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Anticholinesterase insecticide action at the murine male reproductive system
Bioorganic & Medicinal Chemistry Letters 23 (2013) 5434–5436
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Anticholinesterase insecticide action at the murine male reproductive system Yuki Noro, Motohiro Tomizawa ⇑, , Yuki Ito, Himiko Suzuki, Keisuke Abe, Michihiro Kamijima Department of Occupational and Environmental Health, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan
a r t i c l e
i n f o
Article history: Received 1 June 2013 Revised 1 July 2013 Accepted 12 July 2013 Available online 19 July 2013
a b s t r a c t The present report describes for the first time that anticholinesterase type insecticides specifically inhibit the fatty acid amide hydrolase and/or monoacylglycerol lipase, as secondary target(s), in the murine male reproductive system (testis and epididymis cauda), thereby presumably being involved with spermatotoxicity such as deformity, underdevelopment, and reduced motility. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Anticholinesterase insecticides Chlorpyrifos Fatty acid amide hydrolase Monoacylglycerol lipase Organophosphate and methylcarbamate insecticides Spermatotoxicity
Organophosphate (OP) and methylcarbamate (MC) insecticides (Fig. 1) are generally utilized throughout the globe for protecting crops, people, livestock, and companion animals from pest insect attack and disease transmission. These compounds are nerve poisons acting at the cholinergic neurons by inhibiting acetylcholinesterase (AChE) as the primary target.1 The anticholinesterase agents phosphorylate or carbamoylate the serine OH residue at the AChE catalytic triad. On the other hand, the anticholinesterases may also react with many other serine hydrolases to reveal the secondary or unexpected physiological effects.2,3 Interestingly, recent reports have suggested that the human male reproductive system is also a target for OP and MC insecticides.4–8 In rodent, OP insecticides induce spermatotoxicity including sperm deformity or underdevelopment (broken sperm and cytoplasmic droplets) and reduced sperm motility, whereas the molecular target or mechanism causing the spermatotoxicity is not defined yet.9,10 Accordingly, the present investigation detects a possible anticholinesterase target in the murine male reproductive system by activity-based protein profiling (ABPP) approach3 with a phosphonofluoridate chemical probe (FP-TAMRA), thus proposing a hypothesis for the first time that inhibition of fatty acid amide hydrolase (FAAH) and/or monoacylglycerol lipase (MAGL) in the mouse testis and epididymis cauda is relevant to the anticholinesterase-induced spermatotoxicity. ⇑ Corresponding author. Tel.: +81 3 5477 2529; fax: +81 3 5477 2626. E-mail address: [email protected] (M. Tomizawa). Present address: Faculty of Applied Bioscience, Tokyo University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.07.021
In vitro screening of target molecule(s) for OP and MC in the testicular membrane proteome (obtained from male ICR mouse) was performed by ABPP11 with a FP-TAMRA fluorescent probe (Pierce Biotechnology, Rockford, IL).12 In brief, mouse testis membrane preparation (1,000g supernatant, 20,000g pellet) in 50 mM Tris– HCl buffer (pH 8.0) was incubated with the FP-TAMRA probe in competition with an anticholinesterase agent. The reaction mixture was subjected to SDS–PAGE separation ultimately for detecting fluorescence activities by a flatbed scanner FLA-3000 (FUJIFILM, Tokyo, Japan). Relative to animal experiments, the study was carried out in accordance with the guidelines for animal experiments of the Nagoya City University. Relevance of OP exposure to the male reproductive toxicity can be examined by evaluating FAAH/MAGL activity in the membrane preparations of mouse testis and epididymis cauda. Therefore, ICR male mice (9 weeks age, 30–35 g) were treated orally with corn oil (vehicle) and chlorpyrifos (CPF) (35 or 50 mg/kg/day) for 10 days, and afterward the testis and epididymis cauda were obtained. The membrane preparations were used for FAAH and MAGL hydrolysis assays13,14 with the corresponding substrates [14C]anandamide and [14C]mono-oleoylglycerol, respectively (55 mCi/mmol for both substrates, American Radiolabeled Chemicals, Inc., St. Louis, MO). Alternatively, the CPF-treated testicular proteome was incubated with the FP-TAMRA chemical probe to visualize traces of the relevant molecular target(s). The FP-TAMRA chemical probe labels many serine hydrolases in the mouse testicular membrane proteome (Fig. 2). In this in-gel
5435
Y. Noro et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5434–5436 Table 1 Potencies of anticholinesterases as inhibitors of testicular FAAH and MAGL IC50 ± SD, (n = 3) (lM)
Anticholinesterase
CPF oxon Dichlorvos Paraoxon Diazinon oxon Carbaryl Propoxur Carbofuran
Figure 1. Structures of anticholinesterase agents considered in the present investigation. Two chemotypes of anticholinesterases are examined for identification of a potential molecular target(s) in the murine male reproductive system: that is, organophosphates (OP) chlorpyrifos (CPF), its bioactivated metabolite oxon (CPF oxon), and dichlorvos, and methylcarbamate (MC) propoxur. Other OP and MC insecticides used here are listed in Table 1 (see also Supplementary data for chemical structures).
analysis system, OP compound CPF oxon (0.01–1 mM), bioactivated metabolite of CPF, clearly inhibited the testicular serine hydrolase targets (63 and 35 kDa) recognized as FAAH and MAGL, respectively (Fig. 2 and Supplementary data).11 OPs dichlorvos (Fig. 2), paraoxon, and diazinon oxon (Supplementary data) selectively reacted with the FAAH. However, MC insecticides propoxur (Fig. 2), carbaryl, and carbofuran (Supplementary data) failed to inhibit FAAH and MAGL. Several other minor OP-sensitive targets were also detected similar to those in the mouse brain membrane
FAAH
MAGL
0.22 ± 0.02 4.1 ± 0.7 5.3 ± 0.8 40 ± 4.1 160 ± 34 990 ± 68 P1000 (44%)a
0.12 ± 0.01 51 ± 17 21 ± 4.4 110 ± 8.1 410 ± 84 >1000 (27%)a >1000 (26%)a
a Percent inhibition at the indicated concentration. FAAH or MAGL was assayed by hydrolysis of the corresponding substrate [14C]anandamide (FAAH) or [14C]mono-oleoylglycerol (MAGL), respectively.13,14 An IC50 value (molar concentration of a test chemical necessary for 50% inhibition of specific enzyme activity) is determined by iterative least-squares regression using Sigmaplot software. Chemical structures of anticholinesterases are given in Figure 1 and Supplementary data.
proteome.15 Thus, potencies of OP and MC compounds as inhibitors of testicular FAAH or MAGL activity were evaluated by [14C]anandamide or [14C]mono-oleoylglycerol hydrolysis assay, respectively (Table 1). CPF oxon was potent to FAAH and MAGL, while other OPs preferred to inhibit FAAH rather than to act on MAGL. MCs were weak or inactive for the both targets. This inhibitory profile is analogous to that observed in the ABPP approach. Subsequently, FAAH activity in testis or epididymis cauda was unambiguously diminished by the repeated CPF exposure. MAGL was significantly inhibited in testis but not in epididymis cauda (Table 2). Consistently, the ABPP analysis of the CPF-exposed testicular proteome resulted in an exclusive FAAH/MAGL inhibition
Figure 2. In vitro screening of target molecule(s) for anticholinesterases in the mouse testicular membrane proteome. The activity-based protein profiling (ABPP) approach with a phosphonofluoridate fluorescent probe (FP-TAMRA, which compellingly phosphorylates the diverse serine hydrolases: i.e., an in-gel analysis of enzyme activity) revealed that testicular fatty acid amide hydrolase (FAAH) (63 kDa) or monoacylglycerol lipase (MAGL) (35 kDa)11,12 is a potential target for CPF oxon (activated metabolite of CPF) or dichlorvos but not for MC propoxur (see also Supplementary data). A forty milligram testicular membrane protein was incubated with an anticholinesterase agent (0.001–1000 lM) in competition with the FP-TAMRA serine hydrolase probe (1 lM) (Pierce Biotechnology, Rockford, IL) for 30 min at 25 °C, and then the sample was subjected to SDS–PAGE separation for analyzing the fluorescence activity by a flatbed scanner. A representative ABPP gel image is given for each case. Results for other anticholinesterases (paraoxon, diazinonoxon, carbaryl, and carbofuran) are given in Supplementary data.
5436
Y. Noro et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5434–5436
Table 2 Effects of subacute CPF exposure (10 days via oral route) on FAAH and MAGL activities in mouse testis and epididymis cauda Treatment
Activity (pmol/mg/min) (±SD, n = 5–9) FAAH
Vehicle Low High
MAGL
Testis
Epididymis cauda
Testis
Epididymis cauda
180 ± 18 (100%) 98 ± 45⁄⁄ (54%) 95 ± 22⁄⁄ (52%)
54 ± 5.8 (100%) 44 ± 10⁄⁄ (81%) 39 ± 4.2⁄⁄ (71%)
800 ± 20 (100%) 660 ± 120⁄ (83%) 690 ± 57⁄⁄ (86%)
400 ± 18 (100%) 390 ± 12 (98%) 400 ± 25 (100%)
Asterisks indicate significant difference (⁄P < 0.05 or ⁄⁄P < 0.01 based on Dunnet’s multiple post hoc test) between the vehicle (corn oil) and CPF treatment (35 or 50 mg/kg/day). FAAH and MAGL activities were evaluated by [14C]anandamide and [14C]mono-oleoylglycerol hydrolysis assays, respectively. A dose-dependence in the FAAH or MAGL inhibition upon the CPF exposure was ambiguous based on the enzyme assays, whereas the corresponding ABPP analysis showed a proper dose– inhibition relationship (see Fig. 3). The CPF-administered mice (one tenth or three eighth for low or high dose group, respectively) died by accident and/or intoxication during the exposure period. The relative activity (%) is also given in parentheses. AChE activities (nmol/mg/min ± SD) in the mouse brain homogenate were compared between the vehicle and treated groups: that is, vehicle 66 ± 4 (100%); low 17 ± 4⁄⁄ (26%); high 17 ± 2⁄⁄ (26%), respectively (⁄⁄P < 0.01) (the CPF-exposed mice showed several nerve-toxic signs including tremor).
apoptosis of testicular cells such as Sertoli and Leydig cells. In the epididymis cauda and sperm cells, anandamide also regulates sperm motility and viability.17,18,20–22 In contrast, cannabinoid receptor antagonist rimonabant appreciably increases sperm motility and viability.23 Therefore, the present investigation pronounces that inhibition of FAAH (and also MAGL) by OP insecticides in male reproductive tissues, to conceivably modulate the endocannabinoid signaling, may be predictive of spermatotoxicity such as broken sperms, cytoplasmic droplets, and reduced motility and viability. Acknowledgment The project described was supported by JSPS KAKENHI Grant number 24659303 to M.T. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.bmcl.2013.07.021. References and notes 1. 2. 3. 4. 5.
6. 7. 8. 9.
10. Figure 3. Effect of subacute CPF exposure (10 days via oral route) on the mouse testicular serine hydrolase proteome. A testicular membrane preparation (40 mg protein) was reacted with the FP-TAMRA chemical probe for ABPP gel-based analysis. A typical ABPP gel image is given featuring FAAH and MAGL traces (right).
(Fig. 3). The present CPF exposure (in relatively high doses and a short period) clearly illustrates our goal of exploring a possible anticholinesterase target in the murine male reproductive system. This Letter describes for the first time that anticholinesterase insecticide, particularly OP, acts on the FAAH and/or MAGL in murine testis and epididymis cauda. OP insecticides also act on mouse brain FAAH and MAGL, consequently resulting in hypomotility or catalepsy.13–15 FAAH and MAGL are hydrolyzing enzymes for endocannabinoid agonists anandamide and 2-arachidonoylglycerol, respectively, and the endocannabinoid system plays pivotal roles on spermatogenesis and sperm motility acquirement.16–19 Inhibition or down-regulation of FAAH, elevating anandamide levels, in turn overstimulates cannabinoid signal, thereby finally eliciting
11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
Casida, J. E.; Durkin, K. A. Annu. Rev. Entomol. 2013, 58, 99. Casida, J. E.; Quistad, G. B. Chem. Res. Toxicol. 2004, 17, 983. Long, J. Z.; Cravatt, B. F. Chem. Rev. 2011, 111, 6022. Padungtod, C.; Hassold, T. J.; Millie, E.; Ryan, L. M.; Savitz, D. A.; Christiani, D. C.; Xu, X. Am. J. Ind. Med. 1999, 36, 230. Kamijima, M.; Hibi, H.; Gotoh, M.; Taki, K.; Saito, I.; Wang, H.; Itohara, S.; Yamada, T.; Ichihara, G.; Shibata, E.; Nakajima, T.; Takeuchi, Y. J. Occup. Health 2004, 46, 109. Meeker, J. D.; Singh, N. P.; Ryan, L.; Duty, S. M.; Barr, D. B.; Herrick, R. F.; Bennett, D. H.; Hauser, R. Hum. Reprod. 2004, 19, 2573. Sánchez-Peña, L. C.; Reyes, B. E.; López-Carrillo, L.; Recio, R.; Morán-Martı´nez, J.; Cebrián, M. E.; Quintanilla-Vega, B. Toxicol. Appl. Pharmacol. 2004, 196, 108. Perry, M. J.; Venners, S. A.; Barr, D. B.; Xu, X. Reprod. Toxicol. 2007, 23, 113. Okamura, A.; Kamijima, M.; Shibata, E.; Ohtani, K.; Takagi, K.; Ueyama, J.; Watanabe, Y.; Omura, M.; Wang, H.; Ichihara, G.; Kondo, T.; Nakajima, T. Toxicology 2005, 213, 129. Okamura, A.; Kamijima, M.; Ohtani, K.; Yamanoshita, O.; Nakamura, D.; Ito, Y.; Miyata, M.; Ueyama, J.; Suzuki, T.; Imai, R.; Takagi, K.; Nakajima, T. J. Occup. Health 2009, 51, 478. Long, J. Z.; Nomura, D. K.; Cravatt, B. F. Chem. Biol. 2009, 16, 744. Patricelli, M. P.; Giang, D. K.; Stamp, L. M.; Burbaum, J. J. Proteomics 2001, 1, 1067. Quistad, G. B.; Sparks, S. E.; Casida, J. E. Toxicol. Appl. Pharmacol. 2001, 173, 48. Quistad, G. B.; Klintenberg, R.; Caboni, P.; Liang, S. N.; Casida, J. E. Toxicol. Appl. Pharmacol. 2006, 211, 78. Nomura, D. K.; Blankman, J. L.; Simon, G. M.; Fujioka, K.; Issa, R. S.; Ward, A. M.; Cravatt, B. F.; Casida, J. E. Nat. Chem. Biol. 2008, 4, 373. Cobellis, G.; Cacciola, G.; Scarpa, D.; Meccariello, R.; Chianese, R.; Franzoni, M. F.; Mackie, K.; Pierantoni, R.; Fasano, S. Biol. Reprod. 2006, 75, 82. Maccarrone, M. Prog. Lipid Res. 2009, 48, 344. Lewis, S. E.; Maccarrone, M. Pharmacol. Res. 2009, 60, 126. Lewis, S. E. M.; Rapino, C.; Tommaso, M. D.; Pucci, M.; Battista, N.; Paro, R.; Simon, L.; Lutton, D.; Maccarrone, M. PLoS One 2012, 7, e47704. Maccarrone, M.; Finazzi-Agró, A. Cell Death Differ. 2003, 10, 946. Rossi, G.; Gasperi, V.; Paro, R.; Barsacchi, D.; Cecconi, S.; Maccarrone, M. Endocrinology 2007, 148, 1431. Rossi, G.; Cacciola, G.; Altucci, L.; Meccariello, R.; Pierantoni, R.; Fasano, S.; Cobellis, G. Gen. Comp. Endocrinol. 2007, 153, 320. Aquila, S.; Guido, C.; Santoro, A.; Gazzerro, P.; Laezza, C.; Baffa, M. F.; Andò, S.; Bifulco, M. Br. J. Pharmacol. 2010, 159, 831.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Anticholinesterase insecticide action at the murine male reproductive system Yuki Noro, Motohiro Tomizawa ⇑, , Yuki Ito, Himiko Suzuki, Keisuke Abe, Michihiro Kamijima Department of Occupational and Environmental Health, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan
a r t i c l e
i n f o
Article history: Received 1 June 2013 Revised 1 July 2013 Accepted 12 July 2013 Available online 19 July 2013
a b s t r a c t The present report describes for the first time that anticholinesterase type insecticides specifically inhibit the fatty acid amide hydrolase and/or monoacylglycerol lipase, as secondary target(s), in the murine male reproductive system (testis and epididymis cauda), thereby presumably being involved with spermatotoxicity such as deformity, underdevelopment, and reduced motility. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Anticholinesterase insecticides Chlorpyrifos Fatty acid amide hydrolase Monoacylglycerol lipase Organophosphate and methylcarbamate insecticides Spermatotoxicity
Organophosphate (OP) and methylcarbamate (MC) insecticides (Fig. 1) are generally utilized throughout the globe for protecting crops, people, livestock, and companion animals from pest insect attack and disease transmission. These compounds are nerve poisons acting at the cholinergic neurons by inhibiting acetylcholinesterase (AChE) as the primary target.1 The anticholinesterase agents phosphorylate or carbamoylate the serine OH residue at the AChE catalytic triad. On the other hand, the anticholinesterases may also react with many other serine hydrolases to reveal the secondary or unexpected physiological effects.2,3 Interestingly, recent reports have suggested that the human male reproductive system is also a target for OP and MC insecticides.4–8 In rodent, OP insecticides induce spermatotoxicity including sperm deformity or underdevelopment (broken sperm and cytoplasmic droplets) and reduced sperm motility, whereas the molecular target or mechanism causing the spermatotoxicity is not defined yet.9,10 Accordingly, the present investigation detects a possible anticholinesterase target in the murine male reproductive system by activity-based protein profiling (ABPP) approach3 with a phosphonofluoridate chemical probe (FP-TAMRA), thus proposing a hypothesis for the first time that inhibition of fatty acid amide hydrolase (FAAH) and/or monoacylglycerol lipase (MAGL) in the mouse testis and epididymis cauda is relevant to the anticholinesterase-induced spermatotoxicity. ⇑ Corresponding author. Tel.: +81 3 5477 2529; fax: +81 3 5477 2626. E-mail address: [email protected] (M. Tomizawa). Present address: Faculty of Applied Bioscience, Tokyo University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.07.021
In vitro screening of target molecule(s) for OP and MC in the testicular membrane proteome (obtained from male ICR mouse) was performed by ABPP11 with a FP-TAMRA fluorescent probe (Pierce Biotechnology, Rockford, IL).12 In brief, mouse testis membrane preparation (1,000g supernatant, 20,000g pellet) in 50 mM Tris– HCl buffer (pH 8.0) was incubated with the FP-TAMRA probe in competition with an anticholinesterase agent. The reaction mixture was subjected to SDS–PAGE separation ultimately for detecting fluorescence activities by a flatbed scanner FLA-3000 (FUJIFILM, Tokyo, Japan). Relative to animal experiments, the study was carried out in accordance with the guidelines for animal experiments of the Nagoya City University. Relevance of OP exposure to the male reproductive toxicity can be examined by evaluating FAAH/MAGL activity in the membrane preparations of mouse testis and epididymis cauda. Therefore, ICR male mice (9 weeks age, 30–35 g) were treated orally with corn oil (vehicle) and chlorpyrifos (CPF) (35 or 50 mg/kg/day) for 10 days, and afterward the testis and epididymis cauda were obtained. The membrane preparations were used for FAAH and MAGL hydrolysis assays13,14 with the corresponding substrates [14C]anandamide and [14C]mono-oleoylglycerol, respectively (55 mCi/mmol for both substrates, American Radiolabeled Chemicals, Inc., St. Louis, MO). Alternatively, the CPF-treated testicular proteome was incubated with the FP-TAMRA chemical probe to visualize traces of the relevant molecular target(s). The FP-TAMRA chemical probe labels many serine hydrolases in the mouse testicular membrane proteome (Fig. 2). In this in-gel
5435
Y. Noro et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5434–5436 Table 1 Potencies of anticholinesterases as inhibitors of testicular FAAH and MAGL IC50 ± SD, (n = 3) (lM)
Anticholinesterase
CPF oxon Dichlorvos Paraoxon Diazinon oxon Carbaryl Propoxur Carbofuran
Figure 1. Structures of anticholinesterase agents considered in the present investigation. Two chemotypes of anticholinesterases are examined for identification of a potential molecular target(s) in the murine male reproductive system: that is, organophosphates (OP) chlorpyrifos (CPF), its bioactivated metabolite oxon (CPF oxon), and dichlorvos, and methylcarbamate (MC) propoxur. Other OP and MC insecticides used here are listed in Table 1 (see also Supplementary data for chemical structures).
analysis system, OP compound CPF oxon (0.01–1 mM), bioactivated metabolite of CPF, clearly inhibited the testicular serine hydrolase targets (63 and 35 kDa) recognized as FAAH and MAGL, respectively (Fig. 2 and Supplementary data).11 OPs dichlorvos (Fig. 2), paraoxon, and diazinon oxon (Supplementary data) selectively reacted with the FAAH. However, MC insecticides propoxur (Fig. 2), carbaryl, and carbofuran (Supplementary data) failed to inhibit FAAH and MAGL. Several other minor OP-sensitive targets were also detected similar to those in the mouse brain membrane
FAAH
MAGL
0.22 ± 0.02 4.1 ± 0.7 5.3 ± 0.8 40 ± 4.1 160 ± 34 990 ± 68 P1000 (44%)a
0.12 ± 0.01 51 ± 17 21 ± 4.4 110 ± 8.1 410 ± 84 >1000 (27%)a >1000 (26%)a
a Percent inhibition at the indicated concentration. FAAH or MAGL was assayed by hydrolysis of the corresponding substrate [14C]anandamide (FAAH) or [14C]mono-oleoylglycerol (MAGL), respectively.13,14 An IC50 value (molar concentration of a test chemical necessary for 50% inhibition of specific enzyme activity) is determined by iterative least-squares regression using Sigmaplot software. Chemical structures of anticholinesterases are given in Figure 1 and Supplementary data.
proteome.15 Thus, potencies of OP and MC compounds as inhibitors of testicular FAAH or MAGL activity were evaluated by [14C]anandamide or [14C]mono-oleoylglycerol hydrolysis assay, respectively (Table 1). CPF oxon was potent to FAAH and MAGL, while other OPs preferred to inhibit FAAH rather than to act on MAGL. MCs were weak or inactive for the both targets. This inhibitory profile is analogous to that observed in the ABPP approach. Subsequently, FAAH activity in testis or epididymis cauda was unambiguously diminished by the repeated CPF exposure. MAGL was significantly inhibited in testis but not in epididymis cauda (Table 2). Consistently, the ABPP analysis of the CPF-exposed testicular proteome resulted in an exclusive FAAH/MAGL inhibition
Figure 2. In vitro screening of target molecule(s) for anticholinesterases in the mouse testicular membrane proteome. The activity-based protein profiling (ABPP) approach with a phosphonofluoridate fluorescent probe (FP-TAMRA, which compellingly phosphorylates the diverse serine hydrolases: i.e., an in-gel analysis of enzyme activity) revealed that testicular fatty acid amide hydrolase (FAAH) (63 kDa) or monoacylglycerol lipase (MAGL) (35 kDa)11,12 is a potential target for CPF oxon (activated metabolite of CPF) or dichlorvos but not for MC propoxur (see also Supplementary data). A forty milligram testicular membrane protein was incubated with an anticholinesterase agent (0.001–1000 lM) in competition with the FP-TAMRA serine hydrolase probe (1 lM) (Pierce Biotechnology, Rockford, IL) for 30 min at 25 °C, and then the sample was subjected to SDS–PAGE separation for analyzing the fluorescence activity by a flatbed scanner. A representative ABPP gel image is given for each case. Results for other anticholinesterases (paraoxon, diazinonoxon, carbaryl, and carbofuran) are given in Supplementary data.
5436
Y. Noro et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5434–5436
Table 2 Effects of subacute CPF exposure (10 days via oral route) on FAAH and MAGL activities in mouse testis and epididymis cauda Treatment
Activity (pmol/mg/min) (±SD, n = 5–9) FAAH
Vehicle Low High
MAGL
Testis
Epididymis cauda
Testis
Epididymis cauda
180 ± 18 (100%) 98 ± 45⁄⁄ (54%) 95 ± 22⁄⁄ (52%)
54 ± 5.8 (100%) 44 ± 10⁄⁄ (81%) 39 ± 4.2⁄⁄ (71%)
800 ± 20 (100%) 660 ± 120⁄ (83%) 690 ± 57⁄⁄ (86%)
400 ± 18 (100%) 390 ± 12 (98%) 400 ± 25 (100%)
Asterisks indicate significant difference (⁄P < 0.05 or ⁄⁄P < 0.01 based on Dunnet’s multiple post hoc test) between the vehicle (corn oil) and CPF treatment (35 or 50 mg/kg/day). FAAH and MAGL activities were evaluated by [14C]anandamide and [14C]mono-oleoylglycerol hydrolysis assays, respectively. A dose-dependence in the FAAH or MAGL inhibition upon the CPF exposure was ambiguous based on the enzyme assays, whereas the corresponding ABPP analysis showed a proper dose– inhibition relationship (see Fig. 3). The CPF-administered mice (one tenth or three eighth for low or high dose group, respectively) died by accident and/or intoxication during the exposure period. The relative activity (%) is also given in parentheses. AChE activities (nmol/mg/min ± SD) in the mouse brain homogenate were compared between the vehicle and treated groups: that is, vehicle 66 ± 4 (100%); low 17 ± 4⁄⁄ (26%); high 17 ± 2⁄⁄ (26%), respectively (⁄⁄P < 0.01) (the CPF-exposed mice showed several nerve-toxic signs including tremor).
apoptosis of testicular cells such as Sertoli and Leydig cells. In the epididymis cauda and sperm cells, anandamide also regulates sperm motility and viability.17,18,20–22 In contrast, cannabinoid receptor antagonist rimonabant appreciably increases sperm motility and viability.23 Therefore, the present investigation pronounces that inhibition of FAAH (and also MAGL) by OP insecticides in male reproductive tissues, to conceivably modulate the endocannabinoid signaling, may be predictive of spermatotoxicity such as broken sperms, cytoplasmic droplets, and reduced motility and viability. Acknowledgment The project described was supported by JSPS KAKENHI Grant number 24659303 to M.T. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.bmcl.2013.07.021. References and notes 1. 2. 3. 4. 5.
6. 7. 8. 9.
10. Figure 3. Effect of subacute CPF exposure (10 days via oral route) on the mouse testicular serine hydrolase proteome. A testicular membrane preparation (40 mg protein) was reacted with the FP-TAMRA chemical probe for ABPP gel-based analysis. A typical ABPP gel image is given featuring FAAH and MAGL traces (right).
(Fig. 3). The present CPF exposure (in relatively high doses and a short period) clearly illustrates our goal of exploring a possible anticholinesterase target in the murine male reproductive system. This Letter describes for the first time that anticholinesterase insecticide, particularly OP, acts on the FAAH and/or MAGL in murine testis and epididymis cauda. OP insecticides also act on mouse brain FAAH and MAGL, consequently resulting in hypomotility or catalepsy.13–15 FAAH and MAGL are hydrolyzing enzymes for endocannabinoid agonists anandamide and 2-arachidonoylglycerol, respectively, and the endocannabinoid system plays pivotal roles on spermatogenesis and sperm motility acquirement.16–19 Inhibition or down-regulation of FAAH, elevating anandamide levels, in turn overstimulates cannabinoid signal, thereby finally eliciting
11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
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