Detection of Neonicotinoid Insecticides and Their Metabolites in Human Cerebrospinal Fluid

Adela Jing Li,1* Mengya Si,2* Renli Yin,1 Rongliang Qiu,1 Huashou Li,1 Fen Yao,3 Yunjiang Yu,4 Wenhua Liu,5,6 Zhen Wang,5,6 and Xiaoyang Jiao7 College of Natural Resources and Environment, South China Agricultural University, Guangzhou, China First Affiliated Hospital of Shantou University Medical College, Shantou, China Department of Pharmacology, Shantou University Medical College, Shantou, China South China Institute of Environmental Sciences, Ministry of Ecology and Environment, Guangzhou, China Guangdong Provincial Key Laboratory of Marine Disaster Prediction and Prevention, Shantou University, Shantou, China Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou, China Department of Cell Biology and Genetics, Shantou University Medical College, Shantou, China


Introduction
Neonicotinoid insecticides (NEOs) are now the most widely used neurotoxic insecticides on 140 crops in 120 countries. 1 Global use of NEOs has caused growing concerns for decline of pollinators, 2 risk to nontarget species, 3 and potential adverse effects on human health. 4 A systematic review in 2017 reported associations between human chronic NEO exposure and adverse neurological outcomes based on interview data. 4 A previous study in mice reported that NEOs and their metabolites could be distributed to the cerebral cortex, hippocampus, and striatum after oral intake of the NEOs. 5 Validated biomarker investigations of NEOs were generally measured in human urine 4 or mouse blood, 5 which may not be reflective of central nervous system (CNS) exposure.
Cerebrospinal fluid (CSF) is an integral CNS component emerging in parallel with the developing CNS. Several biochemical markers in CSF have been used for diagnosis and evaluation of neurological diseases. 6 The present study aimed to explore whether NEOs and their metabolites could be evident in CSF.

Methods
This study recruited 314 donors from 4,410 patients available for CSF analysis in the First Affiliated Hospital of Shantou University, Shantou, China, from April 2019 to January 2021. CSF specimens were collected by a senior physician using the method of clinical lumbar puncture. A total of 314 CSF samples were collected from patients experiencing symptoms with different diagnoses (mostly viral encephalitis, encephalitis other than viral encephalitis, leukemia, cerebral hemorrhage, cerebral laceration, urinary tract infection, respiratory failure, pulmonary tuberculosis, and posterior circulation ischemia). Informed consent forms were completed by all participants. The research protocol was approved by the clinical research ethical committee from the First Affiliated Hospital of Shantou University Medical College.
The instrument-derived concentrations of NEOs and metabolites below the limits of detection (LODs) were assigned values of the LODs divided by the square root of 2. Data are presented as medians with interquartile ranges (IQRs) given that all data were skewed. Differences in median CSF levels of study analytes among the five age groups were tested by a nonparametric Kruskal-Wallis H-test, whereas differences in median CSF levels between genders were examined by a Mann-Whitney U-test. Data were analyzed using SPSS (version 19.0; SPSS Inc.) with values of p < 0:05 denoting statistical significance.
As a specific metabolite from ACE, N-dm-ACE was found at the highest concentration (median = 0:049 ng=mL) and at the highest frequency of occurrence (85.4%, n = 268) among the 15 analytes tested. Detection rates for the other 14 compounds were in the range of 0.32%-45.5%. Studies done using in vitro models have reported that some metabolites are more potent than the corresponding parent NEO because of their higher affinity to nicotinic a4b2 nicotinic acetylcholine receptors. 8 A recent study reported that N-dm-ACE concentrations in 93% of CSF samples (13/14) collected from children treated for leukemia and lymphomas were within an order of magnitude (median = 0:012 ng=mL) 9 of the concentrations detected in our study. The potential neurotoxic effects of N-dm-ACE to the human CNS should be considered comprehensively.
In our study, the two NIT detections at 102 and 39:2 ng=mL in CSF were considered outliers, with reported concentrations well over three times the IQR. The first participant was a 72-yold male at the sampling time in 2020. The latter participant was a 60-y-old female at the sampling time in 2021. Both were diagnosed with cerebral hemorrhage at the sampling point.
The enrolled 314 patients were from 1 month to 89 years of age, with 180 (57.3%) males (Table 2). Median CSF concentrations of N-dm-ACE, solely, differed significantly among age groups, having an increasing trend with age ( Table 2). A recent study reported that the aging process diminished copper clearance from the CSF of rats by disrupting copper transporting proteins in the choroid plexus. 10 In the present study, no significant difference was found for median CSF concentrations of target analytes between genders (Table 2).
This was an exploratory study to identify detectable concentrations of nine NEOs and six metabolites in the CSF of 314 patients. For continued global use of NEOs, mechanisms of toxicity, especially to the CNS in humans, need to be more rigorously investigated.  Note: Measurements below the LODs were assigned values of the LODs divided by square root of 2 prior to calculating median concentrations. Differences in median CSF levels among the five age groups tested by a non-parametric Kruskal-Wallis H-test. Differences in median CSF levels between genders tested by a Mann-Whitney U-test. ACE, acetamiprid; CSF, cerebrospinal fluid; IMI, imidacloprid; IMZ, imidaclothiz; LOD, limit of detection; N-dm-ACE, N-desmethyl-acetamiprid; NIT, nitenpyram; Of-IMI, olefin-imidacloprid. ** , p = 0:002 denoting statistically significant differences in CSF levels of N-dm-ACE among the five age groups.