anticholinesterase pesticides (metabolism, neurotoxicity, and epidemiology) || apoptosis induced by...

13APOPTOSIS INDUCED BY ANTICHOLINESTERASEPESTICIDES

QING LI

Department of Hygiene and Public Health, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan

13.1 Introduction 165

13.2 What is Apoptosis? 165

13.3 OP Pesticides Induce Apoptosis In Vivo 166

13.4 OP Pesticides Induce Apoptosis In Vitro 167

13.5 OP Pesticides Induce Apoptosis in ImmuneCells In Vitro 169

13.6 Carbamate Pesticides Induce Apoptosis 17013.6.1 Carbamate Pesticides Induce

Apoptosis In Vivo 17013.6.2 Carbamate Pesticides Induce

Apoptosis In Vitro 170

13.7 Conclusions 172

Acknowledgments 172

References 172

13.1 INTRODUCTION

Anticholinesterase pesticides, including organophosphate(OP) and carbamate pesticides, are widely used throughoutthe world as insecticides, herbicides, and fungicides in agri-culture and as agents for eradicating termites around homes.OP and carbamate pesticides have a common mechanism ofaction but their chemical classes are distinctly different; theesters of phosphoric or phosphothioic acid and those of car-bamic acid. The first OP pesticide to be used commerciallywas tetraethylpyrophosphate, and the first pesticidal carbamicacid esters were synthesized in the 1930s and were marketedas fungicides (Ecobichon, 1991). These compounds arepotent inhibitors of serine esterases, such as acetylcholin-esterase (AChE) and serum cholinesterase (ChE). The maintoxicity of OP and carbamate pesticides is neurotoxicity,which is caused by the inhibition of AChE (Ecobichon,1991; Ellenhorn and Barceloux, 1988).

Recent studies have indicated that OP and carbamate pes-ticides affect several biochemical pathways that do notinvolve the modulation of AChE activity. Under bothin vivo and in vitro conditions, OP and carbamate pesticides

have been shown to induce apoptosis in several types ofcells (Akbarsha and Sivasamy, 1997; Kim et al., 2004; Liet al., 2007, 2009; Nakadai et al., 2006).

13.2 WHAT IS APOPTOSIS?

Cell death can be divided into two basic forms, apoptosis andnecrosis, based on the changes in morphology, enzymaticactivity, and adjacent cellular effects (Levin, 1988; Majnoand Joris, 1995). Apoptosis was originally proposed by Kerrin 1972 as a form of programmed cell death in multicellularorganisms, and involves a series of biochemical events lead-ing to a characteristic cell morphology and death, includingblebbing, changes to the cell membrane, such as loss of mem-brane asymmetry and attachment, cell shrinkage, nuclear frag-mentation, chromatin condensation, and chromosomal DNAfragmentation (Kerr et al., 1972; Majno and Joris, 1995).Apoptosis can be evaluated morphologically (Akbarsha andSivasamy, 1997; Carlson et al., 2000; Oral et al., 2006; Royet al., 1998) and with biochemical examinations by determin-ing DNA fragmentation using electrophoresis (Li et al., 2009;

Anticholinesterase Pesticides: Metabolism, Neurotoxicity, and Epidemiology. Edited by Tetsuo Satoh and Ramesh C. GuptaCopyright # 2010 John Wiley & Sons, Inc.

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Nakadai et al., 2006; Fig. 13.1), and by determining intracellu-lar active caspase-3 with antihuman active caspase-3 anti-bodies (Carlson et al., 2000; Li et al., 2007, 2009; Nakadaiet al., 2006; Fig. 13.2). The caspase family of cysteine pro-teases plays a key role in apoptosis and inflammation.Caspase-3 is a key protease that is activated during the earlystages of apoptosis and, like other members of the caspasefamily, is synthesized as an inactive proenzyme that is pro-cessed in cells undergoing apoptosis by self-proteolysisand/or cleavage by another protease (Patel et al., 1996). Inthe early stage of apoptosis, the membrane phospholipid phos-phatidylserine (PS) translocates from the inner to the outerleaflet of the plasma membrane. Once exposed to the extra-cellular environment, the binding sites on PS become avail-able for Annexin-V, a 35- to 36-kDa, Ca2þ-dependent,phospholipid-binding protein with a high affinity for PS.The translocation of PS precedes other apoptotic processessuch as the loss of plasma membrane integrity, DNA fragmen-tation, and chromatin condensation. Therefore, apoptosis inthe early stage can be detected by FITC-Annexin-V stainingusing a flow cytometer (Dong et al., 2005; Li et al., 2007,2009; Nakadai et al., 2006) (Fig. 13.3).

13.3 OP PESTICIDES INDUCE APOPTOSISIN VIVO

Akbarsha and Sivasamy (1997) first reported that an OP pes-ticide, phosphamidon, induced apoptosis in male germinalline cells of rat in vivo as determined morphologically.Hamm et al. (1998) also suggested that diazinon-inducedcell death in vivo involved apoptotic processes in the teleostOryzias latipes. Chronic, low-level dichlorvos (DDVP)

exposure has the potential to disrupt the cellular antioxidantdefense system, which in turn triggers the release of cyto-chrome c from mitochondria into the cytosol as well ascaspase-3 activation, and finally results in oligonucleosomalDNA fragmentation, a hallmark of apoptosis in DDVP-treated rat brain (Kaur et al. 2007). These studies provide evi-dence of impaired mitochondrial bioenergetics and apoptoticneuronal degeneration after chronic, low-level exposure toOP pesticides. Moreover, Oral et al. (2006) reported that sub-chronic administration of DDVP induced apoptosis in theendometrium of rats, as determined by histopathologicaland immunohistochemical examinations for caspase-3 andcaspase-9, whereas administration of vitamins E and Calong with DDVP significantly reduced the histopathologicalchanges and the extent of apoptosis. Yu et al. (2008) also

Figure 13.1 Chlorpyrifos-induced DNA fragmentation in U937cells determined by agarose gel electrophoresis. M: marker of theDNA ladder, C: positive control, camptotecin at 6 mM. The concen-trations of chlorpyrifos were 0, 71, 142, and 284 mM. Reproducedfrom Nakadai, A. et al. (2006). Toxicology 224:202–209. Withpermission from Elsevier Science.

Figure 13.2 DDVP-induced increase in active caspase-3-positiveNK-92CI cells. (a) The shaded histogram shows the control cells(DDVP at 0 ppm) and the open histogram shows the cells treatedwith DDVP at 100 ppm for 24 h and stained with FITC-rabbitanti-human active caspase-3 antibody. (b) Data are presented asthe mean+SD (n ¼ 3 for 16 h, n ¼ 5 for 24 h). �: p , 0.05; ��:p , 0.01; ��: p , 0.001, significantly different from 0 ppm byunpaired t-test. Reproduced from Li, Q., et al. (2007). Toxicology239:89–95. With permission from Elsevier Science.

166 APOPTOSIS INDUCED BY ANTICHOLINESTERASE PESTICIDES

found that chlorpyrifos (CP) induced cell apoptosis, lipid per-oxidation, and DNA damage and reduced the activities ofantioxidant enzymes superoxide dismutase (SOD), catalase,and glutathione peroxidase in the retina of mice. However,pretreatment with a combination of antioxidants vitaminsC and E significantly attenuated these effects of CP, demon-strating that oxidative stress was involved in CP-induced cellapoptosis in mouse retina. The above reports may have impli-cations for the treatment of OP insecticide poisoning with acombination of vitamins C and E.

13.4 OP PESTICIDES INDUCE APOPTOSISIN VITRO

Bagchi et al. (1995) demonstrated that OP pesticides, such asCP and fenthion, induced the production of reactive oxygenspecies (ROS) and oxidative tissue damage in neuroactive

PC-12 cells in vitro. In addition, it was shown that ROSmay serve as common mediators of apoptosis in responseto many toxins and pathological conditions, suggesting thatOP pesticides may induce apoptosis in vitro. OP compounds,such as parathion, paraoxon (the bioactive metabolite of para-thion), phenyl saligenin phosphate (PSP), tri-ortho-tolylphosphate (TOTP), and triphenyl phosphite (TPPi) inducedtime-dependent increases in apoptosis in SH-SY5Y humanneuroblastoma cells in vitro, which were assessed morpho-logically and by biochemical examinations includingcaspase-3 activation and DNA fragmentation. Parathioninduced apoptosis to the greatest extent; followed byTOTP . TPPi . paraoxon . PSP . diisopropylphosphor-ofluoridate, suggesting that the relative capability of an OPcompound to induce apoptosis is inversely related to itscapacity for AChE inhibition (Ehrich et al., 1997).Paraoxon, parathion, PSP, TOTP, and TPPi induced signifi-cant time-dependent increases in caspase-3 activation,

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Figure 13.3 Chlorpyrifos induced apoptosis in Jurkat T cells. (a) Dot plot of FITC-Annexin-V/PI in control Jurkat T cells; (b) dot plot ofFITC-Annexin-V/PI in chlorpyrifos-treated Jurkat T cells. The percentages in quadrants 2 and 3 show FITC-Annexin-Vþ/PIþ (necrosis) andFITC-Annexin-Vþ/PI2 (apoptosis) cells, respectively. (c) Dose- and time-dependent increases in apoptotic cells in chlorpyrifos-treated JurkatT cells. Data are presented as the mean+SE (n ¼ 8 for 0, 50, and 100 ppm, n ¼ 4 for others). �: p , 0.05; ��: p , 0.01, significantly differentfrom 0 ppm by unpaired t-test. Reproduced from Li, Q. et al. (2009). Toxicology 255:53–57. With permission from Elsevier Science.

13.4 OP PESTICIDES INDUCE APOPTOSIS IN VITRO 167

whereas pretreatment with cyclosporin A, which has cytopro-tective effects, prior to the activation of caspase 3 (Okamotoet al., 1999), decreased apoptosis and caspase-3 activationafter parathion, TOTP, paraoxon, and TPPi exposure. Inaddition, pretreatment with caspase-3 and caspase-8 inhibi-tors significantly decreased caspase-3 activation afterexposure to PSP and parathion. Pretreatment with serine pro-tease inhibitor also decreased caspase activation significantlyafter PSP and TOTP exposure. Alteration of OP compound-induced nuclear fragmentation or caspase-3 activation bypretreatment with cyclosporin A, a serine protease, andcaspase-3 and caspase-8 inhibitors suggested that OPcompound-induced apoptosis may be modulated throughmultiple sites, including mitochondrial permeability pores,receptor-mediated caspase pathways, or serine proteases(Carlson et al., 2000). Caughlan et al. (2004) found that CPand chlorpyrifos-oxon (CPO), but not 3,5,6-trichloro-2-pyridinol (TCP; the breakdown product of CP and CPO),induced apoptosis in rat cortical neurons, which wasregulated by a balance between p38 and extracellular signalregulated kinase (ERK)/c-Jun N-terminal kinase (JNK)/mitogen-activated protein (MAP) kinases. It is generallyagreed that CPO is approximately three orders of magnitudemore potent than CP in the inhibition of brain AChE activity.However, this study demonstrated that CPO is only slightlymore potent than CP in inducing apoptosis. This indicatesthat CP-induced apoptosis may occur independently ofAChE inhibition, although AChE activity was not measuredin this study. Masoud et al. (2003) also found that noncholi-nergic doses of malathion (0.01 to 20 mM) induced apoptosisin murine L929 fibroblasts in a dose- and time-dependentmanner as determined using flow cytometric and caspase acti-vation analyses, suggesting that the cytotoxicity of malathionat noncholinergic doses is mediated through caspase-dependent apoptosis but not through AChE inhibition,which supported the findings mentioned by Caughlan et al.(2004) and Carlson et al. (2000). Moreover, Saulsbury et al.(2008) also compared the difference among CP, CPO, andTCP-induced apoptosis in placental cells. They found thatCP, and its metabolite CPO, caused a dose-dependentreduction in cellular viability with CP being more toxicthan its metabolites, supporting the findings of Caughlanet al. (2004). CP-induced toxicity was characterized by theloss of mitochondrial potential, the appearance of nuclearcondensation and fragmentation, downregulation of Bcl-2,and upregulation of tumor necrosis factor (TNF)-alpha andFas mRNA. Pharmacological inhibition of the Fas, nicotinic,and TNF-alpha receptors did not attenuate CP-inducedtoxicity, and atropine exhibited minimal ability to reversetoxicity, suggesting that CP-induced apoptosis may occurindependently of AChE inhibition (Carlson et al., 2000).Furthermore, signal transduction inhibitors failed to attenuatetoxicity; however, an inhibitor of p38-alpha and p38-betaMAP kinases sensitized the cells to CP-induced toxicity.

Pan-caspase inhibitor produced a slight but significantreversal of CP-induced toxicity, indicating that the majorcaspase pathways are not integral to CP-induced toxicity.Furthermore, the mechanism of CP-induced apoptosis in pla-cental cells is different from that of parathion, TOTP, TPPi,paraoxon, and PSP-induced apoptosis in SH-SY5Y humanneuroblastoma cells, in which the major caspase pathwaysare integral to OP pesticide-induced apoptosis (Carlsonet al., 2000). Taken together, these results suggest that CPinduces apoptosis in placental cells through pathways notdependent on Fas/TNF signaling, activation of caspases, orinhibition of ChE (Saulsbury et al., 2008). Wu et al. (2005)examined the neurotoxic effect of paraoxon and the role ofN-methyl-D-aspartate (NMDA) receptors as a mechanism ofaction in cultured cerebellar granule cells, and found thatparaoxon increases apoptosis about 10-fold compared tobasal levels. Broad-spectrum caspase and caspase-9-specificinhibitors protect against paraoxon-mediated apoptosis, para-oxon-stimulated caspase-3 activity, and neuronal cell death,suggesting that paraoxon-induced apoptosis is through path-ways dependent on the activation of caspases 3 and 9. Thisfinding also suggests that the mechanism of paraoxon-induced apoptosis in cerebellar granule cells is different fromthat of CP-induced apoptosis in placental cells, in which themajor caspase pathways are not integral to CP-induced apop-tosis (Saulsbury et al., 2008). Moreover, it was found that theactivation of NMDA receptors protect neurons against para-oxon-induced neurotoxicity by blocking apoptosis initiatedby paraoxon.

Roy et al. (1998) used whole rat embryo culture to studythe effects of CP at the neural tube stage of development.On embryonic day 9.5, embryos were exposed to 0.5, 5, or50 mg/mL CP. After 48 h (embryonic day 11.5), embryoswere examined for dysmorphogenesis and were then pro-cessed for light microscopic examination of the neuroepithe-lium. The forebrain and hindbrain regions revealed reducedand altered mitotic figures, with dispersion and disorientationof the mitotic layer. In addition, cytotoxicity was indicated bycytoplasmic vacuolation, enlargement of intercellular spaces,and the presence of a significant number of apoptotic cells.These alterations were evident even at the lowest concen-tration of CP, which produced no dysmorphogenesis. Theeffects were intensified at higher concentrations, whichwere just at the threshold for dysmorphogenesis; however,the neuroepithelial abnormalities were still present inembryos that were not dysmorphogenic. Greenlee et al.(2004) also found that low-dose CP exposure in vitro signifi-cantly induced apoptosis in mouse preimplantation embryos.These results support the idea that CP specifically targetsbrain development at low concentrations, indicating theneed to reevaluate the safety of this compound for exposurein vivo.

To explore the mechanism of OP pesticide-induced apop-tosis, Gupta et al. (2007) used Drosophila melanogaster


transgenic for heat shock protein (Hsp) 70 to verify thehypothesis that ROS generated by DDVP modulates Hsp70expression and antioxidant defense enzymes, and acts as asignaling molecule for apoptosis in the exposed organism.DDVP, with or without inhibitors of Hsp70, SOD, and cata-lase, was fed to the third instar larvae of D. melanogastertransgenic for Hsp70 to examine Hsp70 expression, oxidativestress, and apoptotic markers. A concentration- and time-dependent significant increase in ROS generation, accom-panied by a significant upregulation of Hsp70, precededchanges in antioxidant defense enzyme activities and the con-tents of glutathione, malondialdehyde, and protein carbonylin the treated organisms. An inhibitory effect on SOD and cat-alase activities significantly upregulated ROS generation andHsp70 expression in the exposed organism, while inhibitionof Hsp70 significantly affected oxidative stress markersinduced by DDVP. ROS generation is correlated positivelywith Hsp70 expression and apoptotic cell death end points,indicating the involvement of ROS in the overall adversitycaused by DDVP to the organism. The study suggests that(1) Hsp70 and antioxidant enzymes work together for cellulardefense against xenobiotic hazards in D. melanogaster and(2) free radicals may modulate Hsp70 expression and apopto-sis in the exposed organism.

13.5 OP PESTICIDES INDUCE APOPTOSISIN IMMUNE CELLS IN VITRO

It has been reported that OP pesticides show immunotoxicityin human and animals both in vivo (Li et al., 2004) andin vitro (Li, 2007; Li and Kawada, 2006; Li et al., 2000,2002, 2005, 2006, 2008). Several investigators have tried toelucidate the mechanism of OP-induced immunotoxicityfrom the aspect of apoptosis (Li et al., 2007, 2009; Nakadaiet al., 2006; Olgun et al., 2004; Saleh et al., 2003a, 2003b).

Saleh et al. (2003a, 2003b) found that paraoxon and para-thion cause apoptosis in a murine EL4 T-lymphocytic leuke-mia cell line through the activation of caspases 3 and 9, butnot caspase-8. Paraoxon triggered a dose- and time-dependent translocation of cytochrome c from mitochondriainto the cytosol and disrupted mitochondrial transmembranepotential, which was dependent on caspase activation. More-over, paraoxon treatment also resulted in a time-dependentupregulation and translocation of the proapoptotic moleculeBax to mitochondria, which was subsequent to activation ofthe caspase cascades. The results indicate that paraoxoninduces apoptosis in EL4 cells through a direct effect on mito-chondria by disrupting the transmembrane potential, causingthe release of cytochrome c into the cytosol and subsequentactivation of caspase-9 (Saleh et al., 2003a). Olgun et al.(2004) also reported that malathion exposure in vitrocaused both apoptotic and necrotic cell death in C57BL/6mouse thymocytes as evaluated by Annexin-V/propidium

iodide (PI) staining and lactate dehydrogenase (LDH) releaseassays. Based on these findings, it is suggested that malathionis a potent immunotoxicant in vitro and that the mechanism ofcytotoxicity observed upon exposure to malathion may, atleast in part, be due to the induction of apoptosis.

In order to explore the mechanism of OP pesticide-induced immunotoxicity, it was also investigated whetherOP pesticides induced apoptosis in human immune cells,and the underlying mechanism examined (Li et al., 2007,2009; Nakadai et al., 2006). Human immune cells, a humanmonocyte-like cell line (U937), were first treated with CP,and it was found that CP induced the cell death of U937 ina dose- and time-dependent manner, as shown by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT) and LDH assays and PI uptake. Then, it was investi-gated whether CP-induced cell death consisted of apoptosis,and the results indicated that CP induced apoptosis in U937cells in a time- and dose-dependent manner, as shown byAnnexin-V staining and DNA fragmentation (Fig. 13.1;Nakadai et al., 2006). To explore the mechanism of CP-induced apoptosis in U937 cells, the intracellular level ofactive caspase-3 in CP-treated cells was examined, and itwas found that CP induced an increase in intracellularactive caspase-3 in U937 cells in a dose-dependent manner,and a caspase-3 inhibitor, Z-DEVD-FMK, significantlyinhibited CP-induced apoptosis. These findings indicatethat CP induced apoptosis in U937 cells, which is mediatedby the activation of caspase-3 (Nakadai et al., 2006). Daset al. (2006) also found that CP significantly induced apopto-sis and necrosis in cultured human peripheral blood lympho-cytes in vitro in a dose-dependent manner, which wasdetected using DNA diffusion assay.

Based on the above-mentioned findings, it was furtherinvestigated whether OP pesticides can induce apoptosis inhuman natural killer (NK) cells. NK-92CI and NK-92MIcells, which are interleukin-2-independent human NK celllines, express CD56, perforin, granzymes A, B, 3/K, andgranulysin and are highly cytotoxic to K562 cells in the chro-mium release assay (Li et al., 2005, 2006, 2008) were treatedwith DDVP or CP in vitro (Li et al., 2007). Apoptosis inducedby DDVP and CP was determined by FITC-Annexin-V stain-ing and the intracellular level of active caspase-3 analyzed byflow cytometry. It was found that DDVP and CP significantlyinduced apoptosis in NK-92 cells in a dose- and time-dependent manner. DDVP also induced an increase in intra-cellular active caspase-3 in NK-92CI cells in a dose- andtime-dependent manner (Fig. 13.2), and a caspase-3 inhibitor,Z-DEVD-FMK, significantly inhibited DDVP-induced apo-ptosis, suggesting that this apoptosis is partially mediatedby the activation of intracellular caspase-3. The pattern ofapoptosis induced by CP differed from that induced byDDVP. CP showed a faster response than DDVP at higherdoses, whereas DDVP showed a slower but stronger apopto-sis-inducing ability than CP at lower doses. Moreover, the

13.5 OP PESTICIDES INDUCE APOPTOSIS IN IMMUNE CELLS IN VITRO 169

response to OP pesticides differed between NK-92CI andNK-92MI cells, and NK-92CI cells were more sensitive toOP pesticides than NK-92MI cells. This is similar to the inhi-bition of NK activity induced by DDVP, in which NK-92CIcells were more easily inhibited by DDVP than NK-92MIcells and strongly suggested a relationship between DDVP-induced apoptosis and the inhibition of cytolytic activity inNK cells. Taken together, these findings suggest that OPpesticide-induced inhibition of NK activity may be at leastpartially mediated by OP pesticide-induced apoptosis inNK cells (Li et al., 2007, 2008). Mattiuzzo et al. (2006)also found that DDVP significantly induced apoptosis andnecrosis in cultured human lymphoblastoid AHH-1 cellsin vitro in a dose-dependent manner, which was detectedusing TUNEL assay.

To explore the mechanism of OP pesticide-induced inhi-bition of cytotoxic T-lymphocyte (CTL) activity, it wasalso investigated whether OP pesticides can induce celldeath/apoptosis in T cells using Jurkat human T cells invitro (Li et al., 2009). It was found that CP induced the celldeath of Jurkat human T cells in a dose- and time-dependentmanner, as shown by MTT and LDH assays. CP also inducedapoptosis in Jurkat T cells in a dose- and time-dependentmanner, as determined by analysis of Annexin-V staining(Fig. 13.3) and DNA fragmentation, suggesting that CP-induced cell death consisted of apoptosis. CP also inducedan increase in intracellular active caspase-3 in Jurkat T cellsin a dose-and time-dependent manner, and Z-DEVD-FMKsignificantly inhibited CP-induced apoptosis (Fig. 13.4).

These findings indicate that CP can induce apoptosis inhuman Jurkat T cells, and this effect is partially mediatedby the activation of intracellular caspase-3 (Li et al., 2009).It is necessary to investigate the relationship between OPpesticide-induced apoptosis in T cells and OP-induced inhi-bition of CTL activity in future studies. Other OP pesticides,such as monocrotophos, profenofos, and acephate also sig-nificantly induced apoptosis and necrosis in cultured humanperipheral blood lymphocytes in vitro in a dose-dependentmanner, detected by DNA diffusion assay (Das et al., 2006).

13.6 CARBAMATE PESTICIDES INDUCEAPOPTOSIS

Similar to OP pesticides, carbanate pesticides also induceapoptosis both in vivo (Moffit et al., 2007; Simpson et al.,2005) and in vitro (Calviello et al., 2006; Ishido, 2007; Jiaand Misra, 2007; Sook Han et al., 2003) in many cell types.

13.6.1 Carbamate Pesticides Induce ApoptosisIn Vivo

Simpson et al. (2005) reported that the herbicide cycloate[carbamothioic acid; ethyl (cyclohexyl)-S-ethyl ester] givenas a single oral dose to rats, caused selective neuronal celldeath in two regions in the rat forebrain, the pyramidal neur-ons of layers II-III throughout the pyriform cortex and ingranule cells of the caudal ventro-lateral dentate gyrus,suggesting that cycloate may cause an upregulation of apop-tosis in selected regions of the adult brain. Carbendazim witha single gavage dose of 40, 67, 100, or 200 mg/kg of bodyweight also induced a significant increase in apoptosis ingerm cell in adult rat testis measured by TUNEL assay(Moffit et al., 2007). Rath et al. (2005) also found thatthiram induced endothelial cell apoptosis in the capillaryvessels of the growth plates of chickens fed a diet containingthiram 100 ppm for 48 h, which was determined by TUNELassay and DNA fragmentation. Exposure to sublethal concen-trations of methyl thiophanate, a systemic carbamete fungi-cide, led to hepatocellular morphological changes, glycogendepletion, and apoptosis in liver of the lizard Podarcis sicula,which was mediated by peroxisome proliferators-activatedreceptors (Buono et al., 2007).

13.6.2 Carbamate Pesticides Induce ApoptosisIn Vitro

Fimognari et al. (1999) first reported that methyl thiophanatesignificantly induced apoptosis in human lymphocytesin vitro determined by flow cytometry and TUNEL assay.Carbofuran (CF), an anticholinesterase carbamate, is one ofthe most widely used N-methylcarbamate esters in insect andnematode control. CF is regarded as a relatively safe chemical

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Figure 13.4 Caspase-3 inhibitor significantly inhibited chlorpyri-fos-induced apoptosis as determined by FITC-Annexin-V stainingdetected by flow cytometry. The concentration of chlorpyrifos was50 ppm. Data are presented as the mean+SE (n ¼ 4). �: p ,

0.05; ��: p , 0.01, significantly different from the treatments with-out inhibitor by paired t-test. Reproduced from Li, Q. et al. (2009).Toxicology 255:53–57. With permission from Elsevier Science.


based on extensive toxicological data. Kim et al. (2004)examined the cytotoxic effects of CF on cultured rat corticalcells (neurons) by LDH assay, Annexin-V/PI staining, andDNA fragmentation and found that CF induces neuronaldeath by apoptosis. On the other hand, Yoon et al. (2001)found that the N-nitroso derivative N-nitrosocarbofuran(NOCF) of CF, but not CF induced apoptosis of CHL cellsand significant G (2)/M cell cycle arrest, which were demon-strated by morphological changes, DNA fragmentation, andflow cytometric analysis, suggesting that NOCF, an impor-tant metabolite of CF, leads to the induction of cell cyclearrest and apoptosis in CHL cells. Jung et al. (2003) alsofound that NOCF, but not CF, induced apoptotic cell deathin brain microvascular endothelial cells determined byAnnexin-V staining and electron microscopy, at least inpart, through the ERK pathway, supporting the findings byYoon et al. (2001). These findings suggested that the mechan-ism of CF inducing apoptosis in cortical cells (neurons; Kimet al., 2004) is different than that in CHL/microvascularendothelial cells, in which CF did not show an apoptoticeffect (Jung et al., 2003; Yoon et al., 2001). Lee et al.(2004) investigated the molecular mechanism of NOCF-induced apoptosis in CHL cells and found that NOCFcaused dose-dependent upregulation of cytosolic factors,such as Bax and Bid, and the release of cytochrome c, whichwas accompanied by activation of caspase-9, caspase-8,and caspase-3 and the subsequent cleavage of poly(ADP-ribose) polymerase, suggesting that the mitochondrial path-way is primarily involved in NOCF-induced apoptosis.

Thiram and ziram also significantly induced apoptosis inPC12 cells in both dose- and time-dependent manners,respectively. Interestingly, both thiram and ziram inducedrapid and sustained increases of intracellular Ca2þ in PC12cells, which were almost completely blocked by flufenamicacid, an inhibitor of nonselective cation channels. BAPTA-AM, an intracellular Ca2þ chelator, inhibited thiram- andziram-induced apoptosis. These results suggest that thiramand ziram induce apoptotic neuronal cell death by Ca2þ

influx through nonselective cation channels. These findingsmay provide clues to understanding the mechanism of neuro-toxicity of thiram and ziram (Sook Han et al., 2003).Marikovsky (2002) found that thiram inhibited DNA syn-thesis and induced apoptosis in cultured bovine capillaryendothelial cells. These effects were prevented by theaddition of antioxidants, indicating the involvement ofROS. Exogenously, addition of Cu2þ impeded specificallyand almost completely the inhibitory effect of thiram inbovine capillary endothelial cells. Moreover, thiram hadmarkedly inhibited human recombinant Cu/Zn SOD enzy-matic activity (85%) in vitro, suggesting that the effects ofthiram are mediated by the inhibition of Cu/Zn SOD activity.On the other hand, Cereser et al. (2001) found that thiram-mediated cell death was not apoptotic but seemed to be ofthe necrotic type in cultured human skin fibroblasts,

suggesting that thiram may show both apoptotic and necroticactivity depending on the cell type.

Mancozeb, a widely used fungicide of the ethylene-bis-dithiocarbamate group, also induced apoptosis in theMCF-7 breast cancer cell line, as determined by flow cyto-metric assays (Lin and Garry, 2000), and showed proapopto-tic effects on RAT-1 fibroblasts cultured in vitro and inperipheral blood mononucleated cells of Wistar rats deter-mined by DNA single strand break formation, oxidative mar-kers of DNA oxidation, and ROS. The proapoptotic effect ofmancozeb suggests its possible relevance in the pathogenesisof neurodegenerative diseases, often related to exposure topesticides (Calviello et al., 2006). Low-dose mancozebexposure in vitro also significantly induced apoptosis inmouse preimplantation embryos (Greenlee et al. 2004).

A number of epidemiological studies have demonstrated astrong association between the incidence of Parkinson dis-ease and pesticide exposure (Thiruchelvam et al., 2002).Parkinsonian symptoms are seen after exposure to the herbi-cide paraquat and the fungicide maneb (Thrash et al., 2007).Exposure to the pesticides endosulfan and zineb, alone and incombination, caused neurodegeneration in vivo. Based on thisbackground, Jia and Misra (2007) hypothesized that thesepesticides cause neurotoxicity, in part by enhancing apoptoticcell death. The SH-SY5Y human neuroblastoma cell line,which retains a catecholaminergic phenotype, was exposedto zineb in vitro. Zineb caused apoptosis in a concentration-dependent manner. Visual evaluation using a DNA ladderassay and Annexin-V/PI staining confirmed the contributionof both apoptotic and necrotic processes. These findingssuggest that the cytotoxicity of zineb is associated with theoccurrence of early and late apoptotic/necrotic processes inSH-SY5Y human neuroblastoma cells and support the con-tention that pesticide-induced neuronal cell death leading toneurodegenerative disease may, at least in part, be associatedwith early and late apoptosis of dopaminergic neurons. Onthe other hand, melatonin, a scavenger of a number of reactiveoxygen and reactive nitrogen species both in vitro and in vivo,inhibits maneb-induced apoptosis in PC12 neural cells byaffecting the process of activation of caspase-3/7 and themitochondrial membrane potential of the neural cells. Theneurotoxicity of maneb on PC12 cells was elicited throughapoptotic cell death, concomitant with aggregation ofalpha-synuclein, a feature of Parkinson’s disease.Furthermore, aggregation of alpha-synuclein by maneb wasalso inhibited by melatonin. Thus, melatonin preventsmaneb-induced neurodegeneration at a nighttime physiologi-cal blood concentration, most likely by inhibiting the aggre-gation of alpha-synuclein as well as preventingmitochondrial dysfunction in PC 12 cells (Ishido, 2007).Dithiocarbamates also induce apoptosis by inhibiting thenuclear factor-kappaB (NF-kappaB) signaling cascade. It isknown that NF-kappaB plays a central role in the immunesystem and is described as a major factor in many human

13.6 CARBAMATE PESTICIDES INDUCE APOPTOSIS 171

cancers mainly because of its protective effects against apop-tosis (Cvek and Dvorak, 2007).

Hammond et al. (2001) investigated the apoptosis-inducing ability of FB642 (methyl-2-benzimidazolecarba-mate; carbendazim), a systemic fungicide, from the aspect ofantitumor activity in p53-positive and -negative tumor and indrug- and multidrug-resistant cell lines, and found that FB642increases the degree of apoptosis in all tumor cell lines exam-ined, may induce G2/M uncoupling, may selectively kill p53abnormal cells, and exhibits antitumor activity in drug- andmultidrug-resistant cell lines. Similarly, Hao et al. (2002)also found that carbendazim (FB642) showed potent antitu-mor activity against both murine B16 melanoma andhuman HT-29 colon carcinoma cell lines in vitro by inducingthe apoptosis of cancer cells.

13.7 CONCLUSIONS

OP pesticides can induce apoptosis both in vivo in mice, rats,the teleost Oryzias latipes, and Drosophila melanogaster andin vitro in many cell types, including neuron cells such asneuroactive PC-12 cells, cerebellar granule cells, neuroblas-toma cells, cortical neurons, placental cells, fibroblasts, andimmune cells such as monocytes, lymphocytes (NK and Tcells), which are mediated by the caspase cascade pathway,regulating the balance between p38 and ERK/JNK MAPkinases, affecting the mitochondrial pathway, as well as bymodulating Hsp70 expression. OP pesticide-induced apopto-sis may occur independently of AChE inhibition.

Carbamate pesticides, such as carbofuran, cycloate, car-bendazim, mancozeb, zineb, thiram, and ziram can induceapoptosis both in vivo and in vitro, which are mediated byaffecting the caspase cascade pathway, by inhibiting theNF-kappaB signaling cascade and Cu/Zn SOD activity aswell as by affecting the mitochondrial pathway.

ACKNOWLEDGMENTS

The parts of experimental data shown in this review and performedby the author were supported by grants from the Ministry ofEducation, Culture, Sports, Science and Technology of Japan (No.09877077, No. 10770178, No. 12770206, No. 15590523, and No.19590602). The author would like to thank Dr. TomoyukiKawada (Professor and Chief) at the Department of Hygiene andPublic Health, Nippon Medical School, for his advice.

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