Evaluating the reproductive toxicology of tannery effluent in male

Science of the Total Environment 648 (2019) 1440–1452

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Evaluating the reproductive toxicology of tannery effluent in male SWISS mice Abraão Tiago Batista Guimarães a, Raíssa de Oliveira Ferreira a, Joyce Moreira de Souza a, Dieferson da Costa Estrela b, André Talvani c, Débora Maria Soares Souza c, Thiago Lopes Rocha d, Guilherme Malafaia a,⁎ a

Post-Graduation Program in Conservation of Cerrado Natural Resources, Goiano Federal Institute of Education, Science and Technology, – Urutaí Campus, GO, Brazil Graduate Program in Zoology, Federal University of Paraná, Brazil c Laboratory of Environmental Biotechnology and Ecotoxicology, Institute of Tropical Pathology and Public Health, Federal University of Goiás, Goiania, GO, Brazil d Inflammation Immunobiology Lab, Federal University of Ouro Preto, Ouro Preto, Brazil b

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Tannery effluent (TE) cause changes in the reproductive performance of male mice. • The direct contact with TE leads to behavioral and histopathological impacts. • TE can cause significant changes in the dynamics of small-mammal populations.

a r t i c l e

i n f o

Article history: Received 4 March 2018 Received in revised form 19 August 2018 Accepted 19 August 2018 Available online 21 August 2018 Editor: D. Barcelo Keywords: Reproduction Rodents Agro-industrial waste Contaminant Environmental impacts

a b s t r a c t The transformation of skin in-natura into leather in tannery industries generates large volumes of organic matter that attract small mammals. i.e., rodents living close to these facilities. Animals foraging in the backyards of such industries get exposed to the effluent produced by them; however, attention has not been given to the impacts of such exposure on the reproductive biology of these animals. Thus, our study assessed whether the direct exposure to this effluent for periods longer than 90 days leads to reproductive loss in male Swiss mice. We assessed animals' sexual behavior at the end of the experimental period and analyzed their testicular histology, as well as semen quality and volume, besides measuring pro-inflammatory markers and assessing the reproductive performance of the exposed animals. Based on the herein collected data, mice exposed to the gross effluent collected in the backyard of a tannery industry, as well as to the effluent diluted in 5% of water, presented behavioral and histological changes in the testes, disorganized germinal cells in the seminiferous tubules and inflammatory process in intertubular spaces. The inflammatory process resulted from increased proinflammatory cytokine (IFNgamma and CCL2) concentrations in the testes, fact that explained the larger number of sperm abnormalities and the reduced number of produced sperms. These factors, along with the previously reported changes, may have led to the low reproductive performance of animals exposed to the tested pollutant, which was assessed

⁎ Corresponding author at: Laboratório de Pesquisas Biológicas, Instituto Federal Goiano – Campus Urutaí, GO, Brasil. Rodovia Geraldo Silva Nascimento, 2.5 km, Zona Rural, Urutaí, GO CEP: 75790-000, Brazil. E-mail address: [email protected] (G. Malafaia).

https://doi.org/10.1016/j.scitotenv.2018.08.253 0048-9697/© 2018 Elsevier B.V. All rights reserved.

A.T.B. Guimarães et al. / Science of the Total Environment 648 (2019) 1440–1452

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through the lethal dominant test. This pioneering article addressed the reproductive impact caused by the direct exposure of small rodents to tannery effluents. The research helped better understanding how these pollutants can influence natural ecosystems. © 2018 Elsevier B.V. All rights reserved.

1. Introduction The socioeconomic aspects linked to shoe, purse, clothing and upholstery production require processing large volumes of bovine skin in tannery facilities, which are mainly located in Asian and South American countries (Aber et al., 2010). The transformation of skin innatura into leather in these companies generates large amounts of residue presenting a large variety of potentially toxic organic and inorganic compounds (Calheiros et al., 2007). Sulfides, sulfates, chlorides, as well as substantial amounts of sodium and calcium, are used in the skin cleaning stage. They generate high alkaline-content liquid residues, as well as solid residues such as hair, meat and fat (Joseph and Nithya, 2009). The drying stage generates residues rich in sodium chloride, mineral and organic acids, aluminum salts and in many toxic metals such as chrome, lead, arsenic, besides solvents used at the final leather-processing stage (Hu et al., 2011; Sabumon, 2016). Therefore, the large residue production turns tannery companies into potential environmental polluters (Agrawal et al., 2006). Previous studies have already shown the damaging effects of this residue on plants, bacteria and invertebrate species such as Allium cepa, Selenastrum capricornutum, Daphnia magna, Ceriodaphnia dubia, Hyalella azteca, Paracentrotus lividius and Sphaerechinus granularis (Oral et al., 2005; Mitteregger et al., 2007). However, studies about TE (Tannery Effluent) impacts on vertebrates are mainly focused on fish (Souza et al., 2016a). Different studies conducted with groups of mammals, mainly with inbred and outbred mice, have shown the damaging effects of the chronic exposure to TE on these models (Siqueira et al., 2011; Moysés et al., 2014; Ferreira et al., 2015; Almeida et al., 2016; Rabelo et al., 2016; Souza et al., 2016b, 2016c, 2017; Guimarães et al., 2016a, 2016b, 2017; Moysés et al., 2017; Mendes et al., 2017). Most of these studies investigated the effect of ingesting TE diluted in water and pointed out that the constituents of this residue affect animals' central nervous system (CNS). A particular aspect of these studies lies on the fact that their experimental designs correlate animals' exposure level to possible situations involving humans who live close to waterbodies where this effluent is discharged and run the risk of drinking contaminated water. These studies provided important subsidies that can be used for mitigation purposes in places subjected to tannery effluent discharge. The ecotoxicological effects of this effluent have been poorly investigated. Many industries, mainly the small ones, have their backyards full of organic matter, which attracts different insect species that are part of the diet of small-rodents (Hogue, 1993) living in sites adjacent to tannery facilities. Such organic matter (hair, fat and meat from leather processing) becomes an attractive food that can be easily accessed by these rodents who, by foraging on it, end up exposed to the toxic effluent (Estrela et al., 2017; Mendes et al., 2017). Tannery residues make contact with the skin, as well as with the respiratory (inhalation of volatile gases) and digestive tracts (intake due to leaching and self-cleaning) of these animals (Estrela et al., 2017). Studies conducted by Estrela et al. (2017) and Mendes et al. (2017), who investigated the possible effects of direct contact with TE on mammals, stand out in the literature. According to Estrela et al. (2017), the dermal exposure to gross TE, even for a brief period-of-time, caused social memory deficit in female Swiss mice. Mendes et al. (2017) indicated changes in the olfactory ability and in the anti-predatory response of male and female C57Bl/6J mice dermally, and orally, exposed to TE. They were the first to report losses that animals exposed to these

residues can face when they forage in the backyards of tannery facilities. These two studies opened new investigation perspectives. Assessing the reproductive aspects of small mammals who forage in these production facilities is one of the ways to evaluate TE impact on the population structure and on the organization of these species. The ecological homeostasis and the dynamics of these populations can be damaged when animals have their reproductive aspects impaired. Reproduction disorders may have negative influence on the balance of ecosystems in certain regions, since small rodents compose the diet of other animals, besides working as seed dispersers, pollinators and organic matter recyclers (Mihalca and Sándor, 2013; Sunyer et al., 2013). Thus, the aim of our study was to investigate possible reproductive impacts of the exposure of male Swiss mice to TE. Our hypothesis is that the direct contact of these animals with TE leads to deficit in their sexual behavior, as well as to histopathological testicular and spermatic changes, besides affecting their reproductive performance. 2. Materials and methods 2.1. Animals and experimental design Our sample comprised 75 nulliparous male Swiss mice in the age group 32 days from the Veterinary Laboratory of Agrodefesa (LABVET) (UFG, Goiania, GO, Brazil). The animals were kept in the biological research vivarium at Goiano Federal Institute of Biological Research – Urutai Campus (Urutaí, GO, Brazil). All mice were housed on ventilated shelves under controlled temperature (22 ± 2 °C), 12/12-h light/dark photoperiod and controlled humidity conditions (58 ± 3%). The models were grouped in polypropylene boxes (dimensions: 45.4 cm × 30.1 cm × 16.7 cm) covered with galvanized grid treated with an antioxidant product. Water and feed (NUVILAB-CR1) were provided ad libitum throughout the experiment. The animals were counter-balanced through biomass, so they would be statistically similar. Next, the following experimental groups were set: i) Control group (C): comprising mice kept in the residence box throughout the experiment. They were not placed in the exposure boxes or exposed to TE; ii) Dry control group (DC): comprising mice who were placed in dry-exposure boxes, i.e., they did not have access to water or TE; iii) Water control group (WC): comprising mice who were placed in boxes without TE, but who had access to the same volume of water that groups exposed to TE did; iv) 5% tannery effluent group (E5): comprising mice who were placed in the boxes filled with TE (5%) diluted in drinking water; v) 100% tannery effluent group (E100): comprising mice who were placed in boxes filled with gross TE. The exposure boxes were made of transparent polyethylene [40 cm length × 30 cm width × 20 cm height] and covered with perforated cap to air to get in and out of the boxes. Each box received 500 mL of gross or diluted effluent (groups E5 and E100, respectively) or of drinking water (group WC) – the necessary volume to keep the box flooded in order to enable the direct contact with the animals' bodies. The aim of our experimental design was to simulate animals' direct contact with TE, just as it happens with small rodents when they forage in the backyards of small tannery facilities. Group E100 simulated

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situations in which mice get direct contact with the gross TE spread in the organic matter left on the backyards of these facilities. Group E5 represented situations in which the effluent is more diluted due to the water used to wash the skin and/or the machinery in the facilities. The exposure conditions adopted in both groups did not prevent oral exposure through effluent intake. The animals were subjected to oral and dermal exposure to the effluent, just as it can happen with wild rodents. The self-cleaning behavior typical of mice leads to the intake of effluent contaminants during dermal exposure. Animal exposure was performed five days a week (from Monday to Friday, 1 h/day) for 90 days. Mice were placed in boxes with clean wood shavings. The boxes were left under heated light after each exposure period to allow the animals to dry. Next, the models were placed in their residence boxes and taken to the vivarium. The option for exposing the animals for five days, rather than throughout the week, for 1 h, was based on the fact that wild rodents are omnivorous and use their natural environment as diversified source of food such as fruits, eggs, seeds, insects, among others (Kerley, 1989; Ramos, 2007; Perini, 2010). Scraps from tannery plants are not the only food source for these animals. We assume that rodents' foraging for organic scraps in tannery facilities can be linked to the scarcity of food resources near these places or to the broad provision of food.

2.4. Locomotor activity evaluation The overall locomotor activity of the animals was assessed through the Basso Mouse Scale (BMS) (Basso et al., 2006), which evaluates biomechanical attributes such as movement and alignment of paws, tail support, and trunk stability and coordination. We assessed these attributes in the first 5 min of the sexual behavior test, while animals remained in the acclimation period. 2.5. Spermatic quality evaluation We also hypothesized that the direct contact with TE could change spermatozoa motility, volume and morphology in the investigated

2.2. Tannery effluent The tannery effluent used in our study was collected in the drying stage of bovine-skin processing (wet blue). It was provided by a tannery company located in Goiás State (Brazil). Such effluent was collected from the skins (under-process) spread in the backyard of this company, based on Estrela et al. (2017) and Mendes et al. (2017). The physicalchemical, and chemical, features of TE (gross and diluted) were determined according to the methodology by the American Public Health Association (APHA, 1997). On the other hand, the organic analysis was conducted through mass spectrometry with ionization by electrospray, as described by Guimarães et al. (2016a, 2016b). Tables S1 and S2 (respectively) and Fig. S1 (check on “Supplementary Material”) show the results of these analyses. 2.3. Sexual behavior evaluation Mice were subjected to the sexual behavior test due to the hypothesis that the direct contact with TE can change the homeostasis of androgen organs and influence these animals' sexual variables after 90 exposure days. The test was conducted during the dark stage of the cycle – time when mice were more active (Olney and Sharpe, 1969; Moore, 1997; Rodrigues-Alves et al., 2008; Park et al., 2009). Male mice were taken from their residence boxes and individually placed in a glass observation box (dimensions: 34 cm length × 24 cm width × 20 cm height). The walls of the box were opaque, only the front wall was transparent. The box floor was covered with clear wood shavings (approximately 2 cm height). The test was conducted in a soundproofing room under controlled temperature (23 ± 2 °C). There were two infrared cameras in the room coupled to a computer placed outside the room. The test-mouse was left in the observation box for 10 min to acclimate. Then, an estrous female – sexually receptive - belonging to the same strain and age of the male mouse was placed in the box. The process to identify the estrous cycle followed the procedures set by Byers et al. (2012). The female was placed in the box where the male was in. Males were video recorded for 10 min after the female was introduced in the box in order to observe the following behavioral categories (Rodrigues-Alves et al., 2008): (i) latency to first mount or to the first attempt to mount; (ii) latency to the first intromission; (iii) frequency of incomplete-mount attempts (i.e., without penis intromission in the vagina); (iv) number of intromissions and (v) total number of ejaculations.

Fig. 1. (A) Body biomass (g); (B) feed (g) and (C) water (mL) intake by male Swiss mice directly exposed, or not, to tannery effluents. Bars represent the mean + standard deviation (n = 15/group). The * in “A” represent significant difference in the body mass of animals, which was measured at the beginning and in the end of the experiment. These data were subjected to two-way ANOVA with Tukey test at 5% probability level. Data in “B” and in “C” were subjected to the Kruskal-Wallis non-parametric test at 5% probability level. C: control group; DC: dry control group; WC: water control group; E%: 5% tannery effluent; and E100: 100% tannery effluent group.


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as well as that each 1 cm3 is equivalent to 1 mL, so the total count of spermatozoa per mL was set by multiplying the mean number of spermatozoa per quadrant at factor 106, based on Silva et al. (2007). Two aliquots (5 mL/animal) from each previously-fixed sample were analyzed to check the morphology of the spermatozoa. The samples were inked in safranin (5 mg/mL in distilled water) and, subsequently, two temporary slides per animal were prepared (Wyrobek and Bruce, 1975). The slides were analyzed in optical microscope (1000× magnitude). Two hundred spermatozoa per animal were randomly analyzed and the frequency of spermatozoa types was determined based on Filler (1993): normal spermatozoa, round head, pinhead, amorphous head, spermatozoon hook, reduced hook, crooked-necked spermatozoa, wounded spermatozoa, coiled flagellum or broken tail. 2.6. Histopathological analysis of the testes The right testes of the mice were collected to assess the possible effects of direct contact with TE on the structure of their testis and on some spermatogenic process-related variables. Samples were fixed in 4% buffered paraformaldehyde solution, dehydrated in ethanol gradients included in paraffin (Paraplast/McCormick), cut (5 μm thickness) and inked with hematoxylin and eosin (HE) (Luna, 1968; Behmer et al., 1976). Three slides were prepared for each animal; each slide had three cuts, and the cut presenting the best technical quality in each slide was selected to be analyzed; therefore, three cuts were counted per animal. Random images of the animals' testes were taken (100× magnitude) and captured in the Bell Capture software, version 3.1.0.0 (BEL Photonics, Piracicaba, SP, Brazil). Next, the relative frequency of the seminiferous tubules was quantified. These tubules presented (i) vacuoles in the seminiferous epithelium, (ii) apical cell detachment

Fig. 2. (A) Latency in the first mount attempt; (B) frequency of mount attempts (incomplete mounts) and (C) number of Leydig cells in the testes of male Swiss mice directly exposed, or not, to tannery effluents. Bars represent the mean + the standard deviation (n = 150 group). Data were subjected to the non-parametric Kruskal-Wallis test; multiple comparisons were performed through the Dunn's test at 5% probability level. Different lowercase letters indicate significant differences between groups. C: control group; DC: Dry control group; WC: water control group; E5: 5% tannery effluent group; and E100: 100% tannery effluent group.

animals. The left vas deferens was washed with 300 mL of saline solution to assess spermatozoa motility; the content was immediately transferred to a pre-heated slide (at 36 °C), according to Llobet et al. (1995). Next, a 50 mL aliquot was collected for immediate spermatozoa counting in optical microscope (400× magnitude). Spermatozoa were classified as: i) progressively mobile; ii) non-progressively mobile or iii) static, based on Tardif et al. (1998) and Slimen et al. (2014). The right vas deferens was washed with 300 mL of saline solution containing 10% formalin in order to quantify and assess spermatozoa morphology. Spermatozoa counting was conducted in Neubauer chamber using the 15 mL aliquot of the previously set samples. Two chambers were filled for each animal; the mean of the two counts was calculated at the end of the experiment. Spermatozoa were counted in optical microscope (400× magnitude); they were found in quadrants 1, 3, 7 and 9 of the Neubauer chamber, based on Slimen et al. (2014). We took into account that each quadrant of the chamber represents a 0.1 mm3 volume,

Fig. 3. (A) Relative number of Sertoli cells (per μm2 of seminiferous epithelium) and (B) percentage of Sertoli cells in relation to the total germinative cells counted in the seminiferous tubules of male Swiss mice directly exposed, or not, to tannery effluents. Bars represent the mean + standard deviation (n = 15/group). Data were subjected to the non-parametric Kruskal-Wallis test. Multiple comparisons were performed through the Dunn's test at 5% probability level. Different lowercase letters indicate significant differences between groups. C: control group; DC: Dry control group; WC: water control group; E5: 5% tannery effluent group; and E100: 100% tannery effluent group.

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aligned around the tubular lumen and around all cell types composing the spermatogenic process (Zorzetto, 2007). The seminiferous tubules were analyzed in the Bell Capture software, version 3.1.0.0 (BEL Photonics, Piracicaba, SP, Brazil), at 400× magnitude. The following parameters were measured: (i) frequency of Sertoli cells, Type A spermatogonia, and round and elongated spermatids in the seminiferous epithelium area, based on Carvalho (2009), and (ii) frequency of Leydig cells close to the blood vessels, or to the tunica albuginea, and counted in the ten best slides (40× magnitude)/testis. The seminiferous tubules and the interstitial intertubular compartments did not present any histological artifact. 2.7. Inflammatory response analysis Cytokines IFN-gamma and JE/CCL2 were measured through EnzymeLinked Immunosorbent Assay (ELISA) in order to check whether the contact with TE caused inflammatory processes in animals' testes. The immunoenzymatic trials used the supernatant of the macerated lefttestis of mice exposed, or not, to TE. The total of 0.04 g of selected tissue

Fig. 4. Relative number of Type A spermatogonia (A) of round (B) and elongated spermatids (C), and total spermatozoa in male Swiss mice directly exposed, or not, to tannery effluents. Bars represent the mean + standard deviation (n = 15/group). Parametric data were subjected to one-way ANOVA (multiple comparisons were performed through the Tukey test at 5% probability level), whereas the non-parametric ones were subjected to the Kruskal-Wallis test (multiple comparisons were performed through the Dunn's test at 5% probability level). Different lowercase letters indicated significant differences between groups. C: control group; DC: dry control group; WC: water control group; E5: 5% effluent group; and E100: 100% tannery effluent group.

(germ cell desquamation) and (iii) disorganized epithelium. Such changes were classified as “mild”, “moderate” or “intense”, based on Carvalho (2009). Ten (10) cross-sections of seminiferous tubules per animal were analyzed based on Verma and Singh (2014). They were randomly found in spermatogenesis phase VII, which is mainly marked by spermatozoa

Fig. 5. (A) Relative number of progressive motile (B) and static (C) spermatozoa of male Swiss mice directly exposed, or not, to tannery effluents. Bars represent the mean + standard deviation (n = 15/group). Data were subjected to the non-parametric Kruskal-Wallis test (multiple comparisons were performed through the Dunn's test at 5% probability level). Different lowercase letters indicate significant differences between groups. C: control group; DC: dry control group; WC: water control group; E5: 5% tannery effluent group; and E100: 100% tannery effluent group.


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in the cross-sectional cut was macerated for 30s in 500 μL of saline solution (PBS) in order to get the supernatant of the macerated testis. The supernatant was obtained after centrifugation (at 1000 rpm) for 5 min, at 4 °C. Commercial kits (Peprotech, New Jersey, USA) were used, after the samples were prepared, to detect the inflammatory markers (IFNgamma and JE/CCL2). Then, 100 μL/well of monoclonal antibodies against inflammatory markers were added to 96-well microplates in order to sensitize them. These markers were diluted in PBS with 0.1% of bovine serum albumin – BSA (Sigma-Aldrich). The plates were incubated overnight at room temperature. Antibodies adsorbed by the plates were discarded through inversion and successive washes in PBS-Tween. Plates were blocked with 300 μL/well of a solution containing 1% PBS-BSA for 1 h at room temperature. Next, they were washed again. After blocking, 100 μL/of the macerated supernatant samples and 100 μL of the patterns for each marker were added. Another washing was performed after 2 incubation hours; 100 μL of biotinylated detection antibodies and anti-cytokine were added to the samples (by using different dilutions in PBS at pH 7.4 with 1% BSA) and incubated for 2 h at room temperature after the wash. Subsequently, 100 μL/well of HRP streptavidin (Peprotech, New Jersey, USA) was added at dilution ratio 1:200 in PBS with 1% BSA and incubated at room temperature for 30 min. The plates were washed again, added with 100 μL of ABTS (Liquid Substrate System 3-ethylbenzothiazoline-6-sulfonic acid – SigmaAldrich, Missouri USA) and incubated for 30 min at room temperature. The optical density was determined by using a microplate reader with 405 nm filter and 650 nm correction. The quantification of inflammatory markers in the samples was based on the optical density, which resulted from the known standard concentrations and was analyzed in the SOFTmax PRO 4.0 software.

The lethal dominant test was conducted to assess whether the exposure to TE would affect these animals' reproductive development. Animals in different groups were placed alone in standard boxes for rodents after the last exposure day (i.e., the 91st experimental day) and paired with a nulliparous female who was not exposed to pollutants at the estrous phase of the ovarian cycle. The animals remained together for 24 h in order to mate, based on Ehling et al. (1978). According to the aforementioned authors, the mating period must be as long as possible in order to provide more information about the specific actions of chemical mutagens at the germ cell stage. Subsequently, males were euthanized to allow evaluating the parameters described in the previous items. Females were subjected to laparotomy 18 days after pregnancy was diagnosed in order to allow evaluating the following parameters based on the methodological procedures described by Oliveira et al. (2014): fertility rate (number of pregnant females × 100/number of females), number of embryonic implantations, post-implantation loss rate (number of implantations – number of living fetuses × 100/number of implantations), number of reabsorptions and lethal dominant frequency (number of reabsorptions × 100/number of implantations).

2.8. Biometry and hematological analysis

2.10. Statistical analyses

The body biomass of the animals was measured in the beginning and at the end of the experimental period. The relative mass of the testes and epididymis was calculated after the exposure and euthanasia procedures in order to check whether the direct contact with TE caused hypertrophy and hypotrophy in these androgen organs. The mass of each organ (g) was divided by the animal's body biomass, which was

The Shapiro-Wilk normality test was initially used to check data distribution. Data presenting normal distribution were subjected to simple analysis of variance (one-way ANOVA), followed by the Tukey post-test, whenever F was significant. Data that did not present normal distribution were subjected to the non-parametric Kruskal-Wallis test. Both tests adopted 5% significance level. The chi-square test (x2) was applied

B

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measured at the day the euthanasia was performed, based on Estrela et al. (2014). Water and food consumption was measured on a daily basis through the daily subtraction between the offered amount and the leftovers collected in the following day. We analyzed the erythrogram and leukogram of the animals through the automatized method ABX – Micros 60, based on the methodology by Estrela et al. (2015). 2.9. Lethal dominant test

C

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Fig. 6. (A) Total sum of spermatozoa in male Swiss mice directly exposed, or not, to tannery effluents, presenting morphological abnormalities. Photomicrographs of the main morphological abnormalities found in animals' spermatozoa. (B) normal spermatozoon; (C) reduced hook; (D) amorphous head; (E) broken tail. Bars represent the mean + standard deviation (n = 15/group). Data were subjected to one-way ANOVA (multiple comparisons were performed through the Tukey test at 5% probability level). Different lowercase letters indicate significant differences between groups. C: control group; DC: dry control group; WC: water control group; E5: 5% tannery effluent group; and E100: 100% tannery effluent group.

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Fig. 7. Relative number of seminiferous tubules (A) with disorganization, (B) vacuoles and (C) apical detachment of germinative cells in male Swiss mice directly exposed, or not, to tannery effluents. Bars represent the mean + standard deviation (n = 15/group). Parametric data were subjected to one-way ANOVA (multiple comparisons were performed through the Tukey test at 5% probability level), whereas the non-parametric ones were subjected to the Kruskal-Wallis tests (multiple comparisons were performed through the Dunn's at 5% probability level). Different lowercase letters indicate significant differences between groups. C: control group; DC: dry control group; WC: water control group; E5: 5% tannery effluent group; and E100: 100% tannery effluent group.

to quantitatively assess the relation between the result of an experiment and the distribution expected for the phenomenon. All statistical analyses and graphics were performed in the GraphPad Prism software (version 7.0). 3. Results ANS discussion We assessed animals' biomass in order to evaluate possible systemic toxicity - the relative masses of some animals changed. Our analyses showed the effect of factor “time” on body biomass. All groups

presented increased biomass at the end of the experiment in comparison to the initial one (Fig. 1A). In consensus with Golub and Germann (2001), this increase is linked to the animals' development/growth, since they were in the puberty phase when the experiment was conducted (32 days old). We believe that the lack of differences in the mean daily food and water intake, and the animals' body masses at the end of the experimental period is associated with the lack of differences between experimental groups (Fig. 1B–C). We did not find differences in the relative testis masses between experimental groups (Fig. S2A–B – check on “Supplementary Material”). These and body biomass data suggested that mice in all experimental groups kept similar use of the available food resources. This outcome indicated that TE – at the herein assessed exposure concentrations and time – did not induce significant changes in the animal's energetic metabolism, fact that suggested lack of systemic intoxication in the studied animals. The relative mass data allowed inferring that the exposure to TE did not cause hypo- or hypertrophy in the assessed androgen organs. We recorded latency in the first mount and in the frequency of mount attempts (incomplete mounts). The other behavioral categories specified in section “Materials and Methods” were not observed in our study. This outcome can be mainly associated with the time males spent interacting with receptive females. The 30-min time we adopted in our study was likely not enough to depict a broader sexual behavior repertoire. The depiction of male rodents' sexual behavior such as latency in the first ejaculation can range from 10 to 115 min (Hull and Dominguez, 2007). This time gap can be different between different mice and rat strains. We cannot neglect that the mice assessed in our study were nulliparous (without any previous sexual experience); they often need longer to show broader sexual behavior repertoire. If on the one hand latency in the first mount attempt was similar between experimental groups (Fig. 2A), on the other hand, mice in groups E5 and E100 were the ones who presented the highest frequency of mount attempts (Fig. 2B). These data ruled out the hypothesis that TE exposure could harm the chemosensitive receptors of the principal and accessory olfactory systems, which are relevant for the emergence of sexual behaviors. It goes against Mendes et al. (2017), who recorded olfactory losses in male and female C57Bl/6 J mice dermally exposed to gross TE for 15 days. In fact, we believe that TE constituents increased the serum testosterone levels – which balance the motivational effects of sexual behavior such as penile erection and libido (Schanbacher and Lunstra, 1976; Batty, 1978). These serum testosterone levels were likely responsible for the frequency of mount attempts observed in animals exposed to TE. Such hypothesis is mainly reinforced by the larger number of Leydig cells (without morphological changes) in the testes of animals exposed to the contaminant (Fig. 2C). According to Russell et al. (1990) and Shima et al. (2013), Leydig cells present remarkable production of androgens synthesized from cholesterol such as testosterone. Assumingly, the xenobiotics found in TE may have increased the synthesis reactions of this hormone and acted in different components that mediate reactions whose mechanisms are yet to be elucidated. There is no consensus in the literature about the effects of exposing different animal models to different contaminants found in the environment on serum testosterone levels (check the literature review by Guillette and Gunderson, 2001). However, studies conducted by Ljungvall et al. (2005) and Kumar et al. (2008) showed that xenobiotics stimulate the synthesis or release of testosterone in rodents. Ljungvall et al. (2005) observed significant Leydig cell-population increase, along with proportional increase in serum testosterone levels, in boars exposed to phthalates (one of the most abundant constituents identified in the herein used TE). Kumar et al. (2008) reported evidences of increased serum testosterone levels in male Wistar rats exposed to different TE concentrations. Other investigations conducted with mice and other experimental nonmammal models (fish and frogs) exposed to other pollutants (Dai et al., 2001) also evidenced that serum testosterone levels can considerably increase in animals exposed to different contaminants (Sangalang and


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B.1

Fig. 8. Photomiographs of seminiferous tubules in male Swiss mice directly exposed, or not, to tannery effluents. (A) control group; (B) E5 effluent group; (B.1) amplification of seminiferous tubule presenting germinative cell detachment in the lumen; (C) E100 group. Des: disorganized germinal epithelium; Va: vacuole; Lu: lumen; EA: elongated spermatid. ER: round spermatid; EG: spermatogonia; S: Sertoli cells; L: Leydif cell; *: germinative cell detachment in the lumen; VS: blood vessel; +: intercellular spaces. E5: 5% tannery effluent group; and E100: 100% tannery effluent group. HE color shade.

Freeman, 1974; Hopkins et al., 1997). These studies reinforce the hypothesis that TE increased the synthesis or release of testosterone in animals from groups E5 and E100. Surprisingly, we also observed a larger number of Sertoli cells in animals exposed to TE, either per μm2 of seminiferous epithelium (Fig. 3A), or by analyzing the percentage of these cells in relation to the total germinative cells in the counted seminiferous tubules (Fig. 3B). According to many studies (Russell, 1980; Griswold, 1995; Walker and Cheng, 2005; Johnson et al., 2008; Kopera et al., 2010), Sertoli cells play a significant role in spermatogenesis. The interaction between these cells and germinative cells is essential, mandatory and complex, since the neonatal period until the beginning of spermatozoa production (in puberty), either physically or biochemically (Griswold, 1995). Previous studies demonstrated that the proliferation of Sertoli cells often happens before the adult phase either in humans or in rodents (Sharpe et al., 2003). Thus, we assume that there may have happened some unbalance in the processes controlling and mediating their proliferation. This process would explain the larger number of these cells in animals exposed to TE. The proliferation of Sertori cells in rodents mainly happens in the fetal and neonatal periods, as well as before the adult or sexual maturation phases (Sharpe et al., 2003). The exposure to TE in our study started in the pubertal phase of mice (at 32 days of life); therefore, the first days of contact

with TE constituents would had been enough to cause changes in the proliferation of Sertoli cells. Another possibility concerns the extension of the Sertoli-cell proliferation period, which was longer in animals exposed to TE than in the ones who were not exposed to the contaminant. It is tempting to speculate that TE constituents have caused changes in the physiological operations of the hypothalamic-pituitary axis, mainly in neuromodulations linked to the production of the hormone responsible for releasing gonadotropin (GnRH). The increased frequency and extension of GnRH secretion pulse, for instance, may have increased the synthesis and/or release of the follicle-stimulating hormone (FSH). Such process led to greater Sertoli cell proliferation in the first TE-exposure days, because this hormone is essential to such proliferation (Orth, 1984). Previous studies suggested important neurotoxic effects on the hypothalamic-pituitary axis due to rodents' exposure to TE (Moysés et al., 2017; Rabelo et al., 2017; Estrela et al., 2017), thus corroborating our hypothesis. The larger number of Leydig cells (Fig. 2C), which was followed by the proportional increase in the number of Sertoli cells (Fig. 3A–B), suggested failures in the negative feedback mechanisms involving the luteinizing hormone (LH) and FSH. These hormones are closely related to the production of these cells in testes (Mendis-Handagama, 1997; Haider, 2004; Walker and Cheng, 2005). Although animals exposed to TE recorded larger Sertoli cell population, the reduced number of type A spermatogonia (Fig. 4A), the round

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and elongated spermatids (Fig. 4B–C), the lower spermatozoa production (Fig. 4D–A), as well as changes in their motility (Fig. 5A–C) and morphology (Fig. 6A), pointed towards possible functional losses in these cells. It also suggested that the larger number of Sertoli cells can be a compensatory response to the increased number of changed spermatozoa. According to Russell et al. (1990), Sertoli cells are more resistant to the action of toxic agents than germinative cells; however, they are very sensitive to functional disorders capable of affecting animals' metabolic and regulatory pathways. Such outcome can rapidly lead to degeneration and malformation of germinative cells. The literature describes many functions performed by Sertoli cells and the role they play in supporting the spermatogenic cells, in the phagocytosis of tubular residual bodies, in the secretion of the fluid with important substances for epididymal function and sperm maturation, as well as in supplying nutrients to germinative cells (Russell et al., 1990; Russell and Griswold, 1993). These cells also act in the primary endocrine regulation of spermatogenesis, mainly via FSH and testosterone (Walker and Cheng, 2005), besides playing an essential role in the regulation of intratubular and intercellular adluminal environments (Russell and Griswold, 1993). Moreover, they enable the formation of a blood-testicular barrier responsible for the development of a specific, immunoprivileged microenvironment, which is essential to the spermatogenic process (Russell et al., 1990; Russell and Griswold, 1993). The proliferation of Sertoli cells can prevent the increased bioaccumulation of pollutants found in TE and in animals' testes. The tubular degeneration featured by the seminiferous tubules with disorganized epithelium (Fig. 7A and 8), tubular vacuolization (Fig. 7C) and apical detachment of germinative cells (Fig. 7C) in animals exposed to TE reinforced the assumption that Sertoli cells in these animals would have their functions impaired, mainly when it comes to the formation of the blood-testicular barrier. The detachment of germinative cells more often observed in animals exposed to TE, for example (Fig. 8B–C), may have happened due to the loss of cell junctions between germinative and Sertoli cells and/or to unbalance in the microtubules, which

resulted in germinative-cell detachment. Such outcome was also reported in studies about toxicity induced by xenobiotics (Boekelheide, 2005; Xia et al., 2005; Flora, 2011; Costa, 2016). The intercellular spaces observed (Fig. 8B) in germinative cells, either in the basal or in the adluminal environment, reinforced the negative action of TE in adhesion junctions. Our results suggested that histopathological changes in the seminiferous tubules can be associated with inflammatory process in the testes of animals exposed to TE (groups E5 and E100), due to increased concentration of proinflammatory cytokines such as IFN-gamma and CCL2 (Fig. 9A–B). Animals in the control group exposed to TE presented infiltrates of mononuclear cells in the intertubular spaces (Fig. 9C). Such infiltrates presented characteristics compatible to an unspecific (from mild to moderate level) multifocal, intertubular inflammation of unknown cause (Fig. 9C). The smaller total number of neutrophils and monocytes in the peripheral blood (Table S3 – Check on “Supplementary Material”) of animals exposed to TE pointed towards the possible migration of these cells to testicular foci. Results also suggested testicular amyloidosis in animals subjected to the TE treatment, which consists of extracellular accumulation of homogeneous, eosinophilic amorphous material in the interstice (Fig. 9C). This outcome is closely related to worsened local inflammatory processes (Gonzales et al. (1983). We assume that the previously suggested inflammatory process can be associated with the inability of Sertoli cells to form efficient bloodtesticular barriers or with disruption in junctional specifications (i.e., ectoplasmic). Such disruption may have been caused by the direct action of products deriving from oxidative stress reactions caused by xenobiotics in TE [which present the potential to cause such effect - Estrela et al., 2017 and Rabelo et al., 2017], as well as by changes in the biochemical and molecular components mediating the structuring/formation of blood-testicular barriers. These events would influence the blood-testicular barrier function by mechanically segregating germinative cell autoantigens. This segregation would allow the leak of these cells to the interstitial space and cause cell-signaling cascades

C A Lu

*

DC

*

Fig. 9. Concentration of proinflammatory cytokines (IFN-gamma) (A) and CCL2 (B) in the testes of male Swiss mice directly exposed, or not, to tannery effluents. (C) Photomicrographs of testes presenting inflammatory infiltrates (arrows), HE color shade. Bars represent the mean + standard deviation (n = 15/group). Parametric data were subjected to one-way ANOVA (multiple comparisons were performed through the Tukey test at 5% probability level), whereas the non-parametric ones were subjected to Kruskal-Wallis test (multiple comparisons were performed through the Dunn's test at 5% probability level). Different lowercase letters indicate significant differences between groups. C: control group; DC: dry control group; WC: water control group; E5: 5% tannery effluent group; E100: 100% tannery effluent group. DC: germinative epithelium detachment; Lu: lumen.


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responsible for attracting mononuclear peripheral blood cells to inflammatory foci. The structural damage in the blood-testicular barrier of rodents exposed to abundant constituents identified in TE – individually assessed – reinforced this assumption [phthalate: Yao et al., 2010 and Shen et al., 2017; chrome: Carette et al., 2013; lead and arsenic: Ramos-Treviño et al., 2017]. Finally, animals exposed to TE had their reproductive performance affected, although the fertility index did not differ between groups [(C: 80%; DC: 80%; WC: 80%; E5: 90% and E100: 80% (χ2 = 5.420; p = 0.246)]. Females who mated with males exposed to TE recorded smaller number of fetal implantations (Fig. 10A), higher post-implantation loss rate (Fig. 10B) and larger number of reabsorptions (Fig. 10C). The lethal dominant frequency was higher in groups E5 and E100 (Fig. 10D). Losses observed in fetal development and post-implantation embryonic losses can be understood as product of the lethal maturation of genetic material, which was transmitted to the offspring of animals exposed to TE (Maxwell and Newell, 1973) through their spermatozoa (Fig. 11). Assumingly, zygotic and fetal deaths resulted from chromosome changes or rearrangements during the spermatogenic process caused by the xenobiotics in TE. Morphological and motility abnormalities in spermatozoa of animals exposed to TE explained the relation between these effects and possible lethal dominant mutations. Such process resulted in reproductive deficit in these animals. Thus, our data completed the ones reported in previous studies and evidenced the neurobehavioral changes in the offspring of parents subjected to the intake of water contaminated with TE (Guimarães et al., 2016a). The aforementioned author was the first researcher to show that this pollutant can also cause pre-natal losses in the offspring of mice dermally subjected to the xenobiotics in this effluent. Finally, the biological changes influencing the reproductive system of the herein investigated animals were complex and not limited to certain biochemical or molecular mechanisms, mainly when these animals were exposed to non-conventional pollutants such as TE, which comprises a complex mixture of chemical products of well-known toxic effect and other components whose toxicity is yet to be described (Bharagava et al., 2017). According to Guillette and Gunderson (2001), contaminants can act in the reproductive system as antagonists or as the antagonists of hormone receptors. They can also stimulate or inhibit the synthesis and/or release of hormones responsible for inhibiting or stimulating the pituitary gland or hypothalamus, besides changing the concentration and functioning of ligand proteins in blood plasma. It changes the serum concentration of different hormones, among others. The presence of organic and inorganic matter of known toxicity in TE, as well as of compounds yet to be investigated (individual- Tables S1 and S2 – check on “Supplementary Material”) allows assuming a large range of possibilities that can clarify the harming effects observed in our study. 4. Conclusion Our study confirmed the hypothesis that the direct exposure to TE has behavioral and histopathological impacts on, and changes the reproductive performance of, male Swiss mice. Therefore, this pollutant can significantly change the dynamics of small-mammal populations living close to tannery facilities. The set of changes shown in our study strongly suggested that different xenobiotics in TE may have acted individually, or in association, in the biological system of the investigated animals. It may have caused endocrine, biochemical, molecular and/or mutagenic disruptions that, in the long-term, may have led to fertility decay in these animals. Results reported in our study only showed the “tip of the iceberg” of losses resulting from the exposure of terrestrial biota to TE, besides reinforcing that new investigations should be developed to help better understanding the magnitude of the impact these pollutants can have on ecosystems. Given the complexity of TE and the pioneering nature of our study about the reproductive toxicology of animals exposed to the pollutant,

Fig. 10. (A) number of fetal implantations, (B) post-implantation loss rate, (C) number of reabsorptions and (D) lethal dominant frequency in mating between females not exposed to TE and male Swiss mice directly exposed, or not, to tannery effluents. Bars represent the mean + standard deviation (n = 15/group). Data were subjected to the non-parametric Kruskal-Wallis test (multiple comparisons were performed through the Dunn's test at 5% probability level). Different lowercase letters represent significant differences between groups. C: Control group; DC: dry control group, WC: water control group; E5: 5% tannery effluent group; E100: 100% tannery effluent group.

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Fig. 11. Pictures depicting variables counted in different experimental groups, (A) control, without visible changes; (B) E100, malformation; (C) E100, black arrows indicate malformation, whereas the white ones indicate reabsorption.

we encourage the development of new investigations. Further studies can help better elucidating the action mechanisms of chemical constituents (TE: chemical mixture), identifying the individual contributions (more or less important) of such constituents, as well as their biological effects (short- and medium-term) on the offspring of exposed animals. Furthermore, the improvement of analytical techniques adopted to identify and quantify non-routine organic compounds (although present in TE) in biological tissues would also be very useful for studies with TE.

Acknowledgment The authors are grateful to the Brazilian National Council for Research (CNPq) (Brazilian research agency) (Proc. No 467801/ 2014-2) and to Instituto Federal Goiano for the financial support (Proc. No 23218.000286/2017-21). Moreover, the authors are grateful to CNPq for granting the scholarship to the student who developed this study. Funding This study was funded by the Brazilian National Council for Research (CNPq) (Brazilian research agency) (Proc. No 467801/2014-2) and by Goiano Federal Institute– Campus Urutaí (GO, Brazil) (Proc. No 23218.000286/2017-21). Conflict of interest The authors declare no conflict of interest. Ethical approval All procedures were approved by The Ethics Committee on Animal Use of Goiano Federal Institute (Comissão de Ética no Uso de Animais do Instituto Federal Goiano), GO, Brazil (protocol No. 2616170516). Meticulous efforts were made to assure that the animals suffered the least possible and to reduce external sources of stress, pain and discomfort. The current study did not exceed the number of animals necessary to produce trustworthy scientific data. This article does not contain any studies with human participants performed by any of the authors. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.08.253.

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