the endocrine system may be the main target for the toxic manifestation of pesticides and may result in reproductive alterations , especially in steroid - hormone - dependent functions [ 1 , 2 ] . endocrine disruptors were defined by kavlock et al .   as exogenous agents that interfere with the production , release , transport , metabolism , binding , action , or elimination of natural hormones responsible for the maintenance of homeostasis and the regulation of developmental processes . these disturbances may potentially cause risk to population by impairing their capacity to reproduce , being that occupational exposure to pesticides has been linked to a reduction in the quality of semen and a greater rate of infertility and miscarriage [ 5 , 6 ] . s - metolachlor [ s-2-chloro - n-(2-ethyl-6-methylphenyl)-n-(2-methoxy-1-methylethyl ) acetamide ] is a chloroacetanilide herbicide widely used in the agricultural sector to control weeds in corn , cotton , and soybean plantations , among other crops . the commercial formula of s - metolachlor comprises primarily of 88% s - enantiomer and 12% r - enantiomer , although only the s - enantiomer is biologically active . compared to metolachlor ( 50% s - enantiomer and 50% r - enantiomer ) , s - metolachlor represents an important reduction in risk to users , consumers , and the environment [ 7 , 8 ] since formulations of s - metolachlor in proportions above 80% have higher herbicide activity and a smaller amount of product may be used . after agricultural application , metolachlor has been observed to persist longer in the subsoil than on its surface . furthermore , great quantities of bonded residues and degradation products have been observed on the soil surface as a result of the increase in absorption of the soil and the biodegradation of metolachlor , because there is more organic material in this layer . however , this mechanism becomes saturated and metolachlor forms bond residues in the soil and is exposed to solar action or it may contaminate ground water [ 10 , 11 ] , or it may even become volatile in the environment . human exposure to chemical contaminants is practically inevitable , and the consequences to human health depend on the levels of environmental exposure . it has been recognized that environmental contaminants are capable of bonding with gonadal steroid receptors , which may imitate the action of steroid hormones and alter their production output . the detection of contaminant residues in human serum , follicular liquid , and seminal plasma [ 13 , 14 ] , together with reports of a supposed drop in semen quality [ 1517 ] , has highlighted the concern that exposure to environmental pollution can affect human fertility . adverse reproductive effects attributed to pesticides , including their effect on fertility , have also been well established in in vivo and in vitro studies [ 1822 ] . one important observation was that metolachlor increases the activity of the aromatase enzyme in jeg-3 cell cultures . this enzyme is responsible for the conversion of testosterone into estradiol , and the increase in it activity might result in alteration of the amount of testosterone , estradiol , and di - hydrotestosterone ( dht ) , since it is also produced from conversion of testosterone . it makes necessary an investigation into whether metolachlor may alter the production of sexual hormones in vivo and cause disturbances in the reproductive male development . based on these evidences and on the interest to determine the toxic effects of metolachlor on the male reproductive endocrinology , we used prepubertal rats as an experimental model , in an experimental design previously described . after weaning , the exposure to metolachlor was initiated and the growth of the animals and the age and weight at puberty was evaluated . at the end of the period of exposure , blood samples of male rats of 53 days old were collected and subjected to analysis of lh , fsh , testosterone , dihydrotestosterone ( dht ) , and estradiol serum concentrations . the testes were submitted to morphometric evaluation of the seminiferous epithelium , through the analysis of histological sections . the weight of the testes and the seminal vesicle ( undrained and drained ) were also recorded to evaluate the effect of the treatment on the reproductive development . the product used was a commercial formulation of dual gold ( syngenta ag ; syngenta protecao de cultivos ltda . , so paulo , brazil ) , with an s - metolachlor base ; this formulation contained 960  g / l ( 96% ) of s - metolachlor formulated as an emulsifiable concentrate . thirty newly weaned male wistar rats were used , born of females that were monitored from the 17th day of pregnancy , in order to determine their offspring 's exact days of birth . on the fourth postnatal day ( pnd4 ) , the litters were culled to eight pups per female and maintained thus until weaning ( pnd21 ) . the rats were kept in a 12:12 hour dark / light photoperiod cycle , at a controlled room temperature ( 23  1c ) , and they were fed with a commercial balanced ration mixture for rats and given water ad libitum . the metolachlor was diluted in a watery suspension and administered once a day , per os ( gavage ) at a volume of 0.25  ml/100  g of body weight , between 7 and 8 am . the toxicological analysis of metolachlor determined the following parameters that we consider relevant for the choice of doses used in this study : dl50 oral toxicity of metolachlor for rats ( 2780  mg / kg ) , maternal and developmental toxicity noels ( 300  mg / kg / day ) , and reproductive noel ( 23.526.0  mg / kg / day ) united states environmental protection agency . therefore , we used the dose of 5  mg / kg / day ( mt5 ) and 50  mg / kg / day ( mt50 ) . the control group was treated in the same manner , but with deionized water instead of metolachlor . the pps is a marker of puberty in male rat and is used in reproductive toxicological protocols . it is a cornifying process that leads to cleavage of the epithelium forming the stratified squamous lining of the prepuce of the penis and is a sign of puberty . this process is an essential prerequisite for acquisition of complete ejaculation   and is dependent of androgen . this method was undertaken from pnd33 and was carried out once a day at the time of balanopreputial separation , by means of gentle tissue manipulation . during this period , the animals were also weighed . in order to evaluate the effect of metolachlor on the reproductive organs development , the testes and seminal vesicle were weighted and the absolute values were transformed into relative weights as mg/100  g of body weight . the seminal vesicle was weighted with fluid ( undrained ) and after fluid removal ( drained ) . the testes were fixed in bouin 's solution for 8 hours , treated with alcohol , embedded in paraffin , and then cuts of 5  m were prepared as stained slides with hematoxylin and eosin as previous described . briefly , the slides were observed initially under 40 magnification , in order to ascertain the general organ architecture . next , 100 magnification was used for a more detailed analysis of the architecture of the seminiferous tubules . this included analyzing the linear morphometry of the seminiferous tubules by determining the tubular diameter ( measured from the basal lamina to the basal lamina in the opposite direction ) , the thickness of the seminiferous epithelium ( from the basal lamina to the neck of the elongated spermatids ) , and luminal diameter . ten fields per cut per animal were selected within the histological cuts , in the transverse direction of the tubules . for each tubule , the averages were calculated for the measurements indicated and , then , the average of each field was also calculated . the measurements for each animal were obtained through the measurements of all the analyzed fields . blood was collected via cardiac puncture in 53-day - old animals between 07:30 and 08:30 am . and was centrifuged and subjected to serum levels of total testosterone and estradiol by radioimmunoassay , using commercial kits ( testosterone total mpbiomedicals ; estradiol coat - a - count , siemens health care diagnostics , los angeles , ca , usa ) , luminex xmap technology for rat lh and fsh from millipore corp . ( milliplex map rat pituitary panel , billerica , ma , usa ) , and elisa kit for rat dihydrotestosterone dht ( uscn life science inc , nc , usa ) . the minimum sensitivity was 0.9  ng / dl for testosterone , 1.57  pg / ml for estradiol , 2.91  pg / ml for lh , 31  pg / ml for fsh , and 13.25  pg / ml for dht . the intrassay coefficient was < 5% for testosterone , < 3% for estradiol , < 2.5% for lh , < 4.7% for fsh , and < 3% for dht . the data were first submitted to kolmogorov - smirnov tests for normality and the bartlett test for homoscedasticity . the analysis of body growth was performed using the multiway analysis of covariance for repeated measures using the weight at weaning as a cofactor ( mancova ) by a general linear model ( glm ) . the weights were compared among different groups and different ages , considering the expected changes with age . the day of pps was compared using nonparametric analyses with the kruskal - wallis test followed by the post hoc dun test . the weights of seminal vesicle ( drained and undrained ) were compared by two - way anova followed by the tukey hsd post hoc test . all other parameters were analyzed by one - way anova followed by the tukey hsd post hoc test . all analyses were performed with statistica 7.0 ( statsoft inc , tulsa , ok , usa ) . the values were expressed as means and the standard error of the mean (  sem ) for parametric and interquartile ranges of nonparametric analysis . the effect of daily exposure to the herbicide on the reproductive endocrinology was assessed by determining the serum concentrations of testosterone , estradiol , dht , lh , and fsh in blood samples from 53-day - old rats . the serum concentration of testosterone was significantly different between the control and treated groups , increasing around 88% in mt50 , as compared to the control and also with regards to mt5 ( one - way anova , p < 0.01 , figure 1(a ) ) . the serum concentrations of estradiol were higher in the mt50 group , at around 300% , compared to control ( one - way anova , p < 0.01 , figure 1(b ) ) . the serum concentrations of dht were slightly reduced in mt5 ( one - way anova , p < 0.05 , figure 1(c ) ) , but is not altered in mt50 . the lh serum concentrations were not altered by the treatment ( one - way anova , p < 0.05 , figure 2(a ) ) , but fsh was elevated in both treated groups , mt5 and mt50 ( one - way anova , p < 0.05 , figure 2(b ) ) . mt5 group presented larger epithelial height and reduced luminal diameter , with no alterations in the tubular diameter . mt50 group also presented increase in the epithelial height and was verified increase in the tubular diameter ( one - way anova , table 1 , figure 3 ) . no significant differences were observed for the weight of seminal vesicles with fluid content ( undrained ) . however , after the drainage of the seminal fluid ( drained ) , the mt5 and mt50 groups presented a greater volume of seminal fluid than the control group ( two - way anova , p < 0.05 , figure 4 ) . the testes were weighted to ascertain possible alterations caused by daily exposure to metolachlor , and this treatment did not significantly alter the relative testicular weight of the animals among the treated groups ( one - way anova , p > 0.05 ) . the separation of preputial mucosa and exposure of the penile glands are parameters for determining the progression of puberty in male rats , and daily exposure to the herbicide metolachlor significantly anticipated the onset of puberty . in relation to age , the mt5 and mt50 groups presented precocious puberty as compared to the control group ( kruskall - wallis , p < 0.05 , figure 5(a ) ) . in relation to body weight at puberty , the mt5 and mt50 groups were lighter than the control group in this phase of sexual development ( one - way anova , p < 0.05 , figure 5(b ) ) . note that the weights differed among groups only because it corresponded to an age at puberty in about 4 days earlier than in control animals . the assessment of daily weight from weaning until puberty was performed to ascertain possible compromised development caused by the herbicide . the body weight at 23 days old was used as cofactor for correcting the initial body weight of animals , and there was no significant difference among the groups undergoing different treatment regimes with metolachlor . only the expected variation pertaining to age was observed , showing that body evolution was not affected by the concentrations used ( mancova , p > 0.05 ) . metolachlor is a widely used herbicide that might be related to effect of endocrine chemical disruptor since it was known to alter the aromatase activity in culture cells . so , the focus of this study was to verify the toxicological effects on the reproductive parameters of a commercial formulation of metolachlor in vivo , using prepubertal male rats as an experimental model . we demonstrated that prepubertal exposure to metolachlor caused changes in the reproductive endocrinology of male rats , with different effects in the two doses used . the mt5 group presented a reduction in the dht serum concentrations and an increase in the fsh serum concentrations , while the mt50 group showed an increase in the testosterone , estradiol , and fsh serum concentrations . there were also observed a higher amount of fluid in the seminal vesicles , precocious puberty , and changes in morphology of the seminiferous epithelium of treated animals in both groups . the gonadotropins are produced and released by the stimulation of hypothalamic gnrh , and testosterone exerts negative feedback on lh production . despite the increased testosterone serum concentrations , the fsh , which stimulates the production of estradiol by sertoli cells , was elevated in the treatments , as well as serum estradiol concentrations . this increase in estradiol may also be due to the stimulation of aromatase by metolachlor , as observed in culture cells , which leads to greater conversion of estradiol from testosterone [ 29 , 30 ] . the dht , converted from testosterone , presented a small reduction in its serum concentrations . the androgenic activity is mainly exerted by these two hormones , being that the dht has greater bioactivity than testosterone . spermatogenesis takes place in the germinative epithelium , a process whose main objective is the production of viable gametes that will be transported by a mature and efficient reproductive system . because sex hormones may influence the spermatogenesis , we evaluated the morphometry of the seminiferous epithelium in histological sections , and metolachlor exposure increased in the height of the germinal epithelium of seminiferous tubules . the epithelium height is an indicator for the process of spermiogenesis   and for the hormonal modifications associated with problems in its architecture [ 3234 ] . thus , the greatest height of the germinative epithelium may be linked to intense activity on the part of the sertoli cell , stimulated by the greater levels of circulating estradiol and testosterone . besides the increased epithelium , the tubular lumen was reduced . the vesicular gland is androgen - dependent tissue and was used as parameters for evaluating the effects of testosterone level variations . the seminal vesicles were lighter and had a greater amount of liquid in both treatments with metolachlor . as the activity of the vesicular glands is dependent on the action of androgens , it is supposed that the gland is subject to the action of greater amounts of circulating androgens . the age at puberty is an important component of reproductive development and is influenced by sex hormones , which can determine its anticipation or delay . in rats , the age at puberty onset can be evaluated by the separation of the preputial skin from the glans of the penis [ balanopreputial separation ( pps ) ] [ 27 , 35 ] and is triggered by the rise of serum testosterone concentrations in the prepubertal period . metolachlor exposure caused an anticipation of the puberty , possibly accomplished for the highest testosterone levels ( because testosterone was increased in the end of the experimental period ) , which may have contributed to the change in the age at puberty onset . because the rats reached puberty at an earlier age , the animals had lower body weights at puberty . however , the animal body weights throughout the experimental period revealed no differences between groups , suggesting that the effects of prepubertal administration of metolachlor are restricted to reproductive development and function . this study shows , for the first time , the effects on the reproductive development of male treated with metolachlor in the prepubertal period . we conclude that the exposure promotes endocrine problems in reproductive parameters and these changes are reflected by altering the serum concentrations of testosterone , dht , estradiol , and fsh as well as by causing morphological alterations in androgen - targeted tissues , since the lowest dose used , which may indicate the potential risk of occupational exposure to this herbicide .
s - metolachlor is a chloroacetanilide herbicide widely used in the agriculture to control weeds and was demonstrated that it increases the activity of the aromatase enzyme in cell cultures , which may culminate as endocrine disruption action in vivo . to investigate this hypothesis , 
 prepubertal wistar male rats were exposed to metolachlor ( 5 or 50  mg / kg / day , noel for reproductive toxicity : 23.526.0 
 mg / kg / day ) from pnd23 ( postnatal day ) to pnd53 . during this period , 
 the growth of the animals and the age and weight at puberty were recorded . in pnd53 , tissues were collected and the analysis of lh , fsh , testosterone , dihydrotestosterone ( dht ) , estradiol serum concentrations , morphometric evaluation of the seminiferous epithelium , and weight of the testes and the seminal vesicle ( undrained and drained ) was performed ( statistical difference : p < 0.05 ) . 
 metolachlor caused an increase in serum concentrations of testosterone , estradiol , and fsh and a reduction in dht but did not alter the lh . 
 there were also observed a higher amount of fluid in the seminal vesicles , precocious puberty , and changes in morphology of the seminiferous epithelium of treated animals . 
 we demonstrated in this paper that prepubertal exposure to s - metolachlor caused changes in reproductive endocrinology of male rats .