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Toxicity of diazinon and its metabolites increases in diabetic rats
Toxicology Letters 170 (2007) 229–237
Toxicity of diazinon and its metabolites increases in diabetic rats Jun Ueyama a,b , Dong Wang b , Takaaki Kondo b , Isao Saito c , Kenji Takagi b , Kenzo Takagi b , Michihiro Kamijima d , Tamie Nakajima d , Ken-Ichi Miyamoto a , Shinya Wakusawa b , Takaaki Hasegawa e,∗ a
d
Department of Medicinal Informatics, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan b Department of Medical Technology, Nagoya University School of Health Sciences, Nagoya, Japan c Food Safety and Quality Research Center, Tokai COOP Federation, Aichi-gun, Aichi, Japan Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya, Japan e Department of Pharmacy and Pharmacokinetics, Aichi Medical University School of Medicine, Nagakute-cho, Aichi-gun, Aichi 480-1195, Japan Received 19 January 2007; received in revised form 19 March 2007; accepted 19 March 2007 Available online 24 March 2007
Abstract The effect of diazinon (DZN) on the activities of cholinesterase (ChE) in plasma and acetylcholinesterase (AChE) in erythrocyte and brain was investigated in normal and streptozotocin-induced diabetic rats. Hepatic drug-metabolizing enzyme activity was also estimated by measuring the systemic clearance of antipyrine, and the expression of hepatic cytochrome P450 (CYP) 3A2 and CYP1A2, which is closely related to the metabolism from DZN to DZN-oxon, a strong inhibitor of both ChE and AChE. No significant differences in the activities of ChE in plasma and AChE in erythrocyte were observed between normal and diabetic rats. Treatment with DZN significantly decreased these activities in diabetic rats more than in normal rats 6 h after injection (6.5 mg/kg). Treatment with DZN significantly decreased the activity of AChE in brain of diabetic rats than normal rats 3 h after injection (65 mg/kg), although no significant difference in the activity was found between normal and diabetic rats. The urinary recovery of diethylphosphate (DEP), a metabolite of DZN-oxon, was significantly increased in diabetic rats, but that of diethylthiophosphate (DETP), a metabolite of DZN, was unchanged. Significant increases in the systemic clearance of antipyrine and protein levels of hepatic CYP1A2, not CYP3A2, were observed in diabetic rats. These results suggest the possibility that a metabolite of DZN, DZN-oxon, causes higher toxicity in diabetic rats due to the enhancement of hepatic CYP1A2-mediated metabolism of DZN. © 2007 Published by Elsevier Ireland Ltd. Keywords: Organophosphorus pesticide; Diazinon; Dialkylphosphate; Hepatic cytochrome P450 (CYP)
1. Introduction Organophosphorus pesticides (OPs) are widely used in agriculture, forestry, horticulture, public health, and ∗ Corresponding author. Tel.: +81 561 63 1011; fax: +81 561 63 1028. E-mail address: [email protected] (T. Hasegawa).
0378-4274/$ – see front matter © 2007 Published by Elsevier Ireland Ltd. doi:10.1016/j.toxlet.2007.03.010
homes, as a result of their broad spectrum of applications, potent toxicity to insects and their relatively inexpensive cost (Bardin et al., 1994). However, it is well known that heavy exposure to OPs in humans and animals causes an acute cholinergic syndromes such as miosis, salivation, seizures, paralysis, neuromuscular and cardiac conduction disorders through the inhibition of acetylcholinesterase (AChE) (Holstege et al., 2002). Moreover,
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a few investigations have demonstrated the possible effect of chronic exposure to OPs on reproductive function (Kamijima et al., 2004; Okamura et al., 2005) and on neuropsychiatric function (Salvi et al., 2003; Thrasher et al., 2002). Exposure to OPs has been analyzed using their metabolites in urine, because only an analytical apparatus, gas chromatography–mass spectrometry (GC–MS), can detect levels of OPs in plasma, which are usually under the detection limit in the general population (Aprea et al., 1999; Barr et al., 2004; Saieva et al., 2004; Ueyama et al., 2006). However, the effect of environmental background levels of OPs exposure in the general population remains unclear. Some OPs containing phosphorothionate or ‘thio’ bond (P S) are metabolized into products containing phosphate ester or oxon (P O), a potent inhibitor of AChE (Tang et al., 2001). OPs such as diazinon (DZN, O,O-diethyl-O-[2-isopropyl-4-methyl6-pyrimidyl] phosphorothionate) are detoxified by either cytochrome P450 (CYP) or A- and B-esterases (Fabrizi et al., 1999; Poet et al., 2004). DZN is one of the OPs commonly used to control a range of crop pests and also as a veterinary ectoparasiticide in Asia including Japan, although it is no longer sold in the USA market. The postulated metabolic pathway of DZN in vivo is illustrated in Fig. 1 (Fabrizi et al., 1999; Poet et al., 2003). DZN absorbed in the body is metabolized to DZN-oxon through oxidative desulfuration by CYPs such as CYP1A2 and CYP3A2, and then DZN-oxon is hydrolyzed by A- and B-esterase such as paraoxonase (PON1) and carboxylesterase into diethylphosphate (DEP) and 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMHP). Moreover, PON1 and carboxylesterase can
mediate detoxification of DZN by hydrolysis, resulting in the production of diethylthiophosphate (DETP) (Hodgson, 2003; Sams et al., 2000, 2004), which is mainly excreted into urine and bile (Garfitt et al., 2002). It is reported that diabetes mellitus can modify drug pharmacokinetics by changing hepatic drugmetabolizing enzyme activity (Dixon et al., 1961; Thummel and Schenkman, 1990) and that blood–brain barrier permeability is also increased in patients with type II diabetes (Starr et al., 2003). Based on these findings, diabetic patients might be more susceptible to pesticide toxicity due to changes in the degree of the action, detoxification or elimination. The purpose of the present study is to investigate the effect of diabetes on the pharmacokinetics and toxicity of DZN and its metabolites using streptozotocin (STZ)-induced diabetic model rats. The urinary recovery of DZN metabolites, the activities of hepatic drug-metabolizing enzymes, and AChE in brain and erythrocyte and cholinesterase (ChE) in plasma were measured in normal and STZ-induced diabetic rats treated with DZN. 2. Materials and methods 2.1. Chemicals DZN, Florisil PR, DTNB (5,5 -dithio-bis-2-nitrobenzoic acid), acetylthiocholine iodide, eserine (physostigmine) hemisulfate salt and glutathione (reduced form) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). DEP and DETP ammonium salt were purchased
Fig. 1. Proposed metabolic pathway of the organophosphorus pesticide diazinon (DZN).
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from Hayashi Pure Chemical Industries Ltd. (Osaka, Japan). Dibutylphosphate (DBP) was purchased from Tokyo Kasei (Tokyo, Japan). STZ and pentafluorobenzyl bromide (PFBBr) were purchased from Sigma Chemical (St. Louis, MO). Citric acid and sodium carboxymethylcellulose (CMC) were purchased from Yoneyama Yakuhin Kogyo Co., Ltd. (Osaka, Japan). Sodium chloride was purchased from Tomita Pharmaceutical (Tokushima, Japan). Sodium sulfate anhydrous (Na2 SO4 ), potassium carbonate (K2 CO3 ), diethyl ether, acetonitrile, n-hexane, toluene, acetone, sodium disulfite (Na2 S2 O5 ) and 6 mol/l hydrochloric acid were purchased from Kanto Chemicals (Tokyo, Japan), and BONDESILPSA from GL Science Inc. (Tokyo, Japan). All other reagents are commercially available and were of analytical grade. 2.2. Animals Nine-week-old male Wistar rats (270–300 g) were purchased from Japan SLC (Hamamatsu, Japan). The rats were housed under controlled environmental conditions (temperature of 22–24 ◦ C and humidity of 55 ± 5%) with a commercial food diet (Crea rodent diet CE-2, CLEA Japan, Tokyo, Japan) and water freely available for at least 3 days before the experiment and surgery. The procedures involving animals and their care conformed to the Principles of Laboratory Animal Care (NIH publication no. 85-23, revised 1985) and the Guiding Principles for the Care and Use of Laboratory Animals of Nagoya University. 2.3. Animal experiments STZ (45 mg/kg) dissolved in 0.05 M citrate buffer solution (pH 4.5) was administered to the jugular vein 1 week before DZN injection. Control rats received an equivalent volume of citrate buffer solution (normal rats). One week after injection of STZ or vehicle, we measured weight of body, liver and brain, and levels of glucose and protein in urine. Concentrations of glucose and protein in urine were measured by O-toluidine spectrophotometric method and urinary test strip (Bayer Medical Ltd, Tokyo, Japan), respectively. Normal and diabetic rats received a single intraperitoneal injection of DZN (6.5 mg/kg and 65 mg/kg, respectively), each of which was dissolved in 0.9% CMC. For analysis of the activity of AChE in brain, the rats under light anesthesia with diethyl ether were killed by exsanguination 3 h after injection of DZN, and the brains were removed. Antipyrine is widely used as an indicator of drugmetabolizing enzyme activity since it is almost completely metabolized by hepatic CYP isozymes and exhibits a low hepatic extraction ratio in animals and humans (Balani et al., 2002; Carcillo et al., 2003). Antipyrine clearance experiments were performed 1 week after injection of STZ (diabetic rats) or vehicle (normal rats). Briefly, antipyrine (20 mg/kg body weight), which was dissolved in saline, was administered intravenously to normal and diabetic rats. Blood samples were
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collected at designated time intervals (0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, and 5 h after injection of antipyrine). Plasma samples were obtained immediately from the blood samples by centrifugation at 1200 × g for 10 min at 4 ◦ C. Urine samples were collected over 48 h after injection of DZN in metabolic cages (Natsume, Tokyo, Japan). Brain, plasma and urine samples were stored at −80 ◦ C until assayed. 2.4. Assay of acetylcholine esterase (AChE) and cholinesterase (ChE) Activity Activities of AChE in brain and erythrocyte and ChE in plasma were measured by the methods reported previously (Voss and Sachsse, 1970). Blood samples were collected just before and 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h after injection of DZN. The activities of ChE in plasma and AChE in erythrocyte were measured within 24 h after blood collection. Brain samples were stored at −80 ◦ C until analysis. 2.5. GC–MS analysis for metabolites of DZN in urine Concentrations of DEP and DETP in urine were measured by a slightly modified version of the gas chromatography–mass spectrometry (GC–MS) method described previously (Ueyama et al., 2006). The apparatus used for GC–MS was a GC–MSEI (PerkinElmer TurboMass Systems) equipped with an auto sampler (Wellesley, MA, USA). Briefly, 1 ml of urine, 4 ml of distilled water, 25 l of internal standard solution (DBP, 100 mg/l), 5 g of NaCl, 1 ml of 6 mol/l HCl, 50 mg of sodium disulfite and 5 ml of diethyl ether–acetonitrile (1:1, vol/vol) were mixed. After vigorous mechanical shaking, the mixed solution was centrifuged at 4000 × g for 5 min. The organic layer was transferred to screw-top test-glass tubes containing 15 mg of K2 CO3 . The extract was then evaporated to dryness under a stream of nitrogen gas at 45 ◦ C. One ml of acetonitrile, 15 mg of K2 CO3 and 50 l of PFBBr were added to the residue and incubated in a water bath at 80 ◦ C for 30 min with occasional swirling. Afterwards, 4.5 ml of distilled water and 4.5 ml of n-hexane were added, and the mixture was shaken vigorously and centrifuged at 4000 × g for 5 min. The upper layer was evaporated to dryness under a stream of nitrogen gas at 45 ◦ C. The residue was reconstituted in 200 l of toluene and 1 l of the solution was injected into GC–MS. DEP and DETP were quantified with the retention time in selected ion mode (SIM) at m/z 258 and 274, respectively. DBP was used as an internal standard (SIM for quantification is m/z 335). This assay was shown to be linear for the concentrations studied (0.05–50 g/ml) with a correlation coefficient of 0.999. The intra- and inter-assay coefficients of variation for this assay were less than 15%. 2.6. Western blot analysis for hepatic CYP1A2 and CYP3A2 The microsomes were prepared according to the standard methods (Omura and Sato, 1964; Ueyama et al., 2004). The
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protein concentration of the microsomal fraction was measured by Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond, CA) using bovine serum albumin (Sigma Chemical Company, St. Louis, MO) as the standard. Western blot analysis for CYP1A2 and CYP3A2 was performed according to the methods described previously (Miyoshi et al., 2005; Ueyama et al., 2004, 2005). Polyvinylidene difluoride (PVDF) membrane (Millipore Company, Bedford, MA, USA), rabbit polyclonal antibody to rat CYP1A2 or CYP3A2 (Daiichi Kagaku Yakuhin, Tokyo, Japan), horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Dako, Glostrup, Denmark) and enhanced chemiluminescence detection system (ECL, GE Healthcare UK Ltd., Buckinghamshire, UK) were used for Western blot analysis. To quantify the relative levels of CYP3A2 and CYP1A2 in each membrane, the intensity of the stained bands was measured with the NIH image program (Bethesda, MD, USA). The levels were expressed as 100% of those in the control group.
2.7. Data and statistical analyses Concentration–time data for antipyrine in each rat were individually analyzed on the basis of model-independent methods. The area under the plasma concentration–time curve (AUC) and the area under the first-moment curve (AUMC) were calculated by the trapezoidal rule with extrapolation to infinity. The systemic clearance (CLSYS ) was calculated as CLSYS = dose/AUC. The mean residence time (MRT) was calculated as MRT = AUMC/AUC. The volume of distribution at steady state (Vss ) was calculated as VSS = CLSYS × MRT. Results were expressed as mean ± standard errors (S.E.M.). Statistical analysis was performed with the SPSS statistical package for Windows version 11.5 (SPSS Inc., Chicago, IL). Comparison between two groups was performed by Student’s t-test. Statistical comparisons of the activity of AChE in brain between normal and diabetic rats were assessed by one-way analysis of variance (ANOVA). When F ratios were significant (P < 0.05), Scheffe post hoc tests between two groups were done. The effect of diabetes on DZN (0 mg/kg, 6.5 mg/kg and 65 mg/kg)-induced inhibition of AChE activity in brain was assessed by two-way ANOVA. P values less than 0.05 were considered statistically significant.
Fig. 2. Systemic clearance of antipyrine in normal and diabetic rats. Each column represents the mean ± S.E.M. of four animals. **Significantly different from normal rats (P < 0.01).
3. Results 3.1. Biochemical examination in normal and STZ-induced diabetic rats As shown in Table 1, no significant differences in the body and liver weights were observed between the normal and diabetic rats. The ratio of liver/body weight in the diabetic rats significantly increased to approximately 1.3-fold of that in normal rats. Glucose was detected in the urine collected from all STZ-induced diabetic rats ranging from 500 mg/dl to 3000 mg/dl. Protein was not detected in the urine of either normal or diabetic rats (the detection limit was 15 mg/dl). 3.2. Effect of diabetes on hepatic drug-metabolizing enzyme activity The systemic clearance (CLSYS ) of antipyrine in normal and diabetic rats is illustrated in Fig. 2. The values of
Table 1 Body and brain weights and glucose concentration in urine of diabetic rats Group
Normal rats Diabetic rats
Body weight before STZ injection
Weight (g) Body
Liver
294 ± 5 290 ± 4
317 ± 5 289 ± 5
9.8 ± 0.6 11.1 ± 0.4
Liver/body (%)
Concentration of glucose in urine (mg/dl)
Urine volume (ml/48 h)
3.1 ± 0.3 3.9 ± 0.1a
ND 2088 ± 372
51 ± 5 281 ± 48a
Each value represents the mean ± S.E.M. of five animals. Diabetic rats a week after intraperitoneal injection of STZ (45 mg/kg) were used. STZ, streptozocin; ND, not detected. a Significantly different from normal rats (P < 0.05).
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CLSYS of antipyrine in diabetic rats (0.59 ± 0.03 l/h kg) were significantly larger than those of normal rats (0.43 ± 0.03 l/h kg). The steady-state volume of distribution (VSS ) of antipyrine in normal and diabetic rats was 0.95 ± 0.06 and 0.98 ± 0.04 l/kg, respectively. Differences in the volume of distribution of antipyrine failed to reach the 5% level of statistical significance, indicating that the increased CLSYS of antipyrine observed in diabetic rats was due to changes in hepatic drugmetabolizing enzyme activity, but not changes in the volume of distribution. 3.3. Expression of hepatic CYP1A2 and CYP3A2 in normal and STZ-induced diabetic rats The expression of hepatic CYP1A2 and CYP3A2 in normal and diabetic rats is represented in Fig. 3. As shown in Fig. 3, the protein levels of hepatic CYP1A2 in diabetic rats significantly increased to approximately 2.3-fold of normal rats. A slight, but not significant, decrease in the expression of hepatic CYP3A2 was observed in diabetic rats. 3.4. Metabolites of DZN in urine of STZ-induced diabetic rats The concentrations of DEP and DETP in the urine collected over 48 h after injection of DZN are illustrated in Fig. 4. We found that the concentrations of DEP and DETP in urine reached maximum levels
Fig. 3. Expression of hepatic CYP1A2 and CYP3A2 in normal and diabetic rats. Lanes 1–4 and 5–8 represent normal and diabetic rats, respectively. Each column represents the mean ± S.E.M. of four animals. **Significantly different from normal rats (P < 0.01).
Fig. 4. Urinary recovery of DEP and DETP for 48 h after injection of DZN (6.5 mg/kg) in normal and diabetic rats. Each column represents the mean ± S.E.M. of eight animals. *Significantly different from normal rats (P < 0.05).
within 8 h after injection of DZN. The concentration of DEP in urine of diabetic rats (1.66 ± 0.52 mol/l) significantly increased to 2-fold that of normal rats (0.81 ± 0.12 mol/l). However, no significant difference in the concentration of DETP in urine was observed between normal (0.79 ± 0.17 mol/l) and diabetic rats (0.75 ± 0.20 mol/l). 3.5. Effect of DZN on activities of ChE and AChE in plasma and erythrocyte of normal and STZ-induced rats Time curves of plasma ChE activity and erythrocyte AChE activity in normal and diabetic rats after injection of DZN (6.5 mg/kg) are illustrated in Fig. 5. There were no significant differences in the initial activity of ChE in plasma and AChE in erythrocyte between normal and diabetic rats (0.112 ± 0.003 mol/ml min and 0.115 ± 0.005 mol/ml min, and 1.295 ± 0.033 mol/ml min and 1.295 ± 0.044 mol/ml min, respectively). The activity of ChE in plasma 6 h after DZN administration in diabetic rats (60% of preadministration) was significantly declined than that in normal rats (70% of pre-administration). The activity of AChE in erythrocyte 6 h after DZN administration in diabetic rats (50% of pre-administration) was also significantly declined than that in normal rats (65% of pre-administration). As shown in Fig. 5, the activity of AChE in erythrocyte had a tendency to lower in DZNtreated diabetic rats than DZN-treated normal rats, and the lower levels were continued over 96 h after injection of DZN.
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rats, DZN at a high dose (65 mg/kg) caused a significant decrease in the activity of AChE in brain of diabetic rats in comparison to that of normal rats. However, twoway ANOVA showed no significant relationship between brain AChE activity and diabetic status. 4. Discussion
Fig. 5. Activities of ChE and AChE in plasma and erythrocyte in normal (open circle) and diabetic (closed circle) rats after injection of DZN (6.5 mg/kg). Each plot represents the mean ± S.E.M. of four animals. *Significantly different from normal rats (P < 0.05).
3.6. Effect of DZN on AChE activity in brain of normal and STZ-induced rats Fig. 6 shows changes in the activity of AChE in brain of normal and diabetic rats 3 h after DZN intraperitoneal administration (6.5 mg/kg or 65 mg/kg). The brain AChE activity of the control groups (DZN-untreated rats) in normal and diabetic rats was 0.19 ± 0.02 mol/ml min and 0.18 ± 0.01 mol/ml min, respectively, and there was no significant difference between the two groups. While DZN at a low dose (6.5 mg/kg) did not affect the brain AChE activity level in either the normal or diabetic
Fig. 6. Activity of AChE in brain of normal and diabetic rats 3 h after injection of DZN (0, 6.5 and 65 mg/kg). Each column represents the mean ± S.E.M. of five or six animals. Significantly different from DZN-untreated normal and diabetic rats (P < 0.05).
Diabetes mellitus (types I and II) is one of the most widespread diseases in the developed countries. The present study aims to examine the effect of the organophosphorus pesticide DZN, which is metabolized to the oxon form (DZN-oxon), on diabetes. It has been reported that the metabolic rate and inactivation of drugs are altered in diabetes, especially in type I (Daintith et al., 1976; Dixon et al., 1961). Based on these studies, we anticipated that alterations in the function of hepatic detoxification (especially phase I metabolism) in diabetic rats might cause changes in the pharmacokinetics of DZN and its toxicity. In this study, STZ-induced diabetic model rats was used as a type I diabetic model, with marked hyperglycemia and without massive damage to the kidney (Table 1). CYP, which is closely related to phase I metabolism, is present mainly in the liver and small intestine (Kolars et al., 1994; Waxman, 1988). As the proposed metabolic fate of DZN (Fig. 1), DZN is metabolized into an active form, DZN-oxon, by CYP-mediated oxidative desulfuration. The detoxification of DZN is also undertaken by CYPs through dearylation reaction, resulting in the production of IMHP and DEP (Fabrizi et al., 1999; Hodgson, 2003). While the esterase is classified into A, B and C types, DZN-oxon is further metabolized to IMHP and DEP by both A-esterase (PON1) and B-eaterases (carboxylesterase, butylcholinesterase and AChE), but not by C-esterase (Poet et al., 2004; Sams and Mason, 1999). First, we investigated the difference in the systemic clearance (CLSYS ) of antipyrine, which is a marker for the ability of hepatic oxidative metabolism in humans and animals (Balani et al., 2002; Nadai et al., 1998), between the normal and diabetic rats. Diabetes significantly increased the systemic clearance (CLSYS ) of antipyrine with no change in the steady-state volume of distribution (VSS ), suggesting that hepatic oxidative function is significantly enhanced in diabetic rats. Antipyrine metabolism in humans is associated with at least six hepatic CYP isozymes (CYP1A2, CYP 2B6, CYP 2C8, CYP 2C9, CYP2C18 and CYP 3A4) (Engel et al., 1996). Because it is reported that the subtypes related to DZN metabolism is CYP1A2, which contributes to DZN desulfuration, and CYP3A4, which contributes to
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DZN desulfuration and dearylation (Hodgson, 2003). Therefore, we measured the protein levels of hepatic CYP1A2 and CYP3A2 (CYP3A4 in humans) in diabetic rats by Western blot analysis. The results showed that the expression of hepatic CYP1A2 dramatically increases in diabetic rats, whereas the expression of hepatic CYP3A2 is unchanged. Patel et al. (1990) reported that the levels of PON1 activity progressively decrease with time in the diabetic rats, being 36% lower than that in normal rats 6 months, but not one week, after injection of STZ. They also reported that there are no significant differences in carboxylesterase activity in serum between normal and diabetic rats. Considering the proposed metabolic pathway of DZN (Fig. 1), these results suggest that the metabolism of DZN to DZN-oxon might be enhanced in diabetic rats. Because no technique is available to measure the levels of DZN-oxon in blood or brain due to its high affinity to ChE or AChE in vivo and difficulty in obtaining DZN-oxon standard, concentration of DEP in urine over 48 h after injection of DZN was measured as an indirect biomarker of DZN-oxon. It was found that the urinary recovery of DEP in diabetic rats was twice as high as in normal rats, while the urinary recovery of DETP was unchanged. There are some reports concerning the pharmacokinetic data of DZN, DZN-oxon, DEP and DETP in normal rats, but not in diabetic rats (Poet et al., 2003, 2004; Wu et al., 1996). Although the urine volume over 48 h was significantly increased in diabetic rats compared with normal rats, there is no data regarding alteration of urinary excretion rate of these compounds. Data obtained from this study suggest that the modification of DZN-oxon formation from DZN is closely associated with STZ-induced diabetes. Measurement of the activities of ChE and AChE in blood is widely used to evaluate the exposure level of OPs. Therefore, we measured the activities of ChE in plasma and AChE in erythrocyte of normal and diabetic rats after injection of DZN (6.5 mg/kg). The present study was found that DZN dramatically decreases the activities of ChE in plasma and AChE in erythrocyte of normal and diabetic rats and that these activities at early phase after injection of DZN are lower in diabetic rats than normal rats. Results of the present study may provide information that even acute exposure to DZN at low dose induces neurotoxicity in diabetic patients and the DZN-induced neurotoxicity continues for long time, although the data obtained in this study cannot be extrapolated directly to diabetic patients. To investigate the effect of DZN on the central nervous system (CNS), the AChE activity in brain was measured after injection of DZN. The inhibition of AChE in brain is a critical target that correlates well with observed toxicological
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response in laboratory animals, while the activities of ChE in plasma and AChE in erythrocyte are important as biomarkers for assessing exposure. In our preliminary experiment, DZN decreased maximally the activity of AChE in brain of diabetic rats 3 h after DZN injection (data not shown). Therefore, the effect of DZN on the activity of AChE in brain was determined 3 h after DZN injection. In this experiment, the used dose of DZN (6.5 or 65 mg/kg) had no effect on the substantial muscarine and/or nicotine action either in normal or diabetic rats. As shown in Fig. 6, DZN at a dose of 6.5 mg/kg had no effect on the activity of AChE in the brain of either normal or diabetic rats. In diabetic rats treated with 65 mg/kg of DZN, the activity of AChE in brain was significantly decreased 3 h after DZN injection compared with untreated diabetic rats. However, the precise mechanism by which DZN decreases the activity of AChE in brain remains unclear at present. It has been reported that the organophosphorus pesticide dichlorvos induces blood–brain barrier dysfunction in mice but not in rats (Sinha and Shukla, 2003), suggesting the possibility that exposure to DZN does not induce blood–brain barrier dysfunction in rats. It is reported that blood–brain barrier permeability increases in diabetic patients (Horani and Mooradian, 2003; Starr et al., 2003) and STZ-induced rats (Huber et al., 2006; Mooradian et al., 2005). Based on these observations, we assume that DZN-induced decrease in the brain AChE activity in diabetic rats is caused by increase in the penetration of DZN and/or DZN-oxon into the brain, probably due to increase in hepatic drug-metabolizing enzyme activity such as CYP1A2 and/or increase in blood–brain barrier permeability. In conclusion, the present study suggests the possibility that DZN-oxon, a metabolite of DZN, causes higher toxicity in STZ-induced diabetic rats by accelerating hepatic CYP1A2-mediated metabolism of DZN. It is also likely that STZ-induced diabetic rats are more vulnerable to the toxic effects of DZN than normal rats. Further study is warranted to investigate the effect of OPs including DZN exposure on hyperglycemia with some therapeutics and type II diabetes.
Acknowledgements This work was supported in part by a Health and Labor Sciences Research Grant (Research on Risk of Chemical Substances) from the Ministry of Health, Labor and Welfare of Japan, and a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.
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J. Ueyama et al. / Toxicology Letters 170 (2007) 229–237 Ueyama, J., Kitaichi, K., Nadai, M., Iwase, M., Tomyo, N., Kanazawa, H., Suzuki, R., Takagi, K., Takagi, K., Hasegawa, T., 2004. Effect of pioglitazone on endotoxin-induced decreases in hepatic drugmetabolizing enzyme activity and expression of CYP3A2 and CYP2C11. Eur. J. Pharmacol. 498, 257–265. Ueyama, J., Nadai, M., Kanazawa, H., Iwase, M., Nakayama, H., Hashimoto, K., Yokoi, T., Baba, K., Takagi, K., Takagi, K., Hasegawa, T., 2005. Endotoxin from various gramnegative bacteria has differential effects on function of hepatic cytochrome P450 and drug transporters. Eur. J. Pharmacol. 510, 127–134. Ueyama, J., Saito, I., Kamijima, M., Nakajima, T., Gotoh, M., Suzuki, T., Shibata, E., Kondo, T., Takagi, K., Miyamoto, K., Takamatsu, J., Hasegawa, T., Takagi, K., 2006. Simultaneous determination of uri-
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Toxicity of diazinon and its metabolites increases in diabetic rats Jun Ueyama a,b , Dong Wang b , Takaaki Kondo b , Isao Saito c , Kenji Takagi b , Kenzo Takagi b , Michihiro Kamijima d , Tamie Nakajima d , Ken-Ichi Miyamoto a , Shinya Wakusawa b , Takaaki Hasegawa e,∗ a
d
Department of Medicinal Informatics, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan b Department of Medical Technology, Nagoya University School of Health Sciences, Nagoya, Japan c Food Safety and Quality Research Center, Tokai COOP Federation, Aichi-gun, Aichi, Japan Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya, Japan e Department of Pharmacy and Pharmacokinetics, Aichi Medical University School of Medicine, Nagakute-cho, Aichi-gun, Aichi 480-1195, Japan Received 19 January 2007; received in revised form 19 March 2007; accepted 19 March 2007 Available online 24 March 2007
Abstract The effect of diazinon (DZN) on the activities of cholinesterase (ChE) in plasma and acetylcholinesterase (AChE) in erythrocyte and brain was investigated in normal and streptozotocin-induced diabetic rats. Hepatic drug-metabolizing enzyme activity was also estimated by measuring the systemic clearance of antipyrine, and the expression of hepatic cytochrome P450 (CYP) 3A2 and CYP1A2, which is closely related to the metabolism from DZN to DZN-oxon, a strong inhibitor of both ChE and AChE. No significant differences in the activities of ChE in plasma and AChE in erythrocyte were observed between normal and diabetic rats. Treatment with DZN significantly decreased these activities in diabetic rats more than in normal rats 6 h after injection (6.5 mg/kg). Treatment with DZN significantly decreased the activity of AChE in brain of diabetic rats than normal rats 3 h after injection (65 mg/kg), although no significant difference in the activity was found between normal and diabetic rats. The urinary recovery of diethylphosphate (DEP), a metabolite of DZN-oxon, was significantly increased in diabetic rats, but that of diethylthiophosphate (DETP), a metabolite of DZN, was unchanged. Significant increases in the systemic clearance of antipyrine and protein levels of hepatic CYP1A2, not CYP3A2, were observed in diabetic rats. These results suggest the possibility that a metabolite of DZN, DZN-oxon, causes higher toxicity in diabetic rats due to the enhancement of hepatic CYP1A2-mediated metabolism of DZN. © 2007 Published by Elsevier Ireland Ltd. Keywords: Organophosphorus pesticide; Diazinon; Dialkylphosphate; Hepatic cytochrome P450 (CYP)
1. Introduction Organophosphorus pesticides (OPs) are widely used in agriculture, forestry, horticulture, public health, and ∗ Corresponding author. Tel.: +81 561 63 1011; fax: +81 561 63 1028. E-mail address: [email protected] (T. Hasegawa).
0378-4274/$ – see front matter © 2007 Published by Elsevier Ireland Ltd. doi:10.1016/j.toxlet.2007.03.010
homes, as a result of their broad spectrum of applications, potent toxicity to insects and their relatively inexpensive cost (Bardin et al., 1994). However, it is well known that heavy exposure to OPs in humans and animals causes an acute cholinergic syndromes such as miosis, salivation, seizures, paralysis, neuromuscular and cardiac conduction disorders through the inhibition of acetylcholinesterase (AChE) (Holstege et al., 2002). Moreover,
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a few investigations have demonstrated the possible effect of chronic exposure to OPs on reproductive function (Kamijima et al., 2004; Okamura et al., 2005) and on neuropsychiatric function (Salvi et al., 2003; Thrasher et al., 2002). Exposure to OPs has been analyzed using their metabolites in urine, because only an analytical apparatus, gas chromatography–mass spectrometry (GC–MS), can detect levels of OPs in plasma, which are usually under the detection limit in the general population (Aprea et al., 1999; Barr et al., 2004; Saieva et al., 2004; Ueyama et al., 2006). However, the effect of environmental background levels of OPs exposure in the general population remains unclear. Some OPs containing phosphorothionate or ‘thio’ bond (P S) are metabolized into products containing phosphate ester or oxon (P O), a potent inhibitor of AChE (Tang et al., 2001). OPs such as diazinon (DZN, O,O-diethyl-O-[2-isopropyl-4-methyl6-pyrimidyl] phosphorothionate) are detoxified by either cytochrome P450 (CYP) or A- and B-esterases (Fabrizi et al., 1999; Poet et al., 2004). DZN is one of the OPs commonly used to control a range of crop pests and also as a veterinary ectoparasiticide in Asia including Japan, although it is no longer sold in the USA market. The postulated metabolic pathway of DZN in vivo is illustrated in Fig. 1 (Fabrizi et al., 1999; Poet et al., 2003). DZN absorbed in the body is metabolized to DZN-oxon through oxidative desulfuration by CYPs such as CYP1A2 and CYP3A2, and then DZN-oxon is hydrolyzed by A- and B-esterase such as paraoxonase (PON1) and carboxylesterase into diethylphosphate (DEP) and 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMHP). Moreover, PON1 and carboxylesterase can
mediate detoxification of DZN by hydrolysis, resulting in the production of diethylthiophosphate (DETP) (Hodgson, 2003; Sams et al., 2000, 2004), which is mainly excreted into urine and bile (Garfitt et al., 2002). It is reported that diabetes mellitus can modify drug pharmacokinetics by changing hepatic drugmetabolizing enzyme activity (Dixon et al., 1961; Thummel and Schenkman, 1990) and that blood–brain barrier permeability is also increased in patients with type II diabetes (Starr et al., 2003). Based on these findings, diabetic patients might be more susceptible to pesticide toxicity due to changes in the degree of the action, detoxification or elimination. The purpose of the present study is to investigate the effect of diabetes on the pharmacokinetics and toxicity of DZN and its metabolites using streptozotocin (STZ)-induced diabetic model rats. The urinary recovery of DZN metabolites, the activities of hepatic drug-metabolizing enzymes, and AChE in brain and erythrocyte and cholinesterase (ChE) in plasma were measured in normal and STZ-induced diabetic rats treated with DZN. 2. Materials and methods 2.1. Chemicals DZN, Florisil PR, DTNB (5,5 -dithio-bis-2-nitrobenzoic acid), acetylthiocholine iodide, eserine (physostigmine) hemisulfate salt and glutathione (reduced form) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). DEP and DETP ammonium salt were purchased
Fig. 1. Proposed metabolic pathway of the organophosphorus pesticide diazinon (DZN).
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from Hayashi Pure Chemical Industries Ltd. (Osaka, Japan). Dibutylphosphate (DBP) was purchased from Tokyo Kasei (Tokyo, Japan). STZ and pentafluorobenzyl bromide (PFBBr) were purchased from Sigma Chemical (St. Louis, MO). Citric acid and sodium carboxymethylcellulose (CMC) were purchased from Yoneyama Yakuhin Kogyo Co., Ltd. (Osaka, Japan). Sodium chloride was purchased from Tomita Pharmaceutical (Tokushima, Japan). Sodium sulfate anhydrous (Na2 SO4 ), potassium carbonate (K2 CO3 ), diethyl ether, acetonitrile, n-hexane, toluene, acetone, sodium disulfite (Na2 S2 O5 ) and 6 mol/l hydrochloric acid were purchased from Kanto Chemicals (Tokyo, Japan), and BONDESILPSA from GL Science Inc. (Tokyo, Japan). All other reagents are commercially available and were of analytical grade. 2.2. Animals Nine-week-old male Wistar rats (270–300 g) were purchased from Japan SLC (Hamamatsu, Japan). The rats were housed under controlled environmental conditions (temperature of 22–24 ◦ C and humidity of 55 ± 5%) with a commercial food diet (Crea rodent diet CE-2, CLEA Japan, Tokyo, Japan) and water freely available for at least 3 days before the experiment and surgery. The procedures involving animals and their care conformed to the Principles of Laboratory Animal Care (NIH publication no. 85-23, revised 1985) and the Guiding Principles for the Care and Use of Laboratory Animals of Nagoya University. 2.3. Animal experiments STZ (45 mg/kg) dissolved in 0.05 M citrate buffer solution (pH 4.5) was administered to the jugular vein 1 week before DZN injection. Control rats received an equivalent volume of citrate buffer solution (normal rats). One week after injection of STZ or vehicle, we measured weight of body, liver and brain, and levels of glucose and protein in urine. Concentrations of glucose and protein in urine were measured by O-toluidine spectrophotometric method and urinary test strip (Bayer Medical Ltd, Tokyo, Japan), respectively. Normal and diabetic rats received a single intraperitoneal injection of DZN (6.5 mg/kg and 65 mg/kg, respectively), each of which was dissolved in 0.9% CMC. For analysis of the activity of AChE in brain, the rats under light anesthesia with diethyl ether were killed by exsanguination 3 h after injection of DZN, and the brains were removed. Antipyrine is widely used as an indicator of drugmetabolizing enzyme activity since it is almost completely metabolized by hepatic CYP isozymes and exhibits a low hepatic extraction ratio in animals and humans (Balani et al., 2002; Carcillo et al., 2003). Antipyrine clearance experiments were performed 1 week after injection of STZ (diabetic rats) or vehicle (normal rats). Briefly, antipyrine (20 mg/kg body weight), which was dissolved in saline, was administered intravenously to normal and diabetic rats. Blood samples were
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collected at designated time intervals (0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, and 5 h after injection of antipyrine). Plasma samples were obtained immediately from the blood samples by centrifugation at 1200 × g for 10 min at 4 ◦ C. Urine samples were collected over 48 h after injection of DZN in metabolic cages (Natsume, Tokyo, Japan). Brain, plasma and urine samples were stored at −80 ◦ C until assayed. 2.4. Assay of acetylcholine esterase (AChE) and cholinesterase (ChE) Activity Activities of AChE in brain and erythrocyte and ChE in plasma were measured by the methods reported previously (Voss and Sachsse, 1970). Blood samples were collected just before and 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h after injection of DZN. The activities of ChE in plasma and AChE in erythrocyte were measured within 24 h after blood collection. Brain samples were stored at −80 ◦ C until analysis. 2.5. GC–MS analysis for metabolites of DZN in urine Concentrations of DEP and DETP in urine were measured by a slightly modified version of the gas chromatography–mass spectrometry (GC–MS) method described previously (Ueyama et al., 2006). The apparatus used for GC–MS was a GC–MSEI (PerkinElmer TurboMass Systems) equipped with an auto sampler (Wellesley, MA, USA). Briefly, 1 ml of urine, 4 ml of distilled water, 25 l of internal standard solution (DBP, 100 mg/l), 5 g of NaCl, 1 ml of 6 mol/l HCl, 50 mg of sodium disulfite and 5 ml of diethyl ether–acetonitrile (1:1, vol/vol) were mixed. After vigorous mechanical shaking, the mixed solution was centrifuged at 4000 × g for 5 min. The organic layer was transferred to screw-top test-glass tubes containing 15 mg of K2 CO3 . The extract was then evaporated to dryness under a stream of nitrogen gas at 45 ◦ C. One ml of acetonitrile, 15 mg of K2 CO3 and 50 l of PFBBr were added to the residue and incubated in a water bath at 80 ◦ C for 30 min with occasional swirling. Afterwards, 4.5 ml of distilled water and 4.5 ml of n-hexane were added, and the mixture was shaken vigorously and centrifuged at 4000 × g for 5 min. The upper layer was evaporated to dryness under a stream of nitrogen gas at 45 ◦ C. The residue was reconstituted in 200 l of toluene and 1 l of the solution was injected into GC–MS. DEP and DETP were quantified with the retention time in selected ion mode (SIM) at m/z 258 and 274, respectively. DBP was used as an internal standard (SIM for quantification is m/z 335). This assay was shown to be linear for the concentrations studied (0.05–50 g/ml) with a correlation coefficient of 0.999. The intra- and inter-assay coefficients of variation for this assay were less than 15%. 2.6. Western blot analysis for hepatic CYP1A2 and CYP3A2 The microsomes were prepared according to the standard methods (Omura and Sato, 1964; Ueyama et al., 2004). The
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protein concentration of the microsomal fraction was measured by Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond, CA) using bovine serum albumin (Sigma Chemical Company, St. Louis, MO) as the standard. Western blot analysis for CYP1A2 and CYP3A2 was performed according to the methods described previously (Miyoshi et al., 2005; Ueyama et al., 2004, 2005). Polyvinylidene difluoride (PVDF) membrane (Millipore Company, Bedford, MA, USA), rabbit polyclonal antibody to rat CYP1A2 or CYP3A2 (Daiichi Kagaku Yakuhin, Tokyo, Japan), horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Dako, Glostrup, Denmark) and enhanced chemiluminescence detection system (ECL, GE Healthcare UK Ltd., Buckinghamshire, UK) were used for Western blot analysis. To quantify the relative levels of CYP3A2 and CYP1A2 in each membrane, the intensity of the stained bands was measured with the NIH image program (Bethesda, MD, USA). The levels were expressed as 100% of those in the control group.
2.7. Data and statistical analyses Concentration–time data for antipyrine in each rat were individually analyzed on the basis of model-independent methods. The area under the plasma concentration–time curve (AUC) and the area under the first-moment curve (AUMC) were calculated by the trapezoidal rule with extrapolation to infinity. The systemic clearance (CLSYS ) was calculated as CLSYS = dose/AUC. The mean residence time (MRT) was calculated as MRT = AUMC/AUC. The volume of distribution at steady state (Vss ) was calculated as VSS = CLSYS × MRT. Results were expressed as mean ± standard errors (S.E.M.). Statistical analysis was performed with the SPSS statistical package for Windows version 11.5 (SPSS Inc., Chicago, IL). Comparison between two groups was performed by Student’s t-test. Statistical comparisons of the activity of AChE in brain between normal and diabetic rats were assessed by one-way analysis of variance (ANOVA). When F ratios were significant (P < 0.05), Scheffe post hoc tests between two groups were done. The effect of diabetes on DZN (0 mg/kg, 6.5 mg/kg and 65 mg/kg)-induced inhibition of AChE activity in brain was assessed by two-way ANOVA. P values less than 0.05 were considered statistically significant.
Fig. 2. Systemic clearance of antipyrine in normal and diabetic rats. Each column represents the mean ± S.E.M. of four animals. **Significantly different from normal rats (P < 0.01).
3. Results 3.1. Biochemical examination in normal and STZ-induced diabetic rats As shown in Table 1, no significant differences in the body and liver weights were observed between the normal and diabetic rats. The ratio of liver/body weight in the diabetic rats significantly increased to approximately 1.3-fold of that in normal rats. Glucose was detected in the urine collected from all STZ-induced diabetic rats ranging from 500 mg/dl to 3000 mg/dl. Protein was not detected in the urine of either normal or diabetic rats (the detection limit was 15 mg/dl). 3.2. Effect of diabetes on hepatic drug-metabolizing enzyme activity The systemic clearance (CLSYS ) of antipyrine in normal and diabetic rats is illustrated in Fig. 2. The values of
Table 1 Body and brain weights and glucose concentration in urine of diabetic rats Group
Normal rats Diabetic rats
Body weight before STZ injection
Weight (g) Body
Liver
294 ± 5 290 ± 4
317 ± 5 289 ± 5
9.8 ± 0.6 11.1 ± 0.4
Liver/body (%)
Concentration of glucose in urine (mg/dl)
Urine volume (ml/48 h)
3.1 ± 0.3 3.9 ± 0.1a
ND 2088 ± 372
51 ± 5 281 ± 48a
Each value represents the mean ± S.E.M. of five animals. Diabetic rats a week after intraperitoneal injection of STZ (45 mg/kg) were used. STZ, streptozocin; ND, not detected. a Significantly different from normal rats (P < 0.05).
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CLSYS of antipyrine in diabetic rats (0.59 ± 0.03 l/h kg) were significantly larger than those of normal rats (0.43 ± 0.03 l/h kg). The steady-state volume of distribution (VSS ) of antipyrine in normal and diabetic rats was 0.95 ± 0.06 and 0.98 ± 0.04 l/kg, respectively. Differences in the volume of distribution of antipyrine failed to reach the 5% level of statistical significance, indicating that the increased CLSYS of antipyrine observed in diabetic rats was due to changes in hepatic drugmetabolizing enzyme activity, but not changes in the volume of distribution. 3.3. Expression of hepatic CYP1A2 and CYP3A2 in normal and STZ-induced diabetic rats The expression of hepatic CYP1A2 and CYP3A2 in normal and diabetic rats is represented in Fig. 3. As shown in Fig. 3, the protein levels of hepatic CYP1A2 in diabetic rats significantly increased to approximately 2.3-fold of normal rats. A slight, but not significant, decrease in the expression of hepatic CYP3A2 was observed in diabetic rats. 3.4. Metabolites of DZN in urine of STZ-induced diabetic rats The concentrations of DEP and DETP in the urine collected over 48 h after injection of DZN are illustrated in Fig. 4. We found that the concentrations of DEP and DETP in urine reached maximum levels
Fig. 3. Expression of hepatic CYP1A2 and CYP3A2 in normal and diabetic rats. Lanes 1–4 and 5–8 represent normal and diabetic rats, respectively. Each column represents the mean ± S.E.M. of four animals. **Significantly different from normal rats (P < 0.01).
Fig. 4. Urinary recovery of DEP and DETP for 48 h after injection of DZN (6.5 mg/kg) in normal and diabetic rats. Each column represents the mean ± S.E.M. of eight animals. *Significantly different from normal rats (P < 0.05).
within 8 h after injection of DZN. The concentration of DEP in urine of diabetic rats (1.66 ± 0.52 mol/l) significantly increased to 2-fold that of normal rats (0.81 ± 0.12 mol/l). However, no significant difference in the concentration of DETP in urine was observed between normal (0.79 ± 0.17 mol/l) and diabetic rats (0.75 ± 0.20 mol/l). 3.5. Effect of DZN on activities of ChE and AChE in plasma and erythrocyte of normal and STZ-induced rats Time curves of plasma ChE activity and erythrocyte AChE activity in normal and diabetic rats after injection of DZN (6.5 mg/kg) are illustrated in Fig. 5. There were no significant differences in the initial activity of ChE in plasma and AChE in erythrocyte between normal and diabetic rats (0.112 ± 0.003 mol/ml min and 0.115 ± 0.005 mol/ml min, and 1.295 ± 0.033 mol/ml min and 1.295 ± 0.044 mol/ml min, respectively). The activity of ChE in plasma 6 h after DZN administration in diabetic rats (60% of preadministration) was significantly declined than that in normal rats (70% of pre-administration). The activity of AChE in erythrocyte 6 h after DZN administration in diabetic rats (50% of pre-administration) was also significantly declined than that in normal rats (65% of pre-administration). As shown in Fig. 5, the activity of AChE in erythrocyte had a tendency to lower in DZNtreated diabetic rats than DZN-treated normal rats, and the lower levels were continued over 96 h after injection of DZN.
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rats, DZN at a high dose (65 mg/kg) caused a significant decrease in the activity of AChE in brain of diabetic rats in comparison to that of normal rats. However, twoway ANOVA showed no significant relationship between brain AChE activity and diabetic status. 4. Discussion
Fig. 5. Activities of ChE and AChE in plasma and erythrocyte in normal (open circle) and diabetic (closed circle) rats after injection of DZN (6.5 mg/kg). Each plot represents the mean ± S.E.M. of four animals. *Significantly different from normal rats (P < 0.05).
3.6. Effect of DZN on AChE activity in brain of normal and STZ-induced rats Fig. 6 shows changes in the activity of AChE in brain of normal and diabetic rats 3 h after DZN intraperitoneal administration (6.5 mg/kg or 65 mg/kg). The brain AChE activity of the control groups (DZN-untreated rats) in normal and diabetic rats was 0.19 ± 0.02 mol/ml min and 0.18 ± 0.01 mol/ml min, respectively, and there was no significant difference between the two groups. While DZN at a low dose (6.5 mg/kg) did not affect the brain AChE activity level in either the normal or diabetic
Fig. 6. Activity of AChE in brain of normal and diabetic rats 3 h after injection of DZN (0, 6.5 and 65 mg/kg). Each column represents the mean ± S.E.M. of five or six animals. Significantly different from DZN-untreated normal and diabetic rats (P < 0.05).
Diabetes mellitus (types I and II) is one of the most widespread diseases in the developed countries. The present study aims to examine the effect of the organophosphorus pesticide DZN, which is metabolized to the oxon form (DZN-oxon), on diabetes. It has been reported that the metabolic rate and inactivation of drugs are altered in diabetes, especially in type I (Daintith et al., 1976; Dixon et al., 1961). Based on these studies, we anticipated that alterations in the function of hepatic detoxification (especially phase I metabolism) in diabetic rats might cause changes in the pharmacokinetics of DZN and its toxicity. In this study, STZ-induced diabetic model rats was used as a type I diabetic model, with marked hyperglycemia and without massive damage to the kidney (Table 1). CYP, which is closely related to phase I metabolism, is present mainly in the liver and small intestine (Kolars et al., 1994; Waxman, 1988). As the proposed metabolic fate of DZN (Fig. 1), DZN is metabolized into an active form, DZN-oxon, by CYP-mediated oxidative desulfuration. The detoxification of DZN is also undertaken by CYPs through dearylation reaction, resulting in the production of IMHP and DEP (Fabrizi et al., 1999; Hodgson, 2003). While the esterase is classified into A, B and C types, DZN-oxon is further metabolized to IMHP and DEP by both A-esterase (PON1) and B-eaterases (carboxylesterase, butylcholinesterase and AChE), but not by C-esterase (Poet et al., 2004; Sams and Mason, 1999). First, we investigated the difference in the systemic clearance (CLSYS ) of antipyrine, which is a marker for the ability of hepatic oxidative metabolism in humans and animals (Balani et al., 2002; Nadai et al., 1998), between the normal and diabetic rats. Diabetes significantly increased the systemic clearance (CLSYS ) of antipyrine with no change in the steady-state volume of distribution (VSS ), suggesting that hepatic oxidative function is significantly enhanced in diabetic rats. Antipyrine metabolism in humans is associated with at least six hepatic CYP isozymes (CYP1A2, CYP 2B6, CYP 2C8, CYP 2C9, CYP2C18 and CYP 3A4) (Engel et al., 1996). Because it is reported that the subtypes related to DZN metabolism is CYP1A2, which contributes to DZN desulfuration, and CYP3A4, which contributes to
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DZN desulfuration and dearylation (Hodgson, 2003). Therefore, we measured the protein levels of hepatic CYP1A2 and CYP3A2 (CYP3A4 in humans) in diabetic rats by Western blot analysis. The results showed that the expression of hepatic CYP1A2 dramatically increases in diabetic rats, whereas the expression of hepatic CYP3A2 is unchanged. Patel et al. (1990) reported that the levels of PON1 activity progressively decrease with time in the diabetic rats, being 36% lower than that in normal rats 6 months, but not one week, after injection of STZ. They also reported that there are no significant differences in carboxylesterase activity in serum between normal and diabetic rats. Considering the proposed metabolic pathway of DZN (Fig. 1), these results suggest that the metabolism of DZN to DZN-oxon might be enhanced in diabetic rats. Because no technique is available to measure the levels of DZN-oxon in blood or brain due to its high affinity to ChE or AChE in vivo and difficulty in obtaining DZN-oxon standard, concentration of DEP in urine over 48 h after injection of DZN was measured as an indirect biomarker of DZN-oxon. It was found that the urinary recovery of DEP in diabetic rats was twice as high as in normal rats, while the urinary recovery of DETP was unchanged. There are some reports concerning the pharmacokinetic data of DZN, DZN-oxon, DEP and DETP in normal rats, but not in diabetic rats (Poet et al., 2003, 2004; Wu et al., 1996). Although the urine volume over 48 h was significantly increased in diabetic rats compared with normal rats, there is no data regarding alteration of urinary excretion rate of these compounds. Data obtained from this study suggest that the modification of DZN-oxon formation from DZN is closely associated with STZ-induced diabetes. Measurement of the activities of ChE and AChE in blood is widely used to evaluate the exposure level of OPs. Therefore, we measured the activities of ChE in plasma and AChE in erythrocyte of normal and diabetic rats after injection of DZN (6.5 mg/kg). The present study was found that DZN dramatically decreases the activities of ChE in plasma and AChE in erythrocyte of normal and diabetic rats and that these activities at early phase after injection of DZN are lower in diabetic rats than normal rats. Results of the present study may provide information that even acute exposure to DZN at low dose induces neurotoxicity in diabetic patients and the DZN-induced neurotoxicity continues for long time, although the data obtained in this study cannot be extrapolated directly to diabetic patients. To investigate the effect of DZN on the central nervous system (CNS), the AChE activity in brain was measured after injection of DZN. The inhibition of AChE in brain is a critical target that correlates well with observed toxicological
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response in laboratory animals, while the activities of ChE in plasma and AChE in erythrocyte are important as biomarkers for assessing exposure. In our preliminary experiment, DZN decreased maximally the activity of AChE in brain of diabetic rats 3 h after DZN injection (data not shown). Therefore, the effect of DZN on the activity of AChE in brain was determined 3 h after DZN injection. In this experiment, the used dose of DZN (6.5 or 65 mg/kg) had no effect on the substantial muscarine and/or nicotine action either in normal or diabetic rats. As shown in Fig. 6, DZN at a dose of 6.5 mg/kg had no effect on the activity of AChE in the brain of either normal or diabetic rats. In diabetic rats treated with 65 mg/kg of DZN, the activity of AChE in brain was significantly decreased 3 h after DZN injection compared with untreated diabetic rats. However, the precise mechanism by which DZN decreases the activity of AChE in brain remains unclear at present. It has been reported that the organophosphorus pesticide dichlorvos induces blood–brain barrier dysfunction in mice but not in rats (Sinha and Shukla, 2003), suggesting the possibility that exposure to DZN does not induce blood–brain barrier dysfunction in rats. It is reported that blood–brain barrier permeability increases in diabetic patients (Horani and Mooradian, 2003; Starr et al., 2003) and STZ-induced rats (Huber et al., 2006; Mooradian et al., 2005). Based on these observations, we assume that DZN-induced decrease in the brain AChE activity in diabetic rats is caused by increase in the penetration of DZN and/or DZN-oxon into the brain, probably due to increase in hepatic drug-metabolizing enzyme activity such as CYP1A2 and/or increase in blood–brain barrier permeability. In conclusion, the present study suggests the possibility that DZN-oxon, a metabolite of DZN, causes higher toxicity in STZ-induced diabetic rats by accelerating hepatic CYP1A2-mediated metabolism of DZN. It is also likely that STZ-induced diabetic rats are more vulnerable to the toxic effects of DZN than normal rats. Further study is warranted to investigate the effect of OPs including DZN exposure on hyperglycemia with some therapeutics and type II diabetes.
Acknowledgements This work was supported in part by a Health and Labor Sciences Research Grant (Research on Risk of Chemical Substances) from the Ministry of Health, Labor and Welfare of Japan, and a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.
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