrm{average}} $$

This normalization is relative semi-quantification and expressed as normalized peak heights.

Quantitative metabolite set enrichment analysis

MSEA is a way to identify biologically meaningful patterns that are significantly enriched in quantitative metabolomics data and was carried out using the tool from MetaboAnalyst [28, 52]. Over-representation analysis was implemented using the hypergeometric test to evaluate whether a particular metabolite set is represented more than expected by chance within the given compound list. One-tailed P values are provided after adjusting for multiple testing.

Glucose-6 phosphatase assay

Fifty milligrams of liver/colon tissue were homogenized in 500 μL of RIPA buffer with protease inhibitors and the final homogenate was collected. Amount of inorganic phosphorus (Pi) released was assayed using Taussky-Shorr method [53]. Briefly, 150 μL of 100 mM Tris buffer (pH: 6.5) was mixed with 100 μL of 200 mM glucose-6 phosphate (Sigma-Aldrich Inc., USA; G7879) and incubated at 37 °C for 5 min. Subsequently, 10 μL of tissue homogenate was added, mixed, and incubated again at 37 °C for 5 min. The reaction was terminated by the addition of 90 μL of 10% TCA and incubation at 25 °C for 5 min. Finally, the mixture was centrifuged at 4000 rpm for 10 min and the supernatant was collected. The amount of Pi released was measured by mixing the supernatant or inorganic Pi solution (Sigma-Aldrich Inc., USA; P3869) with equal volume of Taussky-Shorr color reagent (10% ammonium molybdate prepared in 5 M sulphuric acid 10 mL, ferrous sulfate heptahydrate 5 g in 100 mL of distilled water) and incubated at 25 °C for 6 min. Finally, the absorbance was read at 660 nm. Specific glucose-6 phosphatase (G6Pase) activity was cleared of the contribution of non-specific phosphohydrolase activities by subtracting the activity toward 20 mMβ-glycerophosphate [54] (Sigma-Aldrich Inc., USA; G9422) and finally net G6Pase activity was expressed as μg of Pi released per mg of protein.

Glycogen assay

A total of 100 mg of liver tissue was homogenized in 500 μL of 3% TCA and the homogenate was centrifuged at 3000 rpm for 5 min. Five volumes of cold 95% ethanol were added to the supernatant and left overnight at room temperature to precipitate glycogen. After a short spin for 10 s, the ethanolic supernatant was discarded and the pellet was dissolved in 250 μL of deionized water. Blank and standards (0.5 mg/mL of glucose) were prepared with same volume of deionized water. A total of 1.25 mL of anthrone reagent (anthrone 50 mg, thiourea 1 g, H 2 SO 4 72 mL in 100 mL deionized water) was added to all tubes and incubated at boiling temperature for 15 min. After cooling, the absorbance was measured at 620 nm against the blank. Amount of glycogen (mg/100 g of tissue) = DU/DS × 0.1 × volume of extract/gram of tissue × 100 × 0.9 where DU = absorbance of samples and DS = absorbance of glucose standard [55].

Administration of sodium acetate

The mice were fasted for 4 h prior to the experiment. NaAc (100 mg/ kg body weight) was administered either orally using gavage or by RI continuously for seven days. Before RIs, the mice were handled gently and allowed to defecate and the complete defecation was confirmed by softly pressing at the distal end of the rectum. The mice were handled inversely and NaAc was administered in a maximum volume of 20 μL using 2–20 μL tips via micropipette. Finally, OGTT was performed by standard protocols. The animals were sacrificed a minimum of 24 h after OGTT and the organs were harvested.

Collection of human fecal samples

Fecal samples were collected from the diabetic (n = 60) and control people (n = 60) from the population earlier studied for HbA 1c and OP analysis. People with bowel or stomach-related issues were excluded from the study. The control volunteers were confirmed for absence of obesity, hypertension, dyslipidemia, or other issues. The collection protocol was approved by the internal research and review board, ethical clearance, biosafety, and animal welfare committee of Madurai Kamaraj University. In addition, the project details were explained and their details in previous questionnaire were reconfirmed and new informed consent was obtained (Additional file 6). Subsequently, the next day early morning fecal samples were collected and stored immediately in ice. The samples were transported to the laboratory within 1 h and stored in −80 °C. The analysts performing the fecal parameters were blinded and unaware of the diabetic or OP exposure status of the samples.

Short chain fatty acid quantification in feces

A total of 100 mg of feces was weighed and suspended in 2 mL of sterile PBS and vortexed for 1 min. The mixture was centrifuged at 3000 × g for 10 min. Five microliters of the supernatant was diluted 1:100 with sterile PBS. Five microliters of ethyl butyrate (Sigma-Aldrich; 109959) was added as internal standard to a final concentration of 5 mM. Subsequently, 250 μL of concentrated HCl was added followed by the addition of 1 mL of diethyl ether (Merck; LiChrosolv). The mixture was vortexed for 1 min and centrifuged at 3000 × g for 10 min. A total of 750 μL of upper phase was collected and derivatized with 120 μL of N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) containing 1% tert-butyldimethylchlorosilane (TBDMSCI) (Sigma-Aldrich Inc., USA; 375934) by incubating at 80 °C for 20 min. The mixture was incubated at room temperature for 48 h to ensure complete derivatization. Gas chromatography was executed as described by Frost et al. [56] by flame ionization detector. The GC injector and detector temperatures were set at 275 °C. The oven temperature program was optimized to hold at 63 °C for 3 min and then to increase by 10 °C min−1 up to 190 °C. Helium gas was used as carrier gas. The transfer line temperature was adjusted to 280 °C. External standards for acetate were prepared at concentrations of 25, 12.5, 6.25, 1.25, and 0.625 mM and ethyl butyric acid was used as the internal standard at a concentration of 100 mM. Reported values were normalized according to the weight of original sample used.

Statistics

All statistical analyses were performed using the statistical softwares SPSS version 20.0 and GraphPad Prism version 6.01. For association studies in survey, age and sex adjusted ORs and 95% CIs were calculated. For human studies, the non-parametric Mann–Whitney U test was employed. Pearson correlation and linear regression were performed to demonstrate the strength of relationship between two parameters. Plasma OP residues were categorized in quartiles based on the weighted sample distribution. For each OP, we used logistic regression to estimate ORs and CI levels for diabetes by comparing each quartile with the lowest quartile. We included likely or suspected confounders in models based on previously published data. In each analysis, we also evaluated the significance of the differences of the average proportion of diabetics across the four quartiles of the model by a generalized maximum likelihood Wald χ2 test. Our regression models were fitted with appropriate degrees of adjustment. We adjusted for age, sex, familial diabetic history, and BMI.

The following statistical analyses were used for animal studies: a two-way ANOVA with Bonferroni post-hoc analysis was used to compare between groups in different time-points and one-way ANOVA with Tukey’s post-hoc analysis or unpaired two-sided Student t-test was used to compare either between multiple or between two groups, respectively. The batch difference between replicate/triplicates were studied by a two-way ANOVA with Bonferroni post-hoc analysis. In all relevant panels, symbols, bars, or horizontal lines represent the mean and error bars represent s.e.m. For mouse experiments, cohort sizes match common practice of the described experiments and are repeated twice or thrice. For human experiments, sample size was chosen to validate statistical analyses. No data points were excluded from analyses in mice or human studies. P < 0.05 was considered statistically significant in all analyses. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.