Experimental exposure to 3-monochloropropane-1 , 2-diol from the pre-puberty causes damage in sperm production and motility in adulthood

3-Monochloropropane-1,2-diol (3-MCPD) is a food contaminant that can be formed during the thermic processing of various foodstuffs. Studies of reproductive toxicology of 3-MCPD are mainly concentrated in the evaluation of possible insults caused by exposure of adult animals. However, the prepuberty might be a period of different susceptibility to chemicals. The aim of this study was to evaluate the effects on reproductive endpoints of the 3-MCPD-exposure prepubertal male rats. Wistar male rats were assigned to 4 groups: control and exposed to 2.5; 5 or 10 mg kg day of 3-MCPD for 30 days by gavage. Testis and epididymis were used for sperm counts and histology analysis. Sertoli cell number and dynamic of the spermatogenesis were evaluated. Sperm were collected from the vas deferens for evaluation of the sperm motility and morphology. Number of sperm with progressive movement, number of Sertoli cells and germ cells and relative daily sperm production were decreased in the groups exposed to 5 and 10 mg kg day of 3-MCPD. Sperm morphology, testicular and epididymal histology were comparable among groups. Results show that 3-MCPD-exposure of rats from prepuberty might cause alterations in spermatogenesis and sperm maturation, similarly to exposure in adulthood.

The exact mechanism of 3-MCPD formation is still unknown (Rahn & Yaylayan, 2011).However, the compound may be formed during thermic processing, as a reaction product of triacylglycerols, phospholipids or glycerol and hydrochloric acid in fat-based or fat-containing foods, and refinement process in vegetable fats and oils (Jędrkiewicz, Kupska, Głowacz, Gromadzka, & Namieśnik, 2016).Moreover, domestic processing (e.g.grilling and toasting) and the migration from coating materials treated with epichlorohydrin also can increase the 3-MCPD levels in foods (Crews, Brereton, & Davies, 2001, Hamlet & Sadd, 2009).
The maximum tolerable daily intake of the 3-MCPD is 2 μg kg -1 bw day -1 .According its food occurrence and consumption, the exposure calculated of the compound for general population might range 0.02 -0.7 μg kg -1 bw day -1 .However, high consumers might ingest of 0.06 to 2.3 μg kg -1 bw day -1 of 3-MCPD (Food and Agriculture Organization [FAO] & World Health Organization [WHO], 2007).
The presence of this contaminant in the diet raises concerns for the risks it may pose to health, due potential carcinogenic, effect possibly mediated by mechanisms involving either hormonal disturbances or cytotoxicity (Robjohns, Marshall, Fellows, & Kowalczyk, 2003).Moreover, Cho et al. (2008) indicate kidneys, testis and ovary as potential targets of the 3-MCPD toxicity.
3-MCPD has been associated to reproductive impact in adult rats (Sun et al., 2013, Sawada et al., 2015), but the effects of the exposure from the prepubertal period are unknown.Spermatogenesis and steroidogenesis are not yet fully established during prepuberty, which in rat occurs at postnatal day 36-55 or 60 (Clegg, 1960, Ojeda, Andrews, Advis, & White, 1980).This phase of the postnatal sexual development has been related as a critical period and more susceptible to reproductive impairment caused by chemical agents (Johnson, Welsh, & Wilker, 1997).That is why the possible reproductive insults caused by exposure in prepuberty should be particularly investigated (Perobelli, 2014).
Children and adolescents may be more exposed to the 3-MCPD than the adults (Li, Nie, Zhou, & Xie, 2015).Thus, the aim of this study was to evaluate the effects on reproductive endpoints of the 3-MCPD-exposure from the prepuberty.For this, testicular and epididymal histology and sperm parameters were analyzed, using the reproductive tract of Wistar rats as the experimental model.

Animals
Male (45 days old, n = 40) Wistar rats supplied by the Central Vivarium of Unoeste -Universidade do Oeste Paulista -were housed in the Vivarium of Experimentation at the Unoeste.During the experiment, animals were allocated into polypropylene cages (43 × 30 × 15 cm) with laboratory-grade pine shavings as bedding.Rats were maintained under controlled temperature (23 ± 1ºC) and lighting conditions (12L, 12D photoperiod).Rat chow and filtered tap water were provided ad libitum.The experimental protocol followed the Ethical Principles in Animal Research of the Brazilian College of Animal Experimentation and was approved by the Ethics Committee for Use of Animals at the Unoeste (Protocol # 2055-CEUA).

Experimental design and treatment
The study was conducted according to the experimental design described below.Rats were randomly assigned into four experimental groups.Control animals (n = 10) received vehicle (saline solution 0.9%), for 30 days by gavage.Rats from the 3-MCPD groups (n = 10 group -1 ) received 2.5 (treated A -TA); 5 (treated B -TB) or 10 mg kg -1 bw day -1 (treated C -TC) of 3-MCPD (Sigma Chemical CO, St. Louis, U.S.A.) diluted in saline solution 0.9%, for 30 days by gavage.Rats were weighed three times for week and had your daily intake estimated ration (in g) and water (in milliliter).In addition, clinical signs of toxicity were observed.

Organs collection
At the end of treatment rats from each experimental group were euthanized with sodium thiopental (100 mg kg -1 ), by intraperitoneal administration.
The right testis, epididymis and vas deferens, ventral prostate, seminal vesicle (without the coagulating gland and full of secretion), liver, kidneys and pituitary were removed and their weights (absolute and relative to body weights) were determined.

Sperm analysis
Immediately after euthanasia, the left vas deferens was collected and spermatozoa were obtained with the aid of a syringe and needle, through internal rinsing with 1.0 mL of PBS solution at 34ºC.Warmed Newbauer chamber was loaded with a small aliquot of sperm solution. of a syringe and needle, sperm were removed from the right vas deferens through internal rinsing with 1.0 mL of buffered formalin.Sperm morphology analysis was performed according to Seed et al. (1996) and abnormalities were classified according to Filler (1993).Right testes were decapsulated and caput/corpus and cauda segments of right epididymis were separated.
Homogenization-resistant testicular spermatids (stage 19 of spermiogenesis) and sperm in the caput/corpus epididymis and cauda epididymis were assessed as described previously by Robb, Amann, and Killian (1978), with adaptations of Fernandes et al. (2007).To calculate daily sperm production (DSP) the number of spermatids was divided by 6.1 (number of days that these cells are present in the seminiferous epithelium).The sperm transit time through the epididymis was determined by dividing the number of sperm in each portion by DSP.

Histological procedures and quantitative analysis of spermatogenesis
The left testis and epididymis were collected and fixed in buffered formalin (10%) for 24 hours.After this period, these organs was sectioned and returned to the buffered formalin for additional 24 hours.The pieces were embedded in paraffin wax and sectioned at 5 μm.The sections used for histological evaluation were stained with hematoxylin and eosin (HE), Periodic acid-Schiff (PAS) and Masson's trichrome and examined by light microscopy.
One hundred random tubular sections per animal (n = 10 animals group -1 ) in three nonconsecutive testis cross-sections were classified into four categories, according to the type of germ cell present.The groups of stages I-VI (one spermatids generation), VII-VIII (spermatozoa), IX-XIII (two generations of spermatids) and XIV (secondary spermatocyte) of the seminiferous epithelium cycle (Leblond & Clemont, 1952) were identified under a light microscope (Leica DMLS) at x 200 magnification.
Nucleus of Sertoli cells and germ cells (spermatogonia type A, pachytene primary spermatocyte and round spermatid) were counted in 20 seminiferous tubules per rat (n = 10 animals group -1 ) at stage VII of spermatogenesis, under a light microscope (Leica DMLS), at x 400 magnification.The number was corrected for nucleolus/nucleus size and section thickness, according to Abercrombie (1946).

Statistical analysis
Statistical analyses were conducted by the ANOVA, with a posteriori Tukey test or nonparametric Kruskall-Wallis test, with a posteriori Dunn test, according to the characteristics of each variable.The results were expressed as mean ± standard deviation (SD) or median and quartile 1 and 3 (Q1/Q3).Differences were considered significant when p < 0.05.

Results and discussion
There was no statistical difference (p > 0.05) in the body weight among experimental groups (Table 1) during the treatment period.The feed intake and water consumption were affected by the 3-MCPD-exposure only at the end of the treatment period (data not shown).On the 27 and 30 th of exposure, there was an increase (p < 0.05) of the feed intake in the TA group compared to the control group.It was also observed difference (p < 0.05) between the TA and TB groups on the 27 th day of treatment.On the 30 th day of treatment, there was a lower (p < 0.05) water consumption in the TC group compared to the TA group, but not in relation to other experimental groups.
The absolute and relative weights of reproductive organs, pituitary, liver and kidneys are shown in Table 1.Significant augmentation (p < 0.01) in the absolute weight of the kidneys in rats from TC group in comparison to the control group was observed.However, relative weight of this organ was similar among experimental groups.The weight of the vas deferens was increased (p < 0.05) in TA group, when compare to control group.
Sperm number per gram of testis and daily sperm production per gram of testis were significantly reduced (p < 0.05) in TC and TB groups, when compared to control and TA groups, respectively (Table 2).Nevertheless, sperm number in the caput/corpus epididymis, epididymal transit time and sperm morphology were similar among experimental groups (Table 2).The number of sperm with progressive movement was significantly decreased (p < 0.05) so that, consequently, the percentage of immotile sperm was increased (p < 0.05) in the TB and TC groups in relation to control group (Figure 1).
Table 2. Sperm parameters of rats from control and treated with 2.5 (TA), 5 (TB) and 10 mg kg -1 day -1 (TC) of 3-MCPD groups.The stages of spermatogenesis were not significantly affected by different doses of 3-MCPD exposure (Table 3).On the other hand, the number of Sertoli cells and germ cells was lower in the TB and TC groups, when compared to control and TA groups (Table 3).
3-MCPD is a food contaminant found in various foods that are part of daily diet.Experimental studies with adult rodents have shown toxic effects of this compound, especially on the urinary and reproductive systems (Cho et al., 2008, Bakhiya, Abraham, Gürtler, Appel, & Lampen, 2011, Kim et al., 2014).However, the reproductive studies caused by exposure from prepubertal phase, which could have a relevant exposure, are scarce.
Although the levels of exposure to food contaminated by 3-MCPD is not high for the general population, the growing consumption of some foods, such as frozen meals, which have higher levels of the contaminant, raise a concern (Arisseto, Vicente, Furlani, & Toledo, 2013).Furthermore, the presence of this compound in infant formulas and breast milk (Zelinková et al., 2008, Wöhrlin, Fry, Lahrssen-Wiederholt, & Preiß-Weigert, 2015) also indicates the need for further toxicological research in this area.
The assessment of body weight and nutritional status is extremely important to obtain information on the overall health of the animals exposed to chemicals (Clegg, Perreault, & Klinefelter, 2001).The reproduction is often one of the first functions to be affected when there is inadequate nutrition and loss body weight (Krasnow & Steiner, 2006).Thus, the analysis of these parameters is essential to interpretation of the effects of chemicals on the reproductive system.In the current study, final body weight of rats from different experimental groups was similar; corroborating study of Onami et al. (2014).   of rats from con an (Q1 -Q3).Kr phs of testis secti kg -1 day -1 -B) gr ) and 3-MCPD ( sence of patholo ke also were ed by Yamad o (1995).How rease in the bod after nine (fe osure to 400 p 9 mg kg -1 ).ght of the anim ductive impact    Sperm parameters were impaired by 3-MCPD exposure.Sperm number per gram of testis and daily sperm production per gram of testis were significantly reduced in the rats from groups exposed to higher doses of food contaminant (5 and 10 mg kg -1 bw day -1 ).These data corroborate with the reduction of the germ cells number observed.However, Kim et al. (2012) related that the exposure of adult rats (3, 10, and 30 mg kg -1 bw day -1 3-MCPD for 7 days) no caused changes in the number of sperm head in the testis.
The impact on relative sperm production was not changed the cytoarchitecture of seminiferous tubules and the frequencies of different stages of the spermatogenic process.This absence of histological injury also was observed in study of Kwack et al. (2004).These researchers found impairment on fertility and pregnancy outcome, without histopathological changes in testis and epididymis, after paternal exposure to 5 mg kg -1 bw of 3-MCPD for four weeks.Despite this, Cho et al. (2008) observed degeneration of the seminiferous epithelium in adult mice after subchronic exposure to higher doses of 36.97 and 76.79 mg kg -1 of 3-MCPD.
In the present study, in spite the maintenance of the general morphological characteristics of the testes and absence of evident histological alterations, 3-MCPD exposure affected the number of germ cells per seminiferous tubules.In adult rats, Madhu, Sarkar, Biswas, Behera, and Patra (2011) observed reduction in seminiferous tubular areas, secondary spermatocytes and nuclear diameter of Leydig and Sertoli cells after 45 days of exposure to 1 mg kg -1 bw day -1 of 3-MCPD.Moreover, spermatogonia, primary spermatocytes and Sertoli cells increased at 15 and 45 days of exposure.On the other hand, in the current study the number of Sertoli cells was decreased in seminiferous tubules of rats exposed to 5 and 10 mg kg -1 bw day -1 of the food contaminant when compared to control and TA groups.This result suggests that there was cell death and not a reduction of its proliferation, since the exposure began after the end of Sertoli cell proliferation period (Orth, Gunsalos, & Lampert, 1988).The decrease in Sertoli cell number may have triggered the reduction observed in counts of the spermatogonias type A, pachytene primary spermatocytes and round spermatids.This fact might be related to control on spermatogenesis magnitude exercised by Sertoli cell (Hess & França, 2005).
In the current study, epididymis histology was no impaired by 3-MCPD, corroborating study of Kwack et al. (2004).On the other hand, Kim et al. (2012) found spermatic granuloma, cell debris, epithelial cell vacuolization and oligospermia in proximal caput epididymis of adult rats after treatment with 10 mg kg-1 bw day-1 of 3-MCPD for seven days.
Evaluation of sperm morphology by light microscopy indicated no difference among the experimental groups, as related by Kim et al. (2012).Nevertheless, electron microscopy analysis realized by Madhu et al. (2011), revealed sperm abnormalities, including deglutination of the acrosomal part, loss of head capsules, and fragmentation of tail fibrils.
3-MCPD did not affect the transit time through the epididymis; however, sperm motility was reduced, suggesting changes in the sperm maturation process.Several authors also observed impairment in sperm motility of adult rats after 3-MCPD exposure at different doses (Kwack et al., 2004, Kim et al., 2012).Decrease in H+-ATPase expression in the cauda epididymis (Kwack et al., 2004) and enzymes inhibition of sperm glycoloysis, especially enzymes glyceraldehyde-3phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (Jones & Porter, 1995, Lynch et al., 1998)

Conclusion
These results show that prepubertal rats exposed to 3-MCPD might present alterations, number of Sertoli and germ cells, relative sperm production and motility, with impact on sperm quality, similar to impairment caused by exposure of adult animals reported in the literature.Since chloropropanols are present in the daily diet of population is essential determining their potential risks to human reproductive health.Thus, additional studies are needed to investigate more deeply the mechanisms involved in these reproductive effects observed and the relationship between the changes in sperm parameters and possible impact on fertility capacity.

Table 1 .
Final body weight and reproductive organ weights of rats from control and treated with 2.5 (TA), 5 (TB) and 10 mg kg -1 day -1 (TC) of 3-MCPD groups.

Table 3 .
Stages of spermatogenesis and Sertoli cells and germ cells number in rats from control and treated with 2.5 (TA), 5 (TB) and 10 mg kg -1 day -1 (TC) of 3-MCPD groups.Values expressed as median (Q1 -Q3).Kruskall-Wallis test, with a posteriori Dunn test.b Values expressed as mean ± S.D. ANOVA with a posteriori Tukey test.Different letters indicate statistically significant difference (p<0.05). a have been associated to motility change.