Metabolomic analysis of the serum and urine of rats exposed to diazinon, dimethoate, and cypermethrin alone or in combination

Abstract

Background: Multiple pesticides are often used in combination for plant protection and public health. Therefore, it is important to analyze the physiological changes induced by multiple pesticides exposure. The objective of this study was to investigate the combined toxicity of the widely-used organophosphorus and pyrethroid pesticides diazinon, dimethoate, and cypermethrin.

Methods: Male Wistar rats were administrated by gavage once daily with the three pesticides individual or in combination for consecutive 28 days. The metabolic components of serum and urine samples were detected by using ¹H nuclear magnetic resonance (NMR)-based metabolomics method. Histopathological examination of liver and kidneys and serum biochemical determination were also carried out.

Results: The results showed that after the 28-day subacute exposure, serum glutamic transaminase and albumin were significantly increased and blood urea nitrogen was significantly decreased in the rats exposed to the mixture of the pesticides compared with the control rats, suggesting that the co-exposure impaired liver and kidney function. Metabolomics analysis indicated that the indicators 14 metabolites were statistically significant altered in the rats after the exposure of the pesticides. The increase in 3-hydroxybutyric acid in urine or decrease of lactate and N-acetyl-L-cysteine in serum could be a potentially sensitive biomarker of the subchronic combined effects of the three insecticides. The reduction level of 2-oxoglutarate and creatinine in urine may be indicative of dysfunction of liver and kidneys.

Conclusion: In summary, the exposure of rats to pesticides diazinon, dimethoate, and cypermethrin could cause disorder of lipid and amino acid metabolism, induction of oxidative stress, and dysfunction of liver and kidneys, which contributes to the understanding of combined toxic effects of the pesticides revealed by using the metabolomics analysis of the urine and serum profiles.

Keywords: Metabolomics analysis; Multiple-pesticide exposure; Organophosphate; Pyrethroid; Subacute toxicity.

Fig. 1

Effect of pesticides diazinon (DZN), dimethoate (DIM), and cypermethrin (CYP) alone or in combination on the liver and kidneys of rats. The tissue sections of liver and kidneys of the rats were examined under a microscope. Compared with the control rats showing normal structure of liver (upper panel) and kidney tissues (bottom panel), the rats exposed to DZN (250 mg/kg body weight/day), DIM (300 mg/kg body weight/day), and CYP (500 mg/kg body weight/day) individual or in combination for 28 consecutive days exhibited swelling degeneration of hepatocytes in liver tissue (the red arrows indicate the hepatocyte necrosis) and abnormal glomerular capillary wall in kidney tissue (the black arrows indicate the glomerular lesions). Scale bar: 50 μm. Abbreviation: CTL, control; MIX, the mixture of DZN, DIM, and CYP

Fig. 2

The representative spectrum of 600 MHz ¹H NMR (0.5 ~ 4.5) spectra of urinary samples from the rats exposed to vehicle (a), diazinon (b), dimethoate (c), cypermethrin (d), or mixture of the three pesticides (e) for 28 consecutive days. Abbreviations: 2-OG, 2-oxoglutarate; 3-HB, 3-hydroxybutyric acid

Fig. 3

Partial least squares discriminate analysis (PLS-DA) from ¹H NMR spectra of urine samples of the rats. The animals were exposed to three pesticides diazinon, dimethoate, and cypermethrin alone or in combination for 28 consecutive days, and then the 24 h-urine samples were collected and analyzed by an NMR spectrometer. Scores plot (a) and the loadings plot (b) for these models were used to distinguish which metabolites were the most significant in terms of describing this separation. Abbreviations: CYP, cypermethrin; DIM, dimethoate; DZN, diazinon; MIX, mixture of CYP, DIM, and DZN

Fig. 4

Typical CPMG ¹H NMR spectra of serum from the rats exposed to vehicle (a), diazinon (b), dimethoate (c), cypermethrin (d), or mixture of the three pesticides (e) for 28 consecutive days. Abbreviations: Cho-PCho, choline-phosphocholine; NAC, N-acetyl-L-cysteine; LDL, low density lipoprotein; VLDL, very low density lipoprotein

Fig. 5

Partial least squares discriminate analysis (PLS-DA) from ¹H NMR spectra of serum samples of rats. The animals were exposed to three pesticides diazinon, dimethoate, and cypermethrin alone or in combination for 28 consecutive days, and then the blood samples were collected and the serum was prepared and analyzed by an NMR spectrometer. Scores plot (a) and the loadings plot (b) for these models were used to distinguish which metabolites were the most significant in terms of describing this separation. Abbreviations: CYP, cypermethrin; DIM, dimethoate; DZN, diazinon; MIX, mixture of CYP, DIM, and DZN