Introduction

Sulcotrione is a new kind of triketone-type herbicide, which inhibits the activity of plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) [1]. It has been widely applied to control and prevent wild grass and weeds for crops such as barley, wheat and maize in Europe, the USA and many other countries, including China, since 2000 [2]. HPPD also exists in mammals for the catabolism of tyrosine. Although sulcotrione shows low toxicity in subchronic and chronic toxicity testing, it can also, through the food chain, be continuously transferred to and accumulate in the human body when it is widely used and applied and when part of its residue in the soil is absorbed by and aggregated in plants. Another part of its residue pollutes the water table through surface runoff and the underground permeable layer. Therefore, sulcotrione, as an HPPD inhibitor, has potential risks to human health, and its possible role as an environmental pollutant must raise attention and vigilance. The residual level of sulcotrione in the soil directly affects its accumulation in agricultural products and is closely related to the level of human exposure. Currently, there is a standard for sulcotrione residue in the soil. Therefore, the investigation of the sulcotrione soil residual levels can provide scientific evidence for the rational application of sulcotrione and the establishment of pesticide residue standards. In addition, this investigation also can supply more data for soil pesticide monitoring databases and provide an informative source for scientific research.

HPPD, a target molecule of sulcotrione, exists universally in prokaryotes and eukaryotes. The catabolism of tyrosine is repressed when HPPD is inhibited by sulcotrione. Epinephrine is derived from tyrosine. The detection of tyrosine and epinephrine levels in the blood of rats exposed to sulcotrione can reflect the inhibiting effect of HPPD on epinephrine levels. The regulatory mechanisms for blood glucose are complicated. There are many possible pathways for sulcotrione to interfere with blood glucose levels. The rats were exposed to different doses of sulcotrione for different times to get blood glucose levels, enzyme activity, hormone levels, etc. The correlations between blood glucose levels and different parameters were analyzed in the rats exposed to sulcotrione to establish or exclude the possible interference of sulcotrione with blood glucose regulation.

In vitro experiments have already shown that sulcotrione can specifically, effectively and reversibly inhibit hepatic HPPD activity. However, there are few in vivo reports on this question. In this study, we investigate, the effects of sulcotrione on hepatic enzyme activity, tyrosinemia and cornea damage through subacute and chronic toxicity tests in rats.

2. Materials and methods

2.1 The investigation of sulcotrione soil residual levels

2.1.1 Soil sample collection

The soil samples were collected following the sampling rules of national farmland and the environmental standards for safety, high quality and pollution-free agricultural products GB/T18047.1-2001 from September 2007 to September 2008 within eight regions in the Zhejiang Province, P.R. China. The soil sampling depth was 0 to 20 cm. A single soil sample was a mixture of multiple (5) spots. A total of 200 soil samples were collected. At the same time, a total of 10 samples (from a mixture of 10 different regions) of cultured plants (corn) were collected in Quzhou and Dongyang in the Zhejiang Province.

2.1.2 Sample preparation

One gram samples (air-dried and passed through a 100 mesh sieve) were dissolved in 1 mL of methanol by mixing for 30 seconds. After incubation for 30 minutes, the sample was treated with ultrasound for 10 minutes. Then the sample was centrifuged for 5 minutes at 29000 g and the supernatant was collected and filtered through a membrane for detection.

2.2 Sulcotrione analysis

2.2.1 HPLC chromatographic conditions [3-4]

The mobile phase consisted of methanol:0.1M sodium dihydrogen phosphate triethylamine = 40:60 (v/v). The flow rate was 0.8 mL/min (A pump: 0.1M sodium dihydrogen phosphate triethylamine buffer 0.48 mL/min, B pumb: methanol 0.32 mL/min). The detection wavelength was 254 nm. The column temperature was room temperature. The sensitivity: was 0.001 AUFS. The injection volume was 10 ^l.

2.2.2 Sulcotrione standard curve

2.2.2.1 Preparation of the standard buffer

A 0.0100 g (accuracy to 0.0001 g) sulcotrione standard sample was accurately weighed. It was dissolved in a small volume of methanol which was poured into a 100 mL volumetric flask. It was diluted in methanol to the final scale. It was the standard stock sulcotrione solution. Before use, the stock solution was diluted in methanol to 10 mg/L as the working solution. The sulcotrione standard HPLC profile was made according to the above mentioned chromatographic conditions. The sulcotrione standard analysis sample was provided by Sigma and had a purity of more than 98% (the number is 46318, which is valid until March 2013).

2.2.2.2 Calculating the results

The sample analytic results were extrapolated based on the area external standard method. The concentration calculation was: X = solution volume x C/sample weight, where X is the sulcotrione concentration (mg/kg) and C is the concentration calculated from the standard curve.

2.2.3 Statistical analysis

SPSS 15.0 version was used for the statistical analysis. The results are presented as mean ± SD. SD is the standard deviation.

2.3 Investigation of tyrosine levels in a population who might have had exposure to sulcotrione.

2.3.1 Determining the population

The population consisted of forty males and forty females (80 totals) who work in the sulcotrione industry and another forty males and forty females who do not work in the sulcotrione industry; ten laboratory staff who have had contact with sulcotrione and 10 staff who do not have had contact were also included.

2.3.2 Blood collection.

The blood was centrifuged at 3000 g for 15 minutes. Then the supernatant was collected and cryopreserved at -18oC. The blood sample was prepared for sulcotrione detection as follows: The frozen serum was thawed at room temperature. A certain volume of the serum sample was transferred and mixed with the same volume of methanol. After this, it was treated with ultrasound for 10 minutes. And then the sample was placed on table for 10 minutes. After centrifuging at 29000 g for 5 minutes, the supernatant was passed through the membrane for detection. The blood sample was prepared for tyrosine detection as follows: a certain volume of sample was dissolved in the same volume of 0.59M HClC4. It was mixed for 30 seconds and centrifuged at 29000 g for 5 minutes. The supernatant was collected and passed through the membrane for detection.

2.3.3 Detection methods [5-7]

2.3.3.1 Standard materials

The analytical standard sulcotrione was purchased from Sigma as described as 2.2.2.1. The analytical standard tyrosine was provided by Dikma and had purity > 99.5% (the case No. is 0-110, which is valid until August of 2013).

The chromatographic conditions for tyrosine detection were: a mobile phase of acetonitrile:water = 5:95 (v/v); a 250 mm x 4.6 mm C18 column with particle size 5 ^m; a flow rate of 0.8 mL/ min; and a detection wavelength of 210 nm. The chromatographic conditions for sulcotrione detection were: a mobile phase of methanol:0.1 M sodium dihydrogen phosphate and triethylamine buffer = 40 : 60 (v/v); a 250 mm x 4.6 mm C18 column with particle size 5 ^m; a flow rate of 0.8 mL/min; and a detection wavelength of 254 nm. Tyrosine and sulcotrione were quantified using the extrapolation method. Under the chromatographic conditions suitable for instrument characteristics, tyrosine and sulcotrione were obtained in a linear correlation curve. The linear regression equation for sulcotrione was y = 20742.17x +655.12, with a correlation coefficient of 0.9993. The linear regression equation for tyrosine was y = 207412.2x-0.1092, with a correlation coefficient of 0.9985.

2.3.3.2 The precision, accuracy and detection limit

The detection precision, accuracy and detection limit for the sulcotrione and tyrosine detection methods were:

parameters

Precision (RSD, %)

Accuracy (recovery, %)

Detection limit

Sulcotrione

4.83-6.79

94.65-109.23

0.044 mg.L-1

Tyrosine

1.70-8.70

89.63-108.22

0.13 umol.L-1

RSD: relative standard deviation.

RSD: relative standard deviation.

According to the requirements of the testing methodology, the relative standard deviation (RSD) is less than 10% and the recovery rate is between 90-110%. The results show that HPLC method for sulcotrione and tyrosine detection meets the above requirement to ensure that the experimental data is accurate and reliable.

2.3.4 Statistical analysis

The statistical software SPSS15.0 was used for the one-way ANOVA analysis, least significant difference method (LSD) analysis and Dunnett's T test. The Pearson method is used for linear correlation analysis. The data are presented as mean ± SD.

2.4.The test of rats exposed to sulcotrione for 28 days to determine the time and response relationship between blood sulcotrione and tyrosine and main toxic response.

The original sulcotrione (purity > 95%) was provided by a domestic corporation. The acceptable daily intake (ADI) of 0.005 mg/kg, extrapolated from the results of a sulcotrione rat chronic toxicity test for no observed adverse effect level (NOAEL), was designated as the low-dose group; the medium-dose group was 0.05 mg/kg and the high-dose group was 0.5 mg/kg. A group without sulcotrione exposure was the control group. There were 28 rats in each group (equal numbers of males and females).

2.4.2 Route of exposure, time and test indicators

The rats were fed with sulcotrione dissolved in cooking oil once a day for 28 continuous days. The control group was fed cooking oil only. Blood was collected from the tail vein at days 0, 7, 14, 21, and 28 after sulcotrione exposure. The rats were not fed on the days of blood collection. At day 28, half the male and female rats were sacrificed and the sulcotrione feeding was discontinued for the remaining rats. At days 35 and 42, blood was collected from the tail vein. During the feeding, the body weight, diet, hair and activity of the rats were routinely checked and recorded. After they were sacrificed, the liver, kidneys, adrenal glands and other organs were collected for pathologic study. The organ and body weight ratio was calculated.

2.4.3 The time of administration, sampling and sample preparation.

Sulcotrione was given to rats at the same time each day (around 16:00). The rats were fasted from 21:00 the day before blood collections. The blood was collected at 8:00 the next day. The blood was centrifuged at 3000 g for 15 minutes. The serum was collected and stored at -18oC for cryopreservation. The blood sample was prepared for sulcotrione detection as follows. The serum was thawed at room temperature. An aliquot of serum was added to the same volume of methanol and mixed for 30 seconds. The mixture was treated with ultrasound for 10 minutes and placed on table for another 10 minutes. Then the sample was centrifuged at 29000 g for 5 minutes. The supernatant was collected and filtered through the membrane for detection. The blood sample was prepared for tyrosine detection as follows. The serum was thawed at room temperature. An aliquot of serum was added to the same volume of 0.59 M HClO4 and mixed for 30 seconds. The sample was centrifuged at 29000g for 5 minutes. The supernatant was collected and filtered through the membrane for detection. Blood glucose was directly measured from tail vein blood sampling.

2.4.4 The analysis method and quality control of detection indicators

The rat blood tyrosine and sulcotrione HPLC detection indicators were established. The accuracy, precision and detection limit for the detection method was under quality control (2.3.1). The blood glucose was detected by the Johnson & Johnson Rapid Blood Glucose Detector (USA). The instrument was calibrated with standard reference liquid before blood glucose detection. The original sulcotrione has more than 98.9% active component. The peak area extrapolation method was used for quantitative analysis of tyrosine and sulcotrione.

2.4.5 Statistical analysis

The statistical software SPSS15.0 was used for one way ANOVA analysis, least significant difference method (LSD) analysis and Dunnett's T test. The Pearson method was used for linear correlation analysis. The data are presented as mean ± SD.

2.5. Effects of sulcotrione on hepatic enzymes involved in tyrosine catabolism, tyrosinemia, and blood glucose in rat.

2.5.1 Animals and treatments

Sulcotrione with a purity of > 95.5% w/w, was supplied by Jia Hua Import & Export Co., Ltd (Zhejiang, China). male Alpk:APfSD (Wistar-derived) rats, aged from 5 to 6 weeks and obtained from Zhejiang Experimental Animal Center [SCXK (zhe) 2008-0033], were housed in stainless steel, wire bottom cages under standard housing conditions (controlled atmosphere with 12:12 h light/dark cycles, 55 ± 5% humidity and an ambient temperature of 22 ± 3°C). The rats were fed on a commercial powdered diet (GB 14924-2001) and given filtered water ad libitum. Rats were acclimatized for 3 days prior to the experiment. Groups of 8 sulcotrione-treated rats and 8 control rats were dosed with either corn oil alone or sulcotrione in corn oil at 5 ml/kg body weight at 0.1, 0.5, and 5 mg/kg/ day for 90 days. Throughout the study, clinical signs were observed, body weight was recorded weekly, and food consumption was monitored twice weekly. Animal care and monitoring were carried out in accordance with strict guidelines issued by the P.R. China legislation. All animal procedures and treatments were performed according to our Institute Animal Care and Use Committee (Certificate No. IACUC-03-001) and animals were terminated when deemed to be under moderate stress or discomfort.

2.5.2 Hepatic enzymes involved in tyrosine catabolism assay

The rats were sacrificed after the 90 days test by inhalation of an overdose of halothane as previously described. Livers were removed and then homogenized with 10 up-down strokes in 30 ml of ice-cold 0.32 M sucrose, and then diluted to give a 20% (w/v) homogenate. The homogenate was then centrifuged at 105,000 g for 60 min at 4°C to remove particulate material. The supernatant (cytosol fraction) was stored in aliquots at -70°C prior to the assay of activities of TAT, HPPD and HGO. The protein content in the supernatant was measured using bovine plasma albumin (BSA) as the internal standard. TAT was assayed in liver cytosol by the method of Schepartz, 1969. HPPD and HGO were measured in liver cytosol by monitoring oxygen consumption after addition of the relevant substrate by the methods described by Ellis et al., 1995, respectively.

2.5.3 Serum epinephrine and tyrosine analysis

After oral glucose tolerance test, femoral artery blood was adopted and then separated the serum. Serum epinephrine and tyrosine were measured by rat epinephrine ELISA kit

(Uscnlife Science & Technology Company, USA) and High Performance Liquid Chromatography (HPLC, SHIMADZU, LC-20AD), respectively.

2.5.4 Fasting blood glucose test and Oral glucose tolerance test

During treatment fasting blood glucose of tail vein in rat were measured in 0, 30, 60, 90 day (Fasting time: from 08:00 to 14:00) using fast blood glucose meter (Johnson & Johnson Services Inc. USA). After final dosing, all rats were fasting for overnight (about 12 hours), and then oral glucose tolerance test was adopted. We first measured blood glucose of tail vein in rat in 0 min, after that immediately giving glucose water solution (2 g/kg b.w ) , then blood glucose were measured in 30, 60, 120 min respectively.

2.5.5 Statistical analysis

The differences between sulcotrione-treated animals and controls were analyzed using SPSS Version 13.0 for Windows (SPSS Inc., Chicago, IL, USA). Changes in the concentration of tissue tyrosine or the enzymes involved in tyrosine catabolism in the time following sulcotrione treatment were analyzed using an analysis of variance followed by the Dunnett t test. A p-value below 0.05 was considered to be statistically significant between experimental groups

3.Results

3.1 Sulcotrione levels in the environment 3.1.1 Sulcotrione soil residual levels

Sulcotrione soil residual levels in the area where the sulcotrione was used in Zhejiang Province are summarized in table1-1 and figure1-1. The mean sulcotrione soil residue was between 0.19-0.47 mg/kg.

area

Quzhou

Wenzhou

Jiande

Dongyang

Haining

Ninghai

Longyou

Jinhua

Total

N

37

20

24

34

10

10

28

37

200

Mean ± sd

0.30±0.18

0.28±0.15

0.35± 0.19

0.33±0.19

0.19± 0.08

0.33±0.18

0.41±0.19

0.47±0.17

0.35±0.19

Table 1.1. Sulcotrione soil residual levels from the sampling area in Zhejiang province (mg/kg)

Table 1.1. Sulcotrione soil residual levels from the sampling area in Zhejiang province (mg/kg)

Figure 1-2 shows a plot of the distribution of the sulcotrione residual levels in the 200 soil samples against the expected normal probability distribution. The scatter graph follows an approximately straight line. So the test data follows a normal distribution. The arithmetic average of the sulcotrione soil residual levels in Zhejiang Province was 0.35 ± 0.19 mg/kg.

3.1.2 Sulcotrione corn residual levels

The sulcotrione residual levels in the 10 corn samples from the Quzhou and Dongyang areas are shown in Table 1-2. The detected values are between 0.02 to 0.10 mg/kg.

Fig. 1-1. The distribution of sulcotrione soil residual levels in Zhejiang Province (mg/kg, n: sample number)

Fig. 1-2. The normal distribution P-P plot for sulcotrione soil residue.

Observed Cum Prob

Fig. 1-2. The normal distribution P-P plot for sulcotrione soil residue.

Sample number

1

2

3

4

5

6

7

8

9

10

Average

0.03

0.06

0.05

0.04

0.02

0.08

0.07

0.10

0.05

0.07

Table 1-2. Sulcotrione corn residual levels (mg/kg)

Table 1-2. Sulcotrione corn residual levels (mg/kg)

Fig. 1-3. The normal distribution P-P plot of sulcotrione coin residue.

Observed Cum Prob

Fig. 1-3. The normal distribution P-P plot of sulcotrione coin residue.

Figure 1-3 shows a plot of the distribution of the sulcotrione residual levels in the 10 corn samples against the expected normal probability distribution. The scatter graph shows an approximately straight line. So the test data follows a normal distribution. The arithmetic average of the sulcotrione soil residual levels in Quzhou and Dongyang was 0.06 ± 0.02 mg/kg.

3.2 Human serum tyrosine level

We had 145 valid blood samples collected from the sulcotrione exposed population and the control population. The serum tyrosine concentration in the sulcotrione exposed population was 94.14 ± 19.67 nmol/ml for males (N = 37) and 98.85 ± 21.66 nmol/ml for females (N = 31). In the control population, the serum tyrosine concentration was 97.60 ± 16.27 nmol/ml for males (N = 35) and 100.2 ± 18.40 nmol/ml for females (N = 35). In laboratory staff who had contact sulcotrione, the serum tyrosine concentration was 82.02 ± 23.26 nmol/ml (male and female, N = 7), compared to 90.36 ± 19.27 nmol/ ml (male and female, N = 10) for laboratory staff who did not have contact with sulcotrione.

3.3 The test of rats exposed to sulcotrione for 28 days to determine the time and response relationship between blood sulcotrione and tyrosine and main toxic response.

3.3.1 The general situation of experimental rats

During the experiment, the body weight, diet, hair, activity in each dose group showed no significant difference from the control group.

3.3.2 The exposed rat liver, kidneys, adrenal glands and body weight ratio and pathological observations.

3.3.2.1 The exposed rat liver, kidney and adrenal gland and body weight ratios.

sex

group

Li ver/body

Kidneys/body

Adrenal glands/body

female

control

2.98

0.69

0.03

Low-dose

2.97

0.68

0.03

Medium-dose

2.90

0.66

0.03

High-dose

2.98

0.67

0.03

male

control

3.14

0.70

0.01

Low dose

2.98

0.68

0.01

Medium dose

3.00

0.71

0.02

High dose

3.08

0.71

0.02

All have p>0.05 in the comparison with the control group.

Table 2-1. The exposed rat liver, kidney and adrenal gland and body weight ratios.

All have p>0.05 in the comparison with the control group.

Table 2-1. The exposed rat liver, kidney and adrenal gland and body weight ratios.

Table 2-1 indicates that in both male and female rats, the liver, kidneys and adrenal glands and the body ratio are not significantly different than those of the controls.

3.3.2.2 Pathological observations on the liver, kidney and adrenal gland in each dose group.

Although there were a few rats in each dose group that showed inflammatory lesions in the liver and kidneys, there was no significant difference in comparison with the control group.

3.3.3 The serum tyrosine level changes in the male and female rats in each dose group after different exposure times.

At different sulcotrione exposure times, the rat serum tyrosine levels in the medium- and high-dose groups were significantly higher than those in the low-dose and control groups. The serum tyrosine levels in the high-dose group were significantly higher than those in the medium-dose group. The serum tyrosine concentrations[figure3-1] in the medium- and high-dose groups increased with prolonged exposure before day 21 (the absorption phase). Then they remained relatively stable (the stable phase). After day 28, when exposure ceased, they began to decrease until day 42 (14 days of no exposure). However, they were still higher than the levels of the control at day 42. There were no significant differences in serum tyrosine levels between male and female rats.

1400 1200 1000 800 600 400 200 0

Control 0. 005mg/kg 0. 05mg/kg 0. 5mg/kg

Î0 7 14 21 28 35 42 ¿ 0 7 14 21 28 35 42 Ti«e (days)

Fig. 3-1. The serum tyrosine level changes in the male and female rats in each dose group after different exposure times.

Control 0. 005mg/kg 0. 05mg/kg 0. 5mg/kg

£0 7 14 21 28 35 42 ¿ 0 7 14 21 28 35 42 Time (days)

Fig. 3-2. The serum sulcotrione level changes in the male and female rats in each dose group after different exposure times.

The serum sulcotrione in the low-dose group was similar to that in the control group. The serum sulcotrione levels in the males and females were both below the detection limit. The serum sulcotrione was detected in the medium- and high-dose groups, and its level increased with increasing exposure over time and had a statistically significant difference from control. The serum sulcotrione concentration in the medium- and high-dose groups increased with prolonged exposure[figure3-2], but decreased significantly after no exposure. There were no significant differences between male and female rats.

Although there existed individual statistical differences in blood glucose levels between the dose groups and the controls, the values of the changes were all within the normal range [figure3-3]. There were no significant differences in blood glucose changes between male and female rats.

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