Urinary Level of Dimethoate, Bisphenol a and Benzo[a]pyrene in the First-year Students of Hohai University From Different Geographical Regions

Background: The objective of this study was to detect the urinary level of dimethoate, benzo(a)pyrene (BaP), and bisphenol A (BPA) in the rst-year Hohai University students of different geographic origins. Methods: First-morning urine samples were collected from 540 healthy freshmen aged 17 to 19. Toxin levels were measured using β-glucuronidase hydrolysis followed by a high-performance liquid chromatography-tandem mass spectrometry-based method. Geometric means (GM) are presented by BMI (Body Mass Index) and different locations for these three toxins in a volume-based and creatinine-standardized way. Results: GM concentration of dimethoate, BPA and BaP were 9.47 µg/L (10.80 µg/g creatinine), 3.54 µg/L (4.04 µg/g creatinine) and 0.34 ng/L (0.39 ng/g creatinine), respectively. The GM concentration of dimethoate in males was signicantly higher than that in females. The BMI higher than 23.9 had a higher GM concentration of dimethoate, BPA, and BaP. The inhabitant in the Southwest of China had a signicantly lower GM concentration of dimethoate, BPA, and BaP than those who live in other locations of China. Conclusion: The average level of environmental toxins accumulation in freshmen is relatively high and differs in the youth who live in different regions. Besides, obesity is correlated to higher toxins levels in youth.


Introduction
Pollution problems of air, water, and food have been increasing as a consequence of global climate change, pesticide misuse, and industry development [1], thus the negative effects of environmental pollution on human health have lately become a great concern. Inevitably, humans are exposed to pollutants such as heavy metals, pesticide residues, polycyclic aromatic hydrocarbons (PAHs), bisphenol A (BPA), and so on through drinking water, food, dust, and ambient air [2] which tightly link to the initiation and progression of multiple diseases [3].
China is one of the largest agricultural countries in the world, with >300,000 tons of agricultural pesticides used annually [4]. Dimethoate, one of the most commonly used organophosphorus pesticides, is wildly used for broad-spectrum control of a wide range of insects including mites, ies, aphids, and plant hoppers [5]. Overuse of dimethoate can lead to massive residues on fruits, vegetables, and grains [6]. Existing research con rmed that even very low levels of dimethoate may have adverse health effects [7,8] which is potentially carcinogenic [9,10]. At the same time, the extensive industrial development model has made China fall into an "environmental pollution-economic development" cycle [11]. PAHs, which are originated from diverse sources including petrochemical products and the combustion of fossil fuels, are pervasive pollutants characterized by their hazardous carcinogenic and mutagenic potential which exist not only in the air but also in food and drinking water [12,13]. Benzo(a)pyrene (BaP) is one of the most studied PAHs classi ed by IARC as a Group 1 carcinogen [14]. Multiple studies have shown that BaP requires metabolic activation to exert its carcinogenic effects [15,16]. It was reported that a higher incidence of forestomach tumors was observed when applied with BaP in food to B6C3F1 mice over a lifetime [17]. BPA is a synthetic plasticizer with more than 8 million pounds produced worldwide each year which can be found everywhere from plastic bottles, the medical devices to the undercoat of the food packages [18]. Likewise, there is growing evidence that BPA may adversely affect human health. Several studies have proved that BPA has negative effects on human reproduction including female fertility [19], male sexual function [20], sperm quality [21], etc. Moreover, BPA has an impact on gene expression processes, such as the enzymatic proteins which have important roles during fetal development [22].
Besides, metabolic syndromes such as type 2 diabetes, non-alcoholic fatty liver disease, and obesity are also associated with BPA [23]. Different subjects originating from distinct parts of the country may carry the pattern of environmental exposure in their regions. It was reported that the blood Pb levels of the populations who live in Wuhan of central China were lower than those in Beijing [24]. It was also reported that the healthy Chinese who live in the mining area of manganese mines or nonferrous metal mines have a signi cantly higher urinary manganese level than those who live in other regions. Another study showed that higher urinary levels for As and Cd in the Wuhan population are observed when compared with other countries [25]. Besides the geographic distributions, a preliminary study has reported that the hair and urinary Al levels in the obese subjects are signi cantly higher by 31% and 46% compared to that in the healthy group, respectively[26].
Hohai University (former Hohai Civil Engineering School of China, established in 1915, HHU) is a national key university under the direct administration of the Ministry of Education. As a comprehensive university with at least 20 colleges, she enrolls more than tens of thousands of students each year from every province in China. Before entering university, the lifestyle of high school students is relatively unitary during nearly 10 years study period at the place of birth. Considering that environmental background may have a signi cant effect on long-term health effects. Meanwhile, these environmental toxin markers can be easily detected in blood and urine. It's meaningful to detect the level of environmental toxin which could re ect the local environment and individual health. Therefore, the objective of the present study was to provide the baseline information of the levels of dimethoate, BaP, and BPA in the urine samples from the rst-year Hohai University students of different geographic origins and to assess the correlation between the level of pollutants exposure with geographic origins and BMI through the baseline. More importantly, it will facilitate the improvement of the overall health level of Chinese people by advocating a healthier lifestyle and provide suggestions for environmental protection policy design.

Study Design and Participants
All procedures including sampling and examination were performed in agreement with the principles set in the Declaration of Helsinki and its later amendments (2013). All examinees were invited to participate and took part in the present study voluntarily. All subjects were informed about the objectives of the study and experimental procedures and signed the informed consent form. The study protocol has been reviewed and approved by the Ethical Review Committee of Sir Run Run Hospital, Nanjing Medical University (2019-SR-018).
A total of 540 freshmen of the HHU originating from East (n = 319), Northeast (n = 10), North (n = 85), Northwest (n = 41), Southwest (n = 43), and South (n = 42) of China were enrolled in the present study (Table 1), in which there were 253 males and 287 females aged from 17 to 19. And the precise geographical location of China was shown in gure 1. Examination and sample collection were performed during the rst medical screening on admission to the university directly after arrival to HHU using noninvasively collected substrates (urine) in September 2019. Only healthy subjects without chronic diseases were involved in the current investigation to avoid side effects and interactions of diseases on the studied parameters.

Sample Processing
Collection of urine samples (second portion) was performed in the morning using plastic Vacuette ® Urine Collection Cups (Greiner Bio-One International AG, Austria).
Evaluation of dimethoate, BPA, and BaP levels in the urine of examinees was performed using Liquid chromatography-mass spectrometry (LC-MS). The levels of omethoate and 3-hydroxypyrene, metabolites of dimethoate and BaP respectively, and BPA, were examined. The 1 µg/mL standard working solutions of dimethoate, BPA, and BaP were prepared with methanol as solvent. After continuous dilution of 10 4 times, it became the standard working solution of 100 pg/mL. Take BaP as an example, different concentrations of benzo (a) pyrene standard working solution were prepared. 50 µL of each BaP standard working solution was prepared and injected into the system. The collected urine samples (2 mL) were ltered with 0.22 µM lter membrane, pH was adjusted to 5.4 by adding acetic acid-sodium acetate buffer (0.5 M), then βglucuronidase/arylsulfatase (10 µL) and vitamin C (5 mg) were added and incubated overnight at room temperature to complete the enzymatic hydrolysis. The samples were extracted, after enzymatic hydrolysis, by solid-phase extraction with SPE column (C18 ENVI 0.25 g).
The extract was eluted with methanol (2 mL) and dried with nitrogen. Finally, methanol (100 µL) was used to re-dissolved the analyte to be determined. 50 µL of the analyte to be tested was transferred to a liquid chromatography bottle with a microsyringe, which was especially used for the injection analysis of BaP levels. The detection methods of dimethoate and BPA were consistent with those of BaP [27].

Statistical Analyses
Statistical treatment of raw data was performed using SPSS 26.0 (IBM Corp., Armonk, NY, USA) software. Geometric median (GM) values were used as descriptive statistics for pollutants levels. T-tests were used to compare GMs between categories. Multiple regression analysis was performed to specify the association among the pollutant levels, BMI, and region of origin. All models were adjusted for age and gender variability. The results of the tests were considered signi cant at p < 0.05.

Results
Urine dimethoate, BPA, BaP were detected in 100% of the recruited people. And the results were presented by volume-based and also by creatinine standardized which was used to eliminate the effect of the time of urine collection, urine concentration, and urine ow rate [28].
Hohai University is a multi-disciplinary comprehensive university located in Jiangsu Province of East China. The freshmen came from all over the country including East, North, South, Northeast, Northwest, and Southwest. We choose East china as the reference category because subjects originating from a distinct part of china carry the patterns of environmental exposure in their domestic region. The obtained data demonstrated that the origin of the students had an important impact on the urine toxins (  The volume-based geometric mean (GM) concentration was 9.47 µg/L ( Table 3). The GM of urine dimethoate in the female group (8.55 µg/L) was signi cantly lower than that in the male group (10.64 µg/L). The GM dimethoate concentration rose signi cantly from 9.12 (µg/L) at the normal BMI (18.5≤BMI≤23.9) to 14.68 (µg/L) at the overweight BMI (BMI>23.9). However, only a moderate change of GM dimethoate concentration was observed between the normal BMI (9.12 µg/L) and the less-weight BMI (BMI<18.5) (8.74 µg/L). The standardized GM of dimethoate concentrations in the overweight male (18.16 µg/g) was signi cantly higher than that in normal (10.60 µg/g) or less weight male (10.33 µg/g). Likewise, overweight females had signi cantly higher standardized GM of dimethoate concentration (14.54 µg/g) than normal (10.28 µg/g) or fewer weight females (9.68 µg/g).
The volume-based GM of urine BPA concentration was 3.54 µg/L (Table 3) Standardizing BPA with urinary creatinine concentrations resulted in a GM BPA concentration of 4.04 µg/g for all recruited people. There was no signi cant difference found between the male group (3.68 µg/g) and the female group (4.39 µg/g). The standardized GM of BPA concentrations in people with an overweight BMI (5.46 µg/g) was signi cantly higher than other BMI groups. The only moderate change was observed between the normal BMI (4.00 µg/g) and the less-weight BMI (3.35 µg/g).
The volume-based GM of urine BaP concentration was 0.34 ng/L ( Table 3). The GM of urine BaP in the female group (0.31 ng/L) was signi cantly lower than that in the male group (0.38 ng/L). People with overweight BMI had a higher GM of urine BaP concentration (0.45 ng/L) compared with the ones with normal (0.34 ng/L) or less weight BMI (0.27 ng/L). Notably, a signi cant change of GM BaP concentration was also observed between the normal BMI and the less-weight BMI.
Males with overweight BMI had signi cantly higher GM BaP concentration (0.52 ng/L) compared with the normal BMI (0.38 ng/L) and less weight BMI (0.29 ng/L). Likewise, females with overweight BMI also had signi cantly higher GM BaP concentration (0.39 ng/L) compared with the normal BMI (0.31 ng/L) and less weight BMI (0.25 ng/L).
Standardizing BaP with urinary creatinine concentrations resulted in a GM BaP concentration of 0.39 ng/g for all recruited people. A signi cant difference was found between the male group (0.40 ng/g) and the female group (0.38 ng/g). The standardized GM of BaP concentrations in people with an overweight BMI (0.50 ng/g) was signi cantly higher than other BMI groups. A signi cant change was also observed between the normal BMI (0.39 ng/g) and the less-weight BMI (0.31 ng/g).
The standardized GM of BaP concentrations in the overweight male (0.54 ng/g) was signi cantly higher than that in normal (0.40 ng/g) or less weight male (0.31 ng/g). Likewise, overweight females had signi cantly higher standardized GM of dimethoate concentration (0.45 ng/g) than normal (0.38 ng/g) or fewer weight females (0.30 ng/g).
Association between urinary toxins levels and BMI, as well as the potential confounding effects of age, gender, and waist, was additionally studied in the regression model (Table 4). Particularly, in this regression model, urinary dimethoate, BPA, and BaP were not associated with age and waist. In turn, gender and BMI were considered as signi cant predictors of the volume-based concentration of urinary dimethoate, BPA, and BaP. Notably, the male gender was positively associated with the volume-based concentration of urinary dimethoate and BaP while inversely associated with urinary BPA. However, after being standardized by urinary creatinine, the male gender was only inversely associated with urinary BPA. It was also notable that BMI was positively associated with both volume-based and standardized concentrations of urinary dimethoate, BaP, and BPA.

Discussion
In this study, we chose 3 kinds of very common pollutants in the Chinese environment to represent the exposure level of pollutants in Chinese young people aged from 17~19. Besides, the detection does not rely on a blood sample, but urinary samples obtained from non-invasive sources which were easily obtained and low cost. Urine could also better re ecting the changes in human metabolism because the metabolite concentration is higher in urine than in human plasma or serum [29] The obtained data demonstrate the freshmen of Hohai University originating from distinct geographic regions of China are characterized by increased pollutants exposure levels. Bushnik, T. reported urinary level of BPA in Canada (1.16 µg/L) is almost 1/3 of that in our data (3.54 µg/L) [30]. This may re ect the differences in the situation of BPA pollution between the Chinese and other countries' environment. Yu., et al reported urinary 1-OHP concentrations increased with increasing concentrations of B[a]P in the industrial area in Lanzhou city [31]. Notably, there is no data report for the urinary level of dimethoate in other countries. So it is meaningful to detect the baseline of these three pollutants to re ect the effects on the human body of the local environment. Furthermore, the male gender shows a signi cantly higher urinary level of BPA than the female gender in the data from Canada, while no signi cant sexual difference of BPA level was observed in our data. This may re ect the differences in pharmacokinetic factors between genders and races, the relevance of which is not currently known [32].
Despite no differences in urinary BPA between genders in our data, the male gender exhibited a signi cantly higher level of urinary dimethoate and BaP than the female gender (Table 3). Moreover, gender was considered as a signi cant predictor of the urinary level of dimethoate and BaP in the regression model. This may re ect the differences in the lifestyle between males and females.
Association of BMI and the urinary level of dimethoate, BaP, and BPA was additionally studied in regression models. In particular, BMI could be considered as a signi cant predictor of the urinary level of these three pollutants no matter whether be standardized by urinary creatinine or not (Table 4). Since most persistent organic pollutants (POPs) are lipophilicity [33], it has been widely studied that POPs could be stored in the adipose tissue [33][34][35]. Moreover, the accumulated POPs could also increase the risks of obesity and diabetes by inducing adipogenesis [36,37] and inhibiting glucose uptake [38]. This re ects that adipose tissue can act as a storage for most pollutants in our bodies. The stored pollutants in the adipose tissue can further enhance adipogenesis and insulin resistance. Together with our data, the increasing BMI and obesity can be a risk factor for higher pollutants accumulation in our body which could further act as the obesogens.
Besides the index of BMI, we found that the students from Southwest China had a signi cantly lower level of all three pollutants compared with East China which was considered as the control group (Table 2). Interestingly, there is no signi cant difference in BMI between students from Southwest China and East China. This re ects that the living environment and lifestyle may determine this difference. [39] Although there are very few reports to study the difference among various provinces in China, it has been reported that air pollution in North China is much worse than in South China [40]. This may partially explain our data and indicates that the living environment can determine the level of accumulated pollutants in our bodies. To investigate the relationship of the accumulated pollutants in our body and our health, further researches are needed to study the detailed differences in the living environment and lifestyle between people from Southwest China and other geographic regions in China. The baseline information of dimethoate, BaP, and BPA level will improve public health awareness and have important implications for health policy formulation.

Abbreviations
BaP: Benzo(a)pyrene; BPA: Bisphenol A; GM: Geometric means; BMI: Body Mass Index; PAHs: Polycyclic aromatic hydrocarbons; HHU: Hohai University; LC-MS: Liquid chromatography-mass spectrometry Declarations Ethics approval and consent to participate All procedures including sampling and examination were performed in agreement with the principles set in the Declaration of Helsinki and its later amendments (2013). All examinees were invited to participate and took part in the present study voluntarily. All subjects were informed about the objectives of the study and experimental procedures and signed the informed consent form. The study protocol has been reviewed and approved by the Ethical Review Committee of Sir Run Run Hospital, Nanjing Medical University (2019-SR-018).

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analysed during this study are included in this published article.
Competing interests