experimental animals : male c57bl/6ncrslc mice ( 8 weeks old ) were purchased 
 from japan slc ( hamamatsu , japan ) . all mice were maintained in 40.5  20.5  18.5 cm 
 individual ventilated cages ( sealsafe plus mouse ; tecniplast , buguggiate , italy ) under 
 controlled temperature ( 23  2c ) and humidity ( 50  10% ) on a 12-hr light / dark cycle at the 
 kobe university life - science laboratory with ad libitum access to a pellet 
 diet ( dc-8 ; clea japan , tokyo , japan ) and filtered water . this study was approved by the 
 institutional animal care and use committee ( permission # 24 - 10 - 03 ) and carried out according 
 to the kobe university animal experimental regulation . ctd purification and hplc analysis : water - soluble dantotsu 
 ( involving 16% of ctd ; sumitomo chemical co. , tokyo , japan ) , donated by sado city ( niigata , 
 japan ) , was washed with 10 times the amount of distilled water to remove the surfactant 
 activating and granulating agents . after being left to stand for at least 48 hr , this step was repeated five times , and then , the white precipitate 
 was collected and air - dried naturally for a week . the content rate of ctd in the white 
 precipitate was measured by a lachrom high - performance liquid chromatography ( hplc ) system 
 ( interface l-7000 , pump l-7100 , auto sampler l-7200 , column oven l-7350 and uv - vis detector 
 l-7420 ; hitachi , tokyo , japan ) using a capcell pak c18 ug120 column ( 5 m 
 particles , 4.6  250 mm ; shiseido , tokyo , japan ) . ctd standard ( > 99.8% ; wako chemical , osaka , japan ) and the obtained white powder were 
 completely dissolved in dimethyl sulfoxide ( dmso ) and then serial - diluted with a mobile 
 phase consisting of 55% acetonitrile in 50 mm potassium phosphate buffer ( ph 3.0 ) , followed 
 by filtration with a 0.20 m syringe driven filter unit ( millex - lg ; 
 millipore , billerica , ma , u.s.a . ) . the column maintained at 40c was eluted with the mobile 
 phase at a flow rate of 1.0 ml / min . after the column was equilibrated , 10 
 l of the samples was injected into the hplc system . we monitored the 
 resultant chromatograph at the wavelength of 260 nm and then calculated the ctd content of 
 the obtained white powder from the calibration curve created by the peak areas and heights 
 of the ctd standard . a single peak was observed in the samples from the white precipitate 
 with the same retention time as that of the ctd standard . a linear calibration curve 
 ( r=0.999 ) created from samples serial - diluted with the ctd standard showed 
 that our purification makes the content rate of ctd increased from 1416% to 9397% by 
 weight . ctd administration and stress exposure : all mice were allowed to acclimate 
 to their home cages for a week prior to the initiation of experiments . we divided the mice 
 into eight groups ( n=5 mice in each cage ) : ctd-0 ( control ) , ctd-10 ( 10 mg / kg / day ) , ctd-50 
 ( 50 mg / kg / day ) and ctd-250 ( 250 mg / kg / day ) with the presence or absence of stress exposure . 
 as a substitute for filtered water for the mice , we used the medigel sucrarose 2 
 oz cup ( clearh2o , portland , me , u.s.a . ) , which is a flavored thermoreversible 
 hydration gel matrix . the amounts of the purified ctd for the respective administration 
 groups were calculated from the ctd purity ( 95% ) , daily gel intake ( 5 g / day / mouse ) , total 
 gel weight ( 60 g ; excluding the package weight ) and average mouse weight ( 24 g ; weighed at 
 initiation of experiments ) . these amounts of ctd were completely dissolved in 600 
 l dmso ( 1% volume of a gel ) and injected into the gels and then 
 double - boiled at 60c followed by shaking to ensure that the ctd was diffused well . for the 
 ctd-0 group , the same volume of dmso without the purified ctd all gels were strapped with cable ties under the grate of the cage lid to prevent 
 contamination with the beddings and the excretions . all gels were weighed daily to estimate 
 the amounts of the putative ctd exposure . in the four stress groups , the mice were subjected 
 to an unpredictable chronic stress procedure as described in our earlier report   with some modifications . briefly , the following six 
 stressors were used : 5 min forced swimming in water at room temperature ( rt ) , 24 hr food and 
 water deprivation , continuous overnight illumination , 30 min horizontal cage shaking ( 80 
 rpm ) , 24 hr switching of cagemates ( being housed with another mouse ) and 24 hr wet bedding . 
 to maximize the unpredictability of this paradigm , the mice were randomly exposed to two 
 mild stressors per day at varying times for 4 weeks . behavioral analysis : on the last day of the 4 week experimental period , an 
 open field test was conducted during the light phase to evaluate the locomotor activity and 
 the anxiety - like behavior of the mice . briefly , the mouse was placed on the corner of an 
 open field ( 60  60  30 cm ) with led illumination . all of the mouse s activities were 
 recorded by a video camera for the subsequent 10 min , and we then analyzed the total 
 distance traveled and the time spent in the center zone ( 30  30 cm ) using smart video 
 tracking software v3.0 ( san diego instruments , san diego , ca , u.s.a . ) . tissue preparation : on the day after the completion of the 4 weeks of 
 combined exposure to ctd and stress , all mice were deeply anesthetized with diethyl ether 
 and transcardially perfused with 0.9% normal saline , followed by perfusion with ice - cold 4% 
 paraformaldehyde in phosphate buffer . the testes were excised , weighed and postfixed with 
 the same fixative overnight at 4c . the testes were dehydrated through a graded series of 
 ethanol followed by xylene and embedded in paraffin . serial sections of testes were then cut 
 at 4 m thickness on a sliding microtome ( sm2000r ; leica microsystems , 
 wetzlar , germany ) and mounted on slide glasses ( platinum pro ; matsunami glass ind . histological and immunohistochemical analyses : for the general 
 histological analysis , testis sections were stained with hematoxylin and eosin ( he ; merck 
 kgaa , darmstadt , germany ) after their deparaffinization and hydration , following the 
 manufacturer s instructions . to detect antioxidant enzymes in the testes the sections were immersed in absolute methanol and 
 0.5% h2o2 for 30 min , respectively , at rt to quench the endogenous 
 peroxidase activity . kyoto , japan ) for 1 hr at rt for protein blocking and then incubated with the rabbit 
 polyclonal anti - gpx4 antibody ( item no . 10005258 ; cayman chemicals , ann arbor , mi , u.s.a . ) 
 diluted 1:8,000 in phosphate buffered saline with 0.05% tween-20 ( pbst ; ph 7.4 ) for 18 hr at 
 4c . after being washed with pbst , the sections were reacted with goat anti - rabbit 
 immunoglobulins conjugated to peroxidase - labeled dextran polymer in tris ( hydroxymethyl ) 
 aminomethane - hcl buffer ( envision+ ; dako , glostrup , denmark ) for 1 hr at rt . immunoreactivity was then detected by incubation with 3,3-diaminobenzidine solution 
 ( envision+ kit / hrp[dab ] , dako ) . the sections were then rinsed with distilled water and 
 counterstained lightly with hematoxylin solution for 1 min . next , the sections were placed 
 in a graded series of ethanol , dehydrated with absolute ethanol , cleared by xylene and 
 coverslipped with eukitt ( o. kindler gmbh , freiburg , germany ) . statistical analysis : statistical analyses were performed with excel 
 statistics 2012 ( ssri version 1.00 , tokyo , japan ) . in the behavioral analyses , outliers more 
 distant than 1.5 interquartile ranges from the upper or lower quartile were omitted . all 
 data were analyzed by two - way anova ( ctd stress ) followed by the tukey - kramer s post hoc 
 test . the results were considered significant when the p - value was less 
 than 0.05 . gel intake , body and testis weights : the daily gel consumption , body 
 weights and testes weights are shown in fig . 1fig . 1.effects of combined exposure on the daily gel intake ( a ) , body weights ( b ) and testis 
 weights ( c ) in the non - stressed ( open column ) and stressed groups ( closed column ) . the 
 two - way anova showed significant main effects for the ctd and stress 
 ( p<0.01 ) , but the interaction effects were not significant in 
 gel intake and body weights . ( a ) ctd dose - dependently inhibited the gel intake , and 
 the inhibition was significant in all ctd - administration groups compared to the ctd-0 
 groups . the stress procedure significantly inhibited the gel intake in each 
 ctd - administration group . ( b ) the body weights of the ctd-250 stress and non - stressed 
 groups were significantly lower than those of the other dose groups , in addition to 
 the significant difference between the ctd-0 and ctd-10 groups . the post hoc analyses 
 also revealed that there were significant differences between the ctd-0 and ctd-250 
 stress and non - stressed groups . ( c ) the two - way anova showed a significant main effect 
 for stress ( p<0.01 ) , but the interaction effect was not 
 significant in the absolute weights of testes . the stress procedure significantly 
 suppressed the absolute testes weights in all stress groups , except the ctd-10 group . ctd significantly suppressed the daily gel intake in all ctd administration 
 groups compared to the non - ctd administration groups ( fig . although the daily intake was significantly inhibited in the stress 
 groups , there was little difference if the values in the days of food deprivation in our 
 stress protocol were omitted ( table 1table 1.body weight , gel intake , putative ctd exposure and testis weightsgroupsctd-0ctd-0 + stressctd-10ctd-10 + stressctd-50ctd-50 + stressctd-250ctd-250 + stressbody weight ( g)28.62  1.6626.48  0.7226.98  0.9925.36  1.2227.50  0.8225.24  0.7825.82 0.9923.76  0.40gel intake / day ( g)6.13  1.375.64  2.804.41  0.842.85  1.774.60  1.593.55  2.093.64  0.642.38  1.42 [ 6.66  2.56 ] [ 3.73  1.59 ] [ 4.65  1.79 ] [ 3.16  1.09 ] putative exposure ( mg / kg / day)008.82  1.685.70  3.5446.0  15.935.5  20.9182  32119  71 [ 7.46  3.18 ] [ 46.5  17.9 ] [ 158  54.5 ] testis weight ( mg)99.6  5.588.2  11.895.7  9.690.5  9.7100.3  9.881.1  8.297.5  7.887.5 the values in square brackets are the 
 averages after the removal of values in the deprivation days from the calculation . ) . as a result , the body weights of mice reflected the same tendency of the gel 
 intake . the body weights were suppressed in all stress groups relative to the non - stressed 
 groups at the same dose of ctd ( fig . the body 
 weights of the ctd-250 groups were significantly lower than those of the other three dose 
 groups . on the other hand , the absolute weights of the testes were decreased by the stress 
 procedure in the ctd-0 , ctd-50 and ctd-250 groups ( fig . effects of combined exposure on the daily gel intake ( a ) , body weights ( b ) and testis 
 weights ( c ) in the non - stressed ( open column ) and stressed groups ( closed column ) . the 
 two - way anova showed significant main effects for the ctd and stress 
 ( p<0.01 ) , but the interaction effects were not significant in 
 gel intake and body weights . ( a ) ctd dose - dependently inhibited the gel intake , and 
 the inhibition was significant in all ctd - administration groups compared to the ctd-0 
 groups . the stress procedure significantly inhibited the gel intake in each 
 ctd - administration group . ( b ) the body weights of the ctd-250 stress and non - stressed 
 groups were significantly lower than those of the other dose groups , in addition to 
 the significant difference between the ctd-0 and ctd-10 groups . the post hoc analyses 
 also revealed that there were significant differences between the ctd-0 and ctd-250 
 stress and non - stressed groups . ( c ) the two - way anova showed a significant main effect 
 for stress ( p<0.01 ) , but the interaction effect was not 
 significant in the absolute weights of testes . the stress procedure significantly 
 suppressed the absolute testes weights in all stress groups , except the ctd-10 group . the values in square brackets are the 
 averages after the removal of values in the deprivation days from the calculation . 2.behavioral effect of combined exposure of ctd and stress in the open field activity 
 in the non - stressed and stressed groups . ( a ) a representative trajectory map of the 
 mice as illustrated by the video tracking software . the exploratory behaviors in the 
 center zone ( 30  30 cm ) of the open field ( 60  60 cm ) were dose - dependently 
 suppressed by ctd compared to the non - administration groups . ( b ) total distances 
 traveled in the open field of the non - stressed ( open columns ) and stressed groups 
 ( closed columns ) . no marked difference was detected by two - way anova in the total 
 distances traveled . ( c ) time spent in the center zone in the open field of the 
 non - stressed ( open columns ) and stressed groups ( closed columns ) . two - way anova showed 
 significant main effects for ctd ( p<0.05 ) and stress 
 ( p<0.01 ) , but the interaction effect was not significant . ctd 
 significantly inhibited the times spent in the center zone in the ctd-250 groups 
 compared to the non - ctd administration groups . a significant anxiogenic effect of the 
 stress procedure was observed between the ctd-0 groups . in the non - stressed condition , there were significant differences in the ctd-10 and ctd-50 groups compared to the 
 ctd-0 group . * p<0.05 , 
 * * p<0.01 . ) revealed that the mice with the combined ctd exposure and stress procedure tended to 
 walk alongside the walls . ctd and the stress procedure had insignificant impacts on the 
 locomotor activities measured by the total distances traveled ( fig . by contrast , the anxiety - like behaviors that avoid being in 
 the center zone were elevated by the stress procedure and were significantly different 
 between the ctd-0 groups ( fig . ctd also 
 enhanced the anxiety - like behaviors in a dose - dependent manner , and in the non - stressed 
 condition , the difference was significant in the ctd-10 and ctd-250 groups compared to the 
 ctd-0 group . behavioral effect of combined exposure of ctd and stress in the open field activity 
 in the non - stressed and stressed groups . ( a ) a representative trajectory map of the 
 mice as illustrated by the video tracking software . the exploratory behaviors in the 
 center zone ( 30  30 cm ) of the open field ( 60  60 cm ) were dose - dependently 
 suppressed by ctd compared to the non - administration groups . ( b ) total distances 
 traveled in the open field of the non - stressed ( open columns ) and stressed groups 
 ( closed columns ) . no marked difference was detected by two - way anova in the total 
 distances traveled . ( c ) time spent in the center zone in the open field of the 
 non - stressed ( open columns ) and stressed groups ( closed columns ) . two - way anova showed 
 significant main effects for ctd ( p<0.05 ) and stress 
 ( p<0.01 ) , but the interaction effect was not significant . ctd 
 significantly inhibited the times spent in the center zone in the ctd-250 groups 
 compared to the non - ctd administration groups . a significant anxiogenic effect of the 
 stress procedure was observed between the ctd-0 groups . in the non - stressed condition , there were significant differences in the ctd-10 and ctd-50 groups compared to the 
 ctd-0 group . histological and immunohistochemical findings : the general histological 
 analyses of the testes by he staining are shown in fig the 
 seminiferous tubules of the control group showed robust spermatogenesis with densely 
 stacked germ cells . multinucleated giant cells ( arrow ) were occasionally observed in 
 the testes of the ctd-0+stress groups . in the ctd groups , dose - dependent vacuoled 
 degeneration ( arrowhead ) of seminiferous tubules was observed . degenerated 
 seminiferous tubules only composed of sertoli cells were present in the ctd-250 
 groups . bar=100 m .. although there were seminiferous tubules densely arranged with germ cells and sperms 
 in the testes of the control group , several testicular signs of toxicity were observed in 
 combined exposure groups . multinucleated giant cells were occasionally observed in the 
 seminiferous tubules of the mice in the stress groups . in addition , variably sized 
 vacuolizations were scattered at the bottom of seminiferous tubules of the ctd - administered 
 groups . in particular , seriously degenerated seminiferous tubules , from which almost all 
 germ cells had dropped off , were found in the ctd-250 groups . there was no marked 
 histological difference between the leydig cells in the testicular interstitial tissues of 
 any of the experimental groups . the 
 seminiferous tubules of the control group showed robust spermatogenesis with densely 
 stacked germ cells . multinucleated giant cells ( arrow ) were occasionally observed in 
 the testes of the ctd-0+stress groups . in the ctd groups , dose - dependent vacuoled 
 degeneration ( arrowhead ) of seminiferous tubules was observed . degenerated 
 seminiferous tubules only composed of sertoli cells were present in the ctd-250 
 groups . bar=100 m . the results of the immunohistochemical analyses of the testes visualizing gpx4 are shown in 
 fig . 4.representative immunohistochemistry for gpx4 of the testis in the non - stressed and 
 stressed groups . gpx4 immunoreactivities were detected in sperms and spermatids in the 
 testes of the control group . in the testes of the stressed groups , reduced 
 immunoreactivity of gpx4 in sperm was observed . in the ctd groups , spermatids showed 
 weakened immunoreactivity of gpx4 . sertoli cells ectopically expressed gpx4 in 
 degenerated seminiferous tubules of the ctd-50+stress group and the ctd-250 groups . bar=100 m .. in the control group , strong gpx4 immunoreactivity was detected in the sperm , and 
 diffuse gpx4 immunoreactivity was seen in the cytoplasm of spermatids . the intensity of gpx4 
 immunoreactivity in the sperm was decreased by stress , and the cytoplasmic immunoreactivity 
 in the spermatids was dose - dependently decreased by ctd . in addition , abnormal 
 immunoreactivity of gpx4 in sertoli cells was detected in the combined exposure groups , and 
 it became strong in degenerated seminiferous tubules in the ctd-50+stress group and the 
 ctd-250 groups . representative immunohistochemistry for gpx4 of the testis in the non - stressed and 
 stressed groups . gpx4 immunoreactivities were detected in sperms and spermatids in the 
 testes of the control group . in the testes of the stressed groups , reduced 
 immunoreactivity of gpx4 in sperm was observed . in the ctd groups sertoli cells ectopically expressed gpx4 in 
 degenerated seminiferous tubules of the ctd-50+stress group and the ctd-250 groups . we verified the combined effects of ctd and stress on the reproductive and behavioral 
 function in mature male mice by a non - invasive administration method . there are several 
 advantages of the gels we used ; compared to baking pellets , the gels can uniformly deliver 
 the test articles to animals without exposing them to high temperature . the gels could not 
 completely cloak the bad palatability of ctd as reported earlier using similar gels mixed 
 with an aromatase inhibitor , anastrozole ; 
 however , the gel consumption observed in combined exposure groups was enough for maintaining 
 the health of the mice . in our preliminary experiments , we initially suspected that the ctd 
 avoidance effect was attributable to any accessory constituents of the commercial pesticide 
 containing ctd , but ctd itself appeared to have some repellent effects on mice . in 
 agricultural fields , ctd - containing pesticides are used at dilutions from 1:20 to 1:4,000 
 for treating seeds and plants . our purification protocol and the water solubility of ctd 
 imply that the white precipitate resulting from the recommended concentration of ctd 
 products is particularly dangerous for pesticide users . in the present study , the unpredictable stress model , which is often used in the assessment 
 of antidepressant medications , evidently raised the anxiety level in the mice . ctd 
 dose - dependently induced anxiety alone , but there was little difference in this measure 
 among the combined exposure groups . to the best of our knowledge , there are only a few reports about the behavioral effects of neonicotinoids on mammals . several parameters of behavioral developmental tests that evaluated the sense of 
 equilibrium , coordinated movement and muscular power of offspring were changed by maternal 
 exposure to low - dose ctd , with a sex difference [ 34 , 
 35 ] . thiamethoxam ( tmx , 100 mg / kg / day ) 
 significantly reduced the locomotor activity of rats in an open field test and induced 
 anxiety in an elevated plus - maze test while suppressing acetylcholinesterase ( ache ) activity 
 in three brain regions . considering that tmx is 
 metabolized to ctd in mammals , our findings regarding the anxiogenic effects of ctd 
 sufficiently agree with the findings of these prior studies . although the mechanisms of the behavioral effects of neonicotinoids remain unclear , 
 cholinergic systems and downstream neurotransmitters are undoubtedly involved . an in 
 vivo study revealed that ctd directly injected into the rat brain evoked a 
 striatal dopamine surge through nachrs . early 
 types of neonicotinoids caused a subsequent desensitization of nachrs as well as immediate 
 neuronal excitation . the behavioral consequences 
 of chronic nicotine intake are also attributed to both the upregulation and desensitization 
 of nachrs . the anxiety - like behaviors in several 
 behavioral tests are part of the phenotype of nachrs subunit 4 knockout mice , implying that the anxiogenic effect of ctd observed 
 in the present study was presumably triggered by the chronic desensitization of nachrs . the chrna7 coding 7 subunit of nachrs contains a glucocorticoid response 
 element , which may explain the decreasing nachrs 
 7 expression by chronic immobilization stress . these observations may support the hypothesis that the behavioral effects of ctd and stress 
 partly share a common mechanism mediating weakened cholinergic activities , such as the 
 desensitization of nachrs , the suppressed level of ache and the disturbance of the release 
 of downstream neurotransmitters . in the ctd-250+stress groups , the stress procedure and the 
 ctd - induced feeding suppression presumably fully suppressed the cholinergic activities , 
 which made the anxiogenic effect of ctd invisible . on the other hand , we found that the chronic exposure to ctd and the stress influenced the 
 testes in different ways . the stress procedure significantly decreased the testicular weight 
 in addition to causing the emergence of the multinucleated giant cells , and both of these 
 are common toxicant observations in testes produced by an abnormal meiotic division of germ 
 cells . ctd did not show an impact on the testicular weight , whereas it dose - dependently 
 degenerated the seminiferous epithelia . in particular , the sertoli cell - only seminiferous 
 tubules found in the ctd-250 groups were unexplainable by ctd - induced chronic nutritional 
 deprivation . in the clinical field , sertoli cell - only syndrome is known to result from 
 partial deletions of the azoospermia factor region on the y - chromosome , which is one of the 
 markers of male infertility . from the toxicological perspective , germ cells are vulnerable 
 to damage from exogenous chemicals , and they are easily phagocytized by sertoli cells . several endocrine - disrupting compounds ( edcs ) including diethylstilbestrol and flutamide 
 induce such a destruction of seminiferous tubules [ 1 , 
 11 , 40 ] . an 
 increasing number of vacuolizations in seminiferous epithelia are also caused by the absence 
 of germ cells , suggesting that they are the first target of ctd in the testes through the 
 blood - testis barrier . this corresponds to the ctd dose - dependent increase of single - stranded 
 dna immunopositive germ cells observed in the testes of quails [ 15 , 36 ] . gpx4 ( also called phospholipid - hydroperoxide glutathione peroxidase as a member of the 
 glutathione peroxidase family ) is an antioxidant enzyme expressed in every mammalian organ . 
 there are three types of transcripts from the same gpx4 gene : 
 mitochondrial , non - mitochondrial and nuclear types were identified . the mitochondrial and 
 non - mitochondrial types in particular are expressed in spermatocytes , and gpx4 is the 
 principal antioxidant enzymes in the testes with 30 times greater expression in the testes 
 compared to other organs . notably , in sperm , gpx4 is responsible for the abilities to 
 scavenge approx . testis - specific gpx4 knockout mice showed a phenotype of infertility with degenerated 
 testicular tissue and morphological aberration of sperm . the antigen of the polyclonal antibody used in this study is a common 
 sequence among the three types of mouse gpx4 , and thus , we could not classify the types of 
 gpx4 immunoreactivity . the weakened gpx4 immunoreactivity in the lumen of seminiferous 
 tubules presumably resulted from the decreases in the number and quality of the sperm . neonicotinoids are known to have strong oxidizing properties that affect the liver and the 
 reproductive and central nervous systems [ 7 , 19 ] . the present report is the first to describe the 
 localized decrease of antioxidant enzymes by ctd in the testes of a mammal . similarly , 
 oxidative effects of psychological stress via stress hormones have been reported [ 23 , 42 ] . although 
 a constant level of reactive oxygen species is necessary for the proliferation of 
 spermatogonial stem cells , the present results 
 also showed that oxidative mechanisms partly explain how stress impacts the reproductive 
 function cooperatively with ctd . although there are no reports of plentiful expressions of gpx4 in sertoli cells , strong 
 ectopic expression of antioxidant enzymes was observed in sertoli cells in degenerated 
 seminiferous tubules in the ctd-250 groups . because gpx4 mrna was not expressed in sertoli 
 cell in rat , investigated whether gpx4 mrna in the testes was 
 altered by several edcs , implying the measurements of oxidative conditions provide an 
 indication of the testicular toxicity by environmental factors . in the present study , although no remarkable interaction effect was detected , we 
 investigated whether ctd and stress affected the behavioral and reproductive functions 
 additively rather than synergistically in adult mice . the endogenous nachrs agonist lynx1 
 was recently reported to serve as a molecular break system for brain plasticity . the developmental effects of neonicotinoids in 
 juvenile and fetal periods remain to be determined . neonicotinoids and their metabolites are 
 detected at high frequency in urine samples from japanese people . human exposure to ctd at a high concentration as high as that of the 
 present ctd-250 groups does not occur naturally , but the precipitates that result from the 
 low water solubility of ctd are very dangerous . in addition , a low concentration of ctd 
 could become harmful under a stressed condition , such as fasting . the finding that one of the metabolites of imidacloprid ( imi ) seriously disturbs mapk / erk 
 signaling compared to imi itself   suggests that 
 the safety coefficient ( 100 ) for calculating the acceptable daily intake is not 
 satisfactory . taking the results of the present study as an example , it is clear that 
 further research is needed to identify the toxicant mechanisms of environmental chemicals in 
 order to protect humans and animals with high sensitivities under stressed conditions .
neonicotinoids , some of the most widely used pesticides in the world , act as agonists to 
 the nicotinic acetylcholine receptors ( nachrs ) of insects , resulting in death from 
 abnormal excitability . 
 neonicotinoids unexpectedly became a major topic as a compelling 
 cause of honeybee colony collapse disorder , which is damaging crop production that 
 requires pollination worldwide . 
 mammal nachrs appear to have a certain affinity for 
 neonicotinoids with lower levels than those of insects ; there is thus rising concern about 
 unpredictable adverse effects of neonicotinoids on vertebrates . 
 we hypothesized that the 
 effects of neonicotinoids would be enhanced under a chronic stressed condition , which is 
 known to alter the expression of targets of neonicotinoids , i.e. , 
 neuronal nachrs . 
 we performed immunohistochemical and behavioral analyses in male mice 
 actively administered a neonicotinoid , clothianidin ( ctd ; 0 , 10 , 50 and 250 mg / kg / day ) , 
 for 4 weeks under an unpredictable chronic stress procedure . vacuolated seminiferous 
 epithelia and a decrease in the immunoreactivity of the antioxidant enzyme glutathione 
 peroxidase 4 were observed in the testes of the ctd+stress mice . in an open field test , 
 although the locomotor activities were not affected , the anxiety - like behaviors of the 
 mice were elevated by both ctd and stress . 
 the present study demonstrates that the 
 behavioral and reproductive effects of ctd become more serious in combination with 
 environmental stress , which may reflect our actual situation of multiple exposure .