Journal of Hazardous Materials 295 (2015) 9–16

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Monitoring tryptophan metabolism after exposure to hexaconazole and the enantioselective metabolism of hexaconazole in rat hepatocytes in vitro Yao Wang a,1 , Wentao Zhu a,1 , Jing Qiu b , Xinru Wang a , Ping Zhang a , Jin Yan a , Zhiqiang Zhou a,∗ a

Department of Applied Chemistry, China Agricultural University, Beijing 100193, China Institute of Quality Standards and Testing Technology for Agro-Products, Key Laboratory of Agro-Product Quality and Safety, Chinese Academy of Agricultural Sciences, Beijing, China b

h i g h l i g h t s • The metabolic process of hexaconazole was enantioselective. • Hexaconazole and its enantiomers caused the down-regulation of tryptophan levels. • Hexaconazole and its enantiomers caused the up-regulation of kynurenine levels.

a r t i c l e

i n f o

Article history: Received 11 July 2014 Received in revised form 24 March 2015 Accepted 2 April 2015 Available online 3 April 2015 Keywords: Hexaconazole Enantioselevtive metabolism Cytotoxicity Tryptophan metabolism

a b s t r a c t In the present study, the enantioselective metabolism, cytotoxicity of hexaconazole and its influence on tryptophan metabolism in rat hepatocytes in vitro were investigated. Following the exposure of primary rat hepatocytes to rac-hexaconazole, the concentrations of its enantiomers in the media were determined by chiral high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). The halflives (t1/2 ) of (+)-hexaconazole and (−)-hexaconazole were 5.17 h and 19.80 h, respectively, indicating that the metabolic process was enantioselective with (−)-hexaconazole enrichment. Using the MTT method, the EC50 values of rac-hexaconazole, (+)-hexaconazole and (−)-hexaconazole after 12 h of exposure were determined to be 71.62, 62.71 and 67.94 ␮M, respectively. Tryptophan metabolism was monitored using metabolomics profiling techniques. Hexaconazole and its enantiomers caused the down-regulation of tryptophan levels and the up-regulation of kynurenine (KYN) levels, suggesting a role for hexaconazole in the activation of the KYN pathway and providing information for the mechanism of its toxicity. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Hexaconazole [(RS)-2-(2,4-dichlorophenyl)-1-(1H-1,2,4triazol-1-yl) hexaconazolean-2-ol] is a triazole fungicide with outstanding activity against a wide variety of diseases, and its preventive, curative, systemic and antisporulant properties provide a useful addition to the range of commercial fungicides [1]. It is known that hexaconazole inhibits cytochrome P-450-mediated oxidative demethylation reactions, which are necessary for sterol synthesis in many fungi [2]. However, in addition to fungi, other

∗ Corresponding author. Tel.: +86 10 62733547; fax: +86 10 62733547. E-mail address: [email protected] (Z. Zhou). 1 These authors contributed equally to this paper. http://dx.doi.org/10.1016/j.jhazmat.2015.04.006 0304-3894/© 2015 Elsevier B.V. All rights reserved.

P450-mediated activities were also affected, resulting in various adverse effects [3]. Hexaconazole has been confirmed to alter the transcription of genes involved in developmental toxicity pathways [4]. Moreover, the US EPA has listed this compound as a Group C-possible human carcinogen [5]. Hexaconazole is extremely persistent, and no apparent degradation of hexaconazole was observed in river water incubated at 20 ◦ C for 3 weeks [6]. In addition, the DT50 for field soil degradation was 225 day [5]. Accordingly, non-target organisms can potentially be exposed to hexaconazole, resulting in adverse health effects. Hexaconazole has an asymmetrically substituted carbon atom and consists of a pair of enantiomers (Fig. 1). The enantiomers of chiral pesticides have been shown to differ in their toxicities, absorption process or biological macromolecule–mediated metabolisms [7]. For example, (+)-hexaconazole was less toxic

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Fig. 1. Chemical structures of hexaconazole enantiomers.

than (−)-hexaconazole against Scenedesmus obliquus [8] and (+)hexaconazole degraded faster than (−)-hexaconazole resulting in an enrichment of (−)-hexaconazole in tomato [9]. Nevertheless, the behaviour of the enantiomers’ in humans and other higher animals has remained unclear. To fully assess the risks of hexaconazole to humans and other higher animals, a careful research on its enantioselective metabolism, toxicity and the possible mechanisms by which this compound affects non-target organisms is necessary. In vitro and in vivo tests are two main methods used in studying toxicity. Due to the large amount of experimental animals needed for regulatory toxicity testing [10], there is increasing need to develop, refine, and establish in vitro screening assays to replace classical in vivo tests [11]. Hepatocytes isolated from animals are valuable models used in short-term predictive assays to establish metabolic routes of various chemicals and to uncover their toxic mechanisms [12]. In chemically defined culture conditions, isolated rat hepatocytes express most of the typical hepatic biochemical functions [13] and have proven to be a valuable model for studying liver metabolism and chemical carcinogenesis [14–16]. Therefore, in the present study, primary rat hepatocytes were used as a model for evaluating the enantioselective metabolism and toxicity of hexaconazole. Toxicology approaches might be incorporated into alternative test systems to further increase the predictivity of these tests and to provide mechanistic insights. Using a cytogenetic assay in human lymphocytes, an unscheduled DNA synthesis test in rat hepatocytes, and a micronucleus test in C57/BL/6J mice bone marrow cells, Yilmaz et al. found that hexaconazole had negative genotoxic effects [17]. Hexaconazole has been shown to be developmentally toxic in vivo [18–20] and affected the reproductive performance of female rats [21]. Because the exploration of metabolic responses is proven to be a good means of understanding toxicity mechanism and disease, the use of this approach has increased rapidly in recent years [22]. Traditional metabolism research is focused on the exogenous metabolism, whereas metabolomics is used to perform a comprehensive and quantitative analysis of wide arrays of metabolites in biological samples. Thus, metabolomics could simultaneously give insight into both the internal and external metabolism. This approach could be widely used to study the mechanisms of action by which pesticides affect non-targeted creatures. Two complementary approaches are used for metabolomic investigations: metabolic profiling and metabolic fingerprinting. Metabolic profiling focuses on the analysis of a group of metabolites that are either related to a specific metabolic pathway or a class of compounds, whereas metabolic fingerprinting classifies samples based on the metabolite patterns or “fingerprints” of metabolites that change in response to disease, toxin exposure, or environmental and genetic alterations [23]. Metabolic profiling analysis has been successfully adopted in the evaluation of tryptophan metabolism [24]. Tryptophan catabolism is involved in the immune

response [25], and the tryptophan metabolism has been proven to be affected in various diseases, including HIV infection [26], Adult T-cell leukaemia [27], Parkinson’s disease [28] and feto-maternal tolerance [29]. Moreover, tryptophan depletion acts as an antimicrobial or antitumoral mechanism [30]. As a possible carcinogen, hexaconazole may have the potential of affecting the immune system. However, to our knowledge few studies have evaluated the effects of fungicides, including hexaconazole, on tryptophan metabolism thus far. A comprehensive evaluation of an organism’s response to a perturbation, will lead to a better understanding of the biological and biochemical mechanisms of complex systems. Therefore, the primary aims of this research include the following: (1) study the enantioselective metabolism of hexaconazole in the primary rat hepatocytes model; (2) investigate the cytotoxicity of hexaconazole; (3) and obtain insight into the effects of hexaconazole on endogenous tryptophan metabolism in relation to the immune response. 2. Materials and methods 2.1. Chemicals and reagents Rac-hexaconazole standard (purity > 99%) was purchased from Sigma–Aldrich (St. Louis, MO). (+)- and (−)-hexaconazole (purity > 99%) were prepared on an Agilent HPLC system with a chiral column containing cellulose-tri(3,5-dimethylphenylcarbamate) (CDMPC-CSP) provided by the Department of Applied Chemistry (China Agricultural University, Beijing). Stock solutions of the racemic standard were prepared in DMSO and were diluted to standard solutions. Rat tail collagen, heparin and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Foetal calf serum (FCS), insulin, N(2-hydroxyethylpiperazine)-N -(2-ethanesulfonic acid) (HEPES), collagenase, antibiotics (penicillin–strepto-mycin mixtures) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from GIBCO (USA). Unlabelled tryptophan, quinolinic acid (QA), xanthurenic acid (XA), serotonin hydrochloride, indole-3-acetic acid (IAA) and tryptamine were obtained from Aladdin Reagent (China). Indole-3-propionic acid (IPA), 3-hydroxyanthranilic acid (HAA), 5-hydroxyindole-3-acetic acid (HIAA), 3-hydroxykynurenine (HK), kynurenine (KYN), kynurenic acid (KA), DL-indole-3-lactic acid (ILA) and melatonin were purchased from Sigma–Aldrich (St. Louis, MO). 2 H5 -tryptophan, 2 H2 -tryptamin, 2 H2 -IAA, 2 H2 -HIAA, 2 H -KA and 2 H -serotonin were purchased from C/D/N Isotopes 5 4 (Canada). 2 H4 -melatonin was obtained from T/R/C (Canada). 2 H -KYN and 2 H -HAA were obtained from Buchem (Apeldoorn, 4 2 the Netherlands). Acetonitrile and methanol (HPLC grade) were purchased from Sinopharm Chemical Reagent (Beijing, China).

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Fig. 2. Representative LC–MS/MS chromatograms of hexaconazole enantiomers extracted from rat hepatocytes incubated with 2 ␮M rac-hexaconazole for 1 h (a) and rat hepatocytes incubated with 2 ␮M rac-hexaconazole for 24 h (b).

All other reagents were of analytical grade and obtained from commercial sources. Water was purified with a Milli-Q system.

2.2. Preparation of the internal standard and stock solutions The preparation of the internal standard and stock solutions were performed in accordance with a previously published method [24]. Stock solutions of each deuterated internal standard were prepared in water with 0.1% (v/v) formic acid and stored at −80 ◦ C. The stock solutions of the unlabelled analytes were prepared in either water with 0.1% formic acid, water with 0.1 M NaOH, methanol or acetonitrile at concentrations of approximately 104 ␮M based on their solubility. The concentrations of the internal standards combined in a working solution were 50 ␮M (2 H4 -KYN, 2 H5 KA, 2 H2 -HIAA, 2 H2 -HAA, 2 H2 -IAA), 200 ␮M (2 H5 -tryptophan) and 10 ␮M (2 H2 -tryptamin, 2 H4 -melatonin, 2 H4 -serotonin). A master mix containing unlabelled standards of tryptophan (2.5 mM) and the other compounds (250 ␮M each) was diluted from 40 ␮M to 25 nM. For calibration, 10 ␮L of the internal standard solution was transferred into a 200-␮L volume micro-insert in a 1.5-mL glass vial (Fisher Scientific) and then diluted to100 ␮L with the respec-

tive aqueous calibration standard. Because stable isotope-labelled reagents were not available for HK, HAA, XA, IPA and ILA, the area ratios for HK, HAA, XA, IPA and ILA were calculated using 2 H -serotonin, 2 H -IAA and 2 H -melatonin, respectively, whereas, 4 2 4 2 H -tryptamin was used to calculate the area ratio for HAA and XA. 2 The use of internal standards helped to provide the best recoveries and precision for these analytes.

2.3. Instrumentation and chromatographic conditions An Agilent 1200 series HPLC (Santa Clara, CA, USA) coupled to an API 2000 triple quadrupole mass spectrometer (AB Sciex Instruments, Foster, CA) with a Turbo Electrospray Ionization (ESI) probe was used. The multiple reaction monitoring (MRM) scan mode was used to detect hexaconazole enantiomers and the target compounds of tryptophan metabolism in the cell culture medium. The Analyst v.1.5.1 software (Applied Biosystems/MDS SCIEX Instruments) was used for the data acquisition and analysis. A Waters (Eschborn, Germany) Atlantis T3 (2.1 × 150 mm i.d. 3 ␮m) reversed-phase column kept at 25 ◦ C was used for the LC separation of tryptophan metabolites. All analytes were detected

Fig. 3. Curves showing the concentration (a) and EF (b) over time for hexaconazole enantiomers after incubation of rat hepatocytes with 2 ␮M rac-hexaconazole. The results are presented as the means ± standard deviations (n = 3).

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Fig. 4. Curves showing the concentration (a) and EF (b) over time for hexaconazole enantiomers after incubation of rat hepatocytes with 1 ␮M (+)-hexaconazole or 1 ␮M (−)-hexaconazole. The results are presented as the means ± standard deviations (n = 3).

in positive ion mode. The chromatographic separation of the analytes was achieved using a linear gradient at a constant flow rate of 0.3 mL/min with mobile phases A (0.1% formic acid in water, v/v) and B (0.1% formic acid in acetonitrile, v/v) as follows: 0–6 min linear increase from 0% to 40% B, 6–8 min from 40% to 95% B, 8–8.1 min from 95% to 0% B, and hold at 0% B for 5 min. The injection volume was 20 ␮L. The enantiomers were separated on a chiral Lux 5 ␮ cellulose1 column (250 × 4.6 mm, Phenomenex, Torrance, CA) with mobile phases 30% (0.1% formic acid in water, v/v) and 70% (0.1% formic acid in acetonitrile, v/v) at a flow rate of 0.5 mL/min. The column was kept at 25 ◦ C and the injection volume was 20 ␮L. The enantiomers were also detected in positive ion mode. All MS parameters were optimised for each analyte. The transitions of m/z 314.1/70.0 was used for the quantitative analysis and 314.1/158.9 for the qualitative analysis. The MRM parameters for tryptophan, its metabolites and hexaconazole are listed and summarised in Appendix A: Supplementary Tables S1 and S2. 2.4. Animals Adult male Sprague–Dawley rats (130–150 g) were supplied by Vital River Laboratory Animal Company (Beijing, China). Animals were housed in a humidity and temperature-controlled room with a 12 h light/dark to acclimate them to the environment. Animals were fed a standard diet and drinking water throughout the study and were fasted 12 h before the experiment. 2.5. Isolation of hepatocytes To anaesthetise the rats, sodium pentobarbital (1%, 40 mg/Kg) was administered to the rat via intraperitoneal injection. Rat hepatocytes were obtained using a two-step collagenase perfusion method, as described in the literature [31]. Firstly, heparin (1000 U/mL) was injected to prevent blood clots and then the liver was perfused in situ with 500 mL of HEPES buffer solution (20 mM) without calcium to remove the blood and to weaken the cell–cell junctions. Secondly, the hepatocytes were separated from each other and released using collagenase (50 mg/100 mL). The recirculation perfusion system was used for 20 min. Finally, the liver was

broken, sifted, and centrifuged at the low speed of 500 × g for 3 min. The isolated hepatocytes were counted using a microscope, suspended in DMEM containing 10% FCS, 10 ␮g/mL insulin, 100 U/mL penicillin, and 100 ␮g/mL streptomycin, and incubated in a 5% CO2 incubator. The viability of the cells was measured using trypan blue exclusion and was found to be greater than 90%. A light microscope and a Countstar automated cell counter (Shanghai Ruiyu Biotech Co., Ltd.) were used to count the cells, study the cellular morphologies and make identifications via comparison to the results from previous studies [32–34]. Light micrographs of primary rat hepatocytes are presented in Appendix A: Supplementary Figs. S1 and S2. 2.6. Metabolism study 1.5 mL of the hepatocyte suspension (106 cell/mL) was plated into each well of a 12-well collagen-I-coated plate. The hepatocytes were then incubated for 18–24 h in a humidified incubator at 37 ◦ C with 5% CO2 to allow the formation of the monolayers. After attachment, the medium was replaced with fresh medium containing the added chemical (2 ␮M rac-hexaconazole, 1 ␮M (+)or (−)-hexaconazole). The exposure dose was chosen based on the results of the cytotoxicity experiments. Three replicate samples (1 mL each) were removed from each treatment to 2 mL tube at different time points (0–24 h). After centrifugation at 10,000 rpm for 2 min, the reaction was immediately quenched and the samples were stored at −20 ◦ C. A mixture of 0.5 mL of acetonitrile and 0.5 mL supernatant was mixed and vortexed for 3 min. Then, the mixture was centrifuged at 12,000 rpm for 4 min at 4 ◦ C and the clear solution was decanted into sample vials. 2.7. Cytotoxicity study 200 ␮L of fresh isolated rat hepatocytes of were seeded in each of the inner 60 wells of a 96-well collagen-I-coated plate at 3 × 104 cells per well. 200 ␮L buffer solution was added to each of the peripheral 36 wells to maintain a moisture balance. Due to the minimal effect of DMSO on the MTT assay, DMSO was diluted and used at a final concentration of 1% in the cell culture medium. 18–24 h

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Fig. 5. Tryptophan concentration (a), KYN concentration (b) and KYN/tryptophan ratio (c) obtained at different time points from the culture supernatants of rat hepatocytes incubated with 2 ␮M rac-hexaconazole, 1 ␮M (+)-hexaconazole or 1 ␮M (−)-hexaconazole. The results are presented as the means ± standard deviations (n = 3). Asterisks represents statistically significant differences compared to the controls: *p < 0.05 and **p < 0.01 levels.

after cell attachment, the medium was replaced with 100 ␮L of fresh DMEM without serum containing the test compounds (30, 50, 70, 100, 120 ␮M for rac-, (+)- and (−)-hexaconazole), blank DMEM without serum (control), and with DMSO (vehicle control). For each control and test concentration, at least three replicate wells were designed. Vehicle control wells only received 1.0% DMSO. After 12 h of exposure to the experimental compounds, ten microliters of MTT solution (5 mg/mL) was added to each well. The plate was incubated at 37 ◦ C for 4 h and the MTT was sufficiently metabolised to generate formazan. Next, the medium was replaced with 100 ␮L of DMSO per well to dissolve the formazan. The degree of the tetrazolium salt MTT that was converted to formazan by mitochondrial succinic dehydrogenase was used to evaluate the mitochondrial function [35]. The plates were shaken for 10 min and the absorbance was read at 570 nm and 630 nm (for background subtraction) with a spectrophotometer (Multiskan MK3, Thermo Scientific, Pittsburgh, PA). The cell viability was calculated as a

percentage of the corresponding vehicle control value. The EC50 values were determined from the survival data using a probit equation with SPSS 16.0.

2.8. Tryptophan metabolism study The cell culture supernatants were thawed in ice. 10 ␮L of the internal standard mix was added to 100 ␮L of the cell culture supernatants and vortexed. The mixture was mixed with 400 ␮L cold methanol and vortexed again. It was then placed at −20 ◦ C to ensure a total precipitation of the protein. After standing for 30 min, the mixture was centrifuged two times for 10 min (10,000 × g; 4 ◦ C) to obtain a clear extract. Finally, the supernatant was dried under flowing nitrogen at 30 ◦ C. The residue was dissolved using 50 ␮L of 0.1% formic acid in water and centrifuged for 5 min (5000 × g; 4 ◦ C) to obtain a clear extract. The sample volume injected was 20 ␮L.

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2.9. Data analysis

3.2. Stability of substrates in medium

The metabolism of both hexaconazole enantiomers appeared to follow a pseudo-first-order kinetic reaction. The degradation rate constants (k) for the experiments were derived from the “In(c0 /c) versus t” plots obtained from curve fitting using Excel 2010 (Microsoft, Inc.). The starting point was the maximum value of the concentration. The half-life (t1/2 , h) was estimated from Eq. (1):

Rac-hexaconazole, (+)- and (−)-hexaconazole were used as substrates in DMEM without hepatocytes to evaluate the degradation of (+)-hexaconazole and (−)-hexaconazole. In the processing of 2 ␮M rac-hexaconazole, for example, the final concentration of rac-hexaconazole, (+)- and (−)-hexaconazole were 1.62, 0.80 and 0.82 ␮M, respectively. The reduced small amount of hexaconazole possibly resulted from the experimental operation was in the error-allowed range. Given the observations, it was concluded that the reduced small amount of hexaconazole in the media without hepatocytes was negligible. This was in contrast to the significant metabolism of hexaconazole in the media containing hepatocytes.

t1/2 =

< MML : MNMATHVARIANT = "NORMAL" > 0.693 k

(1)

The enantiomer fraction (EF) was used as a measure of the stereoselective metabolism of the hexaconazole enantiomers. EF was calculated from the peak areas of (+)-hexaconazole and (−)hexaconazole, as defined in Eq. (2): EF = (−)-hexaconazole/[(+)-hexaconazole + (−)-hexaconazole](2) The EF for a racemic mixture is 0.5, whereas the preferential metabolism of the (−) or (+) enantiomer yields EF < 0.5 or EF > 0.5, respectively. Significant differences were determined using Students’ t-test. P values < 0.05 were considered statistically significant when compared to the controls. The statistical analyses were conducted using SPSS 16.0.

3. Results and discussion 3.1. Assay validation The limit of quantification (LOQ) was defined as the lowest concentration in the calibration curve that provided acceptable precision and accuracy (the acceptance criteria for the LOQ were that the precision and accuracy of the extracted samples had less than 20% variability). The linearity of hexaconazole was evaluated over the range of 0.02–2 ␮M by dilution of the working standard solution of rachexaconazole. Linear calibration curves were obtained by plotting the peak areas of each enantiomer versus the concentrations of the enantiomers. Linear regression analysis was performed using Origin Pro 8.5. Both hexaconazole enantiomers showed excellent linearities, with correlation coefficients of greater than 0.99. The limit of quantification (LOQ) was 0.25 ␮M for tryptophan and 0.025 ␮M for the other analytes involved in tryptophan metabolism.

3.3. Metabolism of hexaconazole The metabolism of hexaconazole and its enantiomers in primary rat hepatocytes was investigated. Fig. 2 shows the representative chromatograms of (+)- and (−)-hexaconazole enantiomers obtained by incubating the hepatocytes with 2 ␮M rac-hexaconazole. The time-dependent metabolism of 2 ␮M rachexaconazole is shown in Fig. 3a. Nonlinear regression analysis was performed using a regression equation that was in accordance with the first-order kinetics equation, in which y = 0.7626e−0.035x and R2 = 0.9931 for (+)-hexaconazole or y = 0.7246e−0.134x and R2 = 0.9989 for (−)-hexaconazole. The t1/2 of (+)-hexaconazole was 5.17 h, whereas t1/2 of (–)-hexaconazole was 19.80 h. Thus, the metabolic kinetic parameters demonstrated the existence of significant enantioselectivity between the two enantiomers. Using the residual concentrations of the two enantiomers, the EF was calculated as 0.53 at 1 h, which increased to 0.91 at 24 h (Fig. 3b) in a time-dependent manner. Overall, hexaconazole was enantioselectively metabolised in primary rat hepatocytes. Furthermore, (+)-hexaconazole and (−)-hexaconazole were used as substrate for hepatocyte cultures to test their configuration stability, separately. Fig. 4a and b shows the concentration-time and EF curves, respectively. No chiral conversion or transformation was observed. This observation is consistent with the chiral aspects of hexaconazole demonstrated in previous studies that used rabbit [36] and rat microsomes [37]. The major biotransformation pathway of hexaconazole has been partially studied. Skidmore et al. demonstrated that temperate cereals metabolised hexaconazole by oxidising the alkyl side chain [38]. Majur and Kenneke showed that the metabolism of hexaconazole differed between rat and rainbow trout hepatic microsomes and purified human CYP 3A4, though all of the phase I

Fig. 6. Representative LC–MS/MS chromatograms of tryptophan and its targeted metabolites for: a standard sample from the linear of tryptophan and its targeted metabolites (a) and the extract from the culture supernatant of rat hepatocytes incubated with 2 ␮M rac-hexaconazole for 24 h (b). Keys: 1, HK; 2, KA; 3, KYN; 4, HAA; 5, tryptophan; 6, tryptamine; 7, XA; 8, KA; 9, HIAA; 10, ILA; 11, melatonin; 12, IAA; 13, IPA.

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metabolites acquired were the oxidised alkyl side chain products [39]. In general, the oxidation of the alkyl side chain is the primary way in which hexaconazole is metabolised. Further studies are therefore needed to identify the metabolites of hexaconazole produced by rat hepatocytes for a more comprehensive environmental risk assessment of hexaconazole. The enantioselective behaviour of hexaconazole in rat hepatocytes may be associated with P450 enzymes, which play important roles in the metabolism. It has been reported that recombinant rat P450 2B1 enantioselectively metabolises PCB 136 into OHPCB metabolites in rat microsomes [40]. Kania et al. demonstrated that the different enantioselective behaviours of heptachlor in rat microsomes depended on different CYP enzymes [41]. Further research should be performed to study the mechanism of enantioselective metabolism. 3.4. Cytotoxicity of hexaconazole In the present study, the MTT assay was used to assess the effects of rac-hexaconazole, (+)- and (−)-hexaconazole on the mitochondrial function of primary rat hepatocytes. Although cytotoxicity investigations are instrumental for environmental risk assessment, few studies have been conducted that directly investigate the cytotoxic effects of a chiral pesticide in an in vitro hepatocyte model. The cytotoxicity of rac-hexaconazole and its two enantiomers was exhibited in a concentration-dependent manner over a range from 0 to 120 ␮M, indicating that hexaconazole dose-dependently diminished the reduction of MTT. There were no significant differences between rac-, (+)- and (−)-hexaconazole (P > 0.05). The EC50 values for these compounds were calculated using the MTT assay and are shown in Table 1. In the comprehensive environmental risk assessment of a given compound, the studies of cytotoxicity and metabolism complement each other [42]. Researching the differences in the metabolism of two enantiomers involves the comparison of their actual toxic effects, while the cytotoxicity investigations provide an overall assessment of the toxic effects of the compound. Thus, a combined approach that investigates the cytotoxicity and metabolism of hexaconazole can provide an integrative and objective risk assessment for this particular compound. 3.5. Tryptophan metabolism Tryptophan and its metabolites are known to affect the immune response via many different pathways. In this study, the levels of tryptophan and its metabolites were measured to determine the effects of hexaconazole on the immune-related responses in primary rat hepatocytes. Of the thirteen target analytes, tryptophan and KYN were the two analytes that were analysed. The other analytes were below the limits of quantification due to the pretty low concentrations of these analytes present in the culture suspension. In the culture supernatants of rat hepatocytes exposed to rac-hexaconazole, (+)- and (−)-hexaconazole, the tryptophan levels clearly decreased (Fig. 5a) whereas the levels of KYN obviously increased (Fig. 5b) compared to the control. Consequently, the KYN/tryptophan ratio displayed a significant time-dependent increase relative to the control (Fig. 5c). Overall, the results of Table 1 Calculated EC50 values of rac-hexaconazole, (+)-hexaconazole and (−)-hexaconazole in primary rat hepatocytes. Chemical Rac-hexaconazole (+)-Hexaconazole (−)-Hexaconazole a

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this study suggested that tryptophan was mainly metabolised via the KYN pathway in primary rat hepatocytes. The representative LC–MS/MS chromatogram of tryptophan and its targeted metabolites extracted from the culture suspension of hepatocytes incubated with 2 ␮M rac-hexaconazole for 24 h is shown in Fig. 6. The first step of tryptophan metabolism consists of a catalytic reaction of TDO (tryptophan 2,3-dioxygenase) or IDO (indoleamine 2,3-dioxygenase) to form N-formylkynurenine [43]. This is the ratelimiting step of tryptopan metabolism. TDO is highly expressed in the liver, whereas IDO is expressed everywhere in the body, except the liver [43–45]. Given this information, we speculated that the effect of hexaconazole on tryptophan metabolism resulted from the activation of TDO, which should be further researched. The KYN/tryptophan ratio is usually regarded as an indicator of TDO. The role that TDO plays in the immune response has been a hot topic in recent research. Schmidt et al. demonstrated that TDO had antimicrobial properties and acted as a mediator of immunoregulatory effects, and the TDO-dependent inhibition of T-cell growth might be related to the immune tolerance [29]. Opitz et al. showed that TDO mediated tumour progression via the generation of KYN, which is an endogenous ligand of the aryl hydrocarbon receptor (AHR) [46]. Schizophrenia research by Miller et al. has confirmed the increased expression of TDO in this disease [47]. Research by Tatsumi et al. revealed that TDO was involved in the process of implantation due to the induction of TDO in the decidualised stromal cells [48]. Moreover, an unexpected expression of TDO was demonstrated in a range of human tumour samples, including hepatic, lung and breast carcinoma [45]. Given the important roles that TDO plays under different pathological conditions, the elevated levels of TDO induced by hexaconazole provided an insight into the mechanism by which hexaconazole exerted its toxicity. KYN is closely related to the immune response. Acting as an endogenous ligand of the human AHR, KYN plays a vital role in the suppression of antitumour responses and thus results in the promotion of tumour-cell survival and motility via AHR [46]. The xenobiotic compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), alters the gene expression in cells via the activation of AHR [49]. Similarly, the up-regulated KYN levels suggested that similar to TCDD, hexaconazole probably exerted a toxic effect via the activation of AHR. Overall, this study suggests an important, but currently overlooked, effect of hexaconazole on tryptophan metabolism in rat hepatocytes. 4. Conclusion In this study, the enantioselective metabolism and cytotoxicity of hexaconazole as well as its effects on the endogenous tryptophan metabolism in relation to the immune response were investigated by exposing primary rat hepatocytes to hexaconazole and its enantiomers. The results demonstrated that the metabolic process was significantly enantioselective, with enrichment of (−)-hexaconazole. The down-regulation of tryptophan levels and up-regulation of KYN levels suggested that hexaconazole and its enantiomers acted as activators of the KYN pathway. The timedependent up-regulation of the KYN/tryptophan ratio provided information for the mechanism by which hexaconazole exerted a toxic effect. The corresponding results stressed the toxic effects of hexaconazole on rat hepatocytes and provided mechanistic insights, which may be useful for assessing the hazards that hexaconazole poses to humans.

EC50 (␮M)a 71.62 ± 11.52 62.71 ± 7.55 67.94 ± 8.52

Values represent the means ± standard deviations (n = 3)

Acknowledgements We gratefully acknowledge the financial support from the National Natural Science Foundation of China (Contract grant

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