Arch Environ Contam Toxicol (2015) 68:74–82 DOI 10.1007/s00244-014-0075-x

Municipal Landfill Leachate-Induced Testicular Oxidative Damage is Associated with Biometal Accumulation and Endocrine Disruption in Rats Isaac A. Adedara • Ifeoluwa O. Awogbindin • Adebayo A. Adesina • Oluwatosin O. Oyebiyi Tajudeen A. Lawal • Ebenezer O. Farombi

•

Received: 8 May 2014 / Accepted: 14 July 2014 / Published online: 2 September 2014 Ó Springer Science+Business Media New York 2014

Abstract Improper management of hazardous wastes adversely impacts the environment and the public health. The present study was aimed at investigating the influence of Olushosun municipal landfill leachate (OMLL) from Ojota in the Lagos State of Nigeria on testicular function by assessing the plasma concentrations of reproductive hormones, testicular biometal levels, and antioxidant levels as well as observing the histological alterations in testes and epididymides of rats after exposure to 0, 12.5, and 25 % OMLL in drinking water for 7 days. Exposure to OMLL significantly decreased the daily fluid intake, but it resulted in testicular biometal accumulation as follows: lead [ cadmium [ nickel [ iron [ copper. Acute exposure to OMLL induced oxidative stress and increased the activities of marker enzymes of testicular function but markedly decreased the circulatory concentrations of luteinizing hormone, follicle-stimulating hormone, prolactin, testosterone, thyroid-stimulating hormone, triiodothyronine, and thyroxine. Testicular and epididymal degeneration with significant decrease in sperm quality and quantity were observed in OMLL-exposed rats. Collectively, the data presented herein indicate that exposure to OMLL-induced testicular dysfunction associated with biometal accumulation and endocrine disruption in rats. If the effects can be extrapolated to humans, OMLL may present significant health implications for individuals exposed to OMLL-contaminated substances.

I. A. Adedara  I. O. Awogbindin  A. A. Adesina  O. O. Oyebiyi  T. A. Lawal  E. O. Farombi (&) Drug Metabolism and Toxicology Research Laboratories, Department of Biochemistry, College of Medicine, University of Ibadan, Ibadan, Nigeria e-mail: [email protected]; [email protected]

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Landfilling, a major method in waste management hierarchy in developing countries, is known to generate leachate that has the potential to contaminate watercourses (Emenike et al. 2012). Because it is an effluent, leachate could migrate laterally and vertically into nearby watercourses either by direct discharge or by gradual seeping through the soil membrane (Fauziah et al. 2013). The presence of toxic substances—such as heavy metals, dissolved organic matter, polychlorinated biphenyls and inorganic macrocompounds—in the leachate are known to cause adverse health effects, such as low birth weight, increased number of birth defects, suppressed immunological function, and certain types of cancers, which have been reported in people living near landfill sites or in solid waste—management workers (Deguchi et al. 2007; Odewabi et al. 2013). The mammalian testis consists of two compartments, namely, the seminiferous tubules and the interstitium. The seminiferous tubules contain androgen-sensitive Sertoli cells and the entire germ line, whereas the interstitium is responsible for blood supply and immunological responses and contains Leydig cells, which mediate endocrine signals of the pituitary to the testis (Wistuba et al. 2007). The process of spermatogenesis requires highly complex endocrine regulation. Testicular function is regulated along an endocrine axis linking the brain, the hypothalamus, the pituitary gland, and the gonads. There are several possible mechanisms by which different environmental contaminants elicit antigonadal actions. Environmental contaminants may elicit a direct inhibitory effect on the testis or act indirectly by altering gonadotrophin homeostasis, which subsequently impairs spermatogenesis. In terms of solid waste management, a landfill of major health concern in Lagos State, the former capital of Nigeria, is the Olushosun landfill. It covers approximately 42 ha of land, with an excavation of approximately 18 m

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deep into the landfill area, and serves as a major site for disposal of waste in the state (Olorunfemi 2011). Olushosun landfill, which was almost a vacant land at creation, is currently surrounded by industries and residential areas known as a flourishing commercial central district in Lagos State. The landfill is not controlled and managed according to international standards of operations (Odewabi et al. 2013; Momodu et al. 2011). Previous studies have linked municipal landfills to the anthropogenic heavy-metal enrichment of the Lagos lagoon and groundwater (Okoye et al. 1991; Farombi et al. 2012). Although previous studies have shown the involvement of oxidative stress in the adverse effects of leachate from Olushosun landfill on sperm and erythrocytes using both in vitro and in vivo techniques (Adedara et al. 2013, 2014), the mechanism underlying adverse reproductive effects due to leachate exposure is not fully known. It remains to be determined if gonad and/or extragonadal alterations contribute to the manifestation of the previously reported spermatotoxic effects of the municipal leachate. Given these gaps in knowledge, the present study was designed to investigate the acute effects of municipal landfill leachate on testicular function by determining sperm output, levels of biometals, antioxidant status, and marker enzymes of testicular function. In addition, determination of plasma hormone levels and histological analyses of testes and epididymides of municipal landfill leachate-exposed rats were performed.

Materials and Methods Chemicals Thiobarbituric acid, 50 ,50 -dithiobis-2-nitrobenzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), hydrogen peroxide (H2O2), glutathione (GSH), and epinephrine were procured from Sigma Chemical (St Louis, Missouri, USA). All other reagents were of analytical grade and were purchased from British Drug Houses (Poole, Dorset, UK). Collection of Leachate The influence of leachate from Olushosun landfill, Lagos State, Nigeria, on testicular function was investigated in the present study. Raw leachate collected from holes in the landfill was thoroughly mixed and filtered to remove debris. The physicochemical characteristics of the leachate used in this study were similar to our previously published data (Farombi et al. 2012). Twenty-five percent leachate was prepared from the homogenous mixture according to a standard procedure (American Society for Testing and Materials 1992) as modified by Adedara et al. (2013).

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Briefly, 250 mL of the raw sample was made up to 1000 mL of distilled water (v/v), mechanically shaken for 1 h, and allowed to settle for 30 min before filtering with a 2.5-lm filter (Whatman no. 42) to remove the suspended particles. The resulting filtrate was finally centrifuged at 6009g for 15 min at room temperature. The pH of the supernatant fluid was measured before storage at 4 °C until use. The sample was labeled Olushosun municipal landfill leachate (OMLL). Animal Model and Experimental Design A total of 30 sexually matured adult male Wistar rats (12 weeks, approximately 200 g) obtained from the Department of Biochemistry, University of Ibadan, Ibadan, Nigeria, were used for the present investigation. The animals were housed in plastic cages placed in a well-ventilated rat house, provided rat chow and water ad libitum, and subjected to natural 12:12-h light-to-dark photoperiod. Ethical regulations were followed according to the national and institutional guidelines for the protection of animal welfare during experiments. The present experiment was performed according to the guidelines and approval of the institutional Animal Ethics Committee. The rats were then randomly divided into 3 groups of 10 rats/group. OMLL was diluted with distilled water and made available as drinking water to the rats at concentrations of 12.5 and 25 % for 7 days (Adedara et al. 2014). Corresponding group of rats drank distilled water for 7 days and served as control. Twenty-four hours after the last treatment, the blood from animals each group was drawn from retro-orbital venous plexus for hormonal estimation before they were killed by cervical dislocation. The testes, epididymides, seminal vesicles, and prostate glands were quickly removed and weighed. The body weights of rats were recorded before exposure and after treatment before they were killed. Determination of Heavy Metals in Testes The left testes from each group were washed in ice-cold 1.15 % KCl solution, blotted, and weighed. They were subsequently homogenized in 4 volumes of homogenizing buffer (50 mM Tris–HCl mixed with 1.15 % KCl [pH adjusted to 7.4]) using a Teflon homogenizer. The resulting homogenate was centrifuged at 10,0009g for 15 min in a Beckman L5-50B centrifuge at 0–4 °C and the supernatants collected for determination of heavy-metal levels according to Farombi et al. (2007). The testes supernatants were digested according to the method described by Hoenig and de Kersabiec (1996). The levels of copper (Cu), cadmium (Cd), iron (Fe), nickel (Ni), and lead (Pb) in the filtrates from each digested sample were then determined with the aid of PerkinElmer Atomic Absorption Spectrophotometer

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A Analyzer 200 (California, USA). An acetylene-air mixture was used as the flame. The working standard for each of the metals were aspirated into the flame in the order of 0.0, 0.2, 0.4, 0.8, and 1.6 ppm before the tissues were aspirated into the flame. Determination of Testicular Antioxidant Status The right testes were homogenized in 50 mM Tris-HCl buffer (pH 7.4) containing 1.15 % potassium chloride. The resulting homogenate was centrifuged at 10,0009g for 15 min at 4 °C and the supernatant thereafter collected for biochemical estimations. Protein concentration was determined by the method of Lowry et al. (1951). Superoxide dismutase (SOD) activity was assayed by the method described by Misra and Fridovich (1972). Catalase (CAT) activity was determined according to the method of Clairborne (1995) using hydrogen peroxide as substrate. Decreased GSH level was determined at 412 nm using the method described by Jollow et al. (1974). Glutathione S-transferase (GST) activity was determined by the method of Habig et al. (1974). Glutathione peroxidase (GPx) activity was assayed by the method of Rotruck et al. (1973), which is based on the reaction between DTNB and GSH remaining after the action of GPx to form a complex that absorbs maximally at 412 nm. H2O2 generation was estimated by the method of Wolff (1994). Lipid peroxidation (LPO) was quantified as malondialdehyde (MDA) according to the method described by Farombi et al. (2000) and expressed as micromoles of MDA per milligram protein. Determination of Activities of Marker Enzymes of Testicular Function Testicular tissue was homogenized in ice-cold phosphate buffer (pH 7.4). The microsomal fraction was used as an enzyme source. Lactate dehydrogenase-X (LDH-X) activity was determined according to the method of Vassault (1983). Gamma-glutamyl transferase (GGT) activity was assayed using L-c-glutamyl-3-carboxyl-4-nitroanilide and glycylglycine as substrates according to the method of Szasz (1974). Glucose-6-phosphate dehydrogenase (G6PD) was evaluated according to the modified method of Dawson et al. (1958) using NADP and glucose-6-phosphate as substrates. Evaluation of Plasma Hormones Commercial enzyme immunoassay kits specific for rats were used to estimate plasma concentrations of testosterone (EIA-5179; DRG Diagnostics), FSH (RPN 2560; Amersham, UK), prolactin (ab113351, Abcam, UK) and LH (RPN 2562, Amersham, UK) in accordance with the manufacturer’s instructions. The sensitivity of the testosterone assay

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was 0.08 ng/ml and with negligible cross-reactivity with other androgen derivatives, such as androstenedione, 5adihydrotestosterone, and methyl testosterone. The intraassay coefficient of variation (CV) for the testosterone assay was 4.4 %. The sensitivity of prolactin was 0.07 ng at 90 %, LH was 0.04 ng at 80 %, whereas FSH sensitivity was 0.07 ng at 98 %. The intra-assay CV was 3.1 % for prolactin, 3.6 % for LH and 3.2 % for FSH. Moreover, according to the manufacturer’s instructions, total plasma triiodothyronine and thyroxine concentrations were determined using commercial enzyme immunoassay kits (DiaSorin, Sauggia, Italy). Intra-assay CVs for total thyroxine were 2.5–3.1 %, whereas total triiodothyronine was 3.8–5.1 %. Sensitivity of the assays was 68 pg/ml for total thyroxine and 254 pg/ml for total triiodothyronine. All of the samples were evaluated on the same day to avoid interassay variation. Total plasma concentrations of triiodothyronine and thyroxine were expressed as nanograms per deciliter and micrograms per deciliter, respectively. Evaluation of Epididymal Sperm Characteristics The motility of the sperm was evaluated according to the method of Zemjanis (1970). Briefly, epididymal sperm was obtained by cutting the cauda epididymis with surgical blades and released onto a sterile clean glass slide. The sperm was then diluted with 29 % sodium citrate dehydrate solution and thoroughly mixed to assess the sperm progressive motility with the aid of a microscope within 2–4 min of their isolation and data expressed as percentages. The sperm were counted by haemocytometer using the improved Neubauer chamber (Deep 1/10 m; LABART, Munich, Germany) according to Pant and Srivastava (2003). A portion of the sperm suspension placed on a glass slide was smeared with another slide and stained with Wells and Awa’s stain (0.2 g eosin and 0.6 g fast green dissolved in distilled water and ethanol in a 2:1 ratio) for morphologic examination and with 1 % eosin and 5 % nigrosine in 3 % sodium citrate dehydrate solution for viability according to Wells and Awa (1970). A total of 400 sperm/rat were used for morphologic examination. Determination of Daily Sperm Production and Testicular Sperm Number Daily sperm production (DSP) was determined using frozen left testes from control and OMLL-treated rats according to Blazak et al. (1993). Briefly, the testis was decapsulated and homogenized in ice-cold physiologic saline containing 0.01 % Triton X-100. Subsequently, an aliquot of the resulting homogenate was transferred to a glass vial and stored on ice. Sample aliquots were then placed on the Neubauer haemocytometer and counted

Arch Environ Contam Toxicol (2015) 68:74–82

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Table 1 Fluid intake, body weight gain, and relative organ weights after OMLL exposure for 7 days in rats Parameter

Control

12.5 % OMLL

25 % OMLL 19.18 ± 1.62*

Fluid intake/d/rat (ml)

28.81 ± 1.41

22.04 ± 1.93*

Body weight gain (g)

7.83 ± 1.21

7.16 ± 0.38

6.71 ± 0.95

Testes (g/100 g bw)

0.52 ± 0.12

0.51 ± 0.1

0.48 ± 0.1

Epididymis (g/100 g bw)

0.11 ± 0.02

0.10 ± 0.2

0.07 ± 0.01*

Seminal vesicle (g/100 g bw)

0.34 ± 0.02

0.37 ± 0.01

0.38 ± 0.09

Prostate gland (g/100 g bw)

0.12 ± 0.04

0.10 ± 0.03

0.08 ± 0.05

Data are expressed as mean ± SD for ten rats per group bw body weight * p \ 0.05 compared with the control

twice at 100 9 magnification under a light microscope to determine the number of sperm heads (stage 19 spermatid head). These values were used to obtain the total number of spermatids per testis and subsequently the spermatids per gram of testes. DSP was calculated by dividing the number of spermatids at stage 19 with those at stage 6.1, which is the duration of the seminiferous cycle in which these spermatids are present in the seminiferous epithelium. Histology Testes and epididymis biopsy specimens were carefully removed and fixed with Bouin’s solution. The samples were thereafter blocked in paraffin after dehydration procedures. Sections of 4–5 lm were prepared by a microtome and stained with hematoxylin and eosin. All slides were coded before examination with light microscope and photographed using a digital camera by investigators who were blinded to the control and leachate-exposed groups. Statistical Analysis Statistical analyses were performed using one-way analysis of variance to compare the experimental groups followed by Bonferroni test to identify significantly different groups (SPSS for Windows, version 17, Copyright Softonic International S. A., Chicago, IL, USA). Values of p \ 0.05 were considered significant.

Results Daily Fluid Intake, Body Weight Gain, and Relative Organ Weights Table 1 lists daily fluid intake, body weight gain, and relative organ weights of rats after exposure to OMLL for 7 days. There was a significant decrease in the daily fluid intake by OMLL-exposed rats compared with the control.

Table 2 Sperm characteristics after OMLL exposure for 7 days in rats End points

Control

12.5 % OMLL

25 % OMLL

ESN (%)

92.17 ± 4.38

65.83 ± 2.77*

61.03 ± 4.63*

Motility (%)

92.67 ± 2.58

76.67 ± 3.16*

54.33 ± 3.53*

96.5 ± 1.64 6.72 ± 0.86

96.5 ± 1.64 10.48 ± 0.85*

95.17 ± 2.93 12.44 ± 0.67*

TSN (106 cells/g testes)

29.03 ± 2.65

27.35 ± 2.51

26.32 ± 1.84

DSP (106 cells/g testes)

1.70 ± 0.27

1.68 ± 0.14

1.57 ± 0.15

Viability (%) Abnormalities (%)

Data are expressed as mean ± SD for 10 rats/group * p \ 0.05 compared with the control

There were no significant (p [ 0.05) differences in body weight gain and relative weights of testes, seminal vesicle, and prostate gland of the control and OMLL-exposed groups. However, the relative weights of the epididymis were significantly decreased in rats exposed to 25 % OMLL compared with the control group. Sperm Characteristics in OMLL-Exposed Rats Compared with the control group, acute exposure to OMLL resulted in a significant decrease in the epididymal sperm number (ESN) and sperm progressive motility at all doses. Conversely, sperm abnormalities were significantly increased in animals exposed to OMLL, whereas sperm viability, testicular sperm number (TSN), and DSP were not significantly affected in OMLL-exposed groups compared with the control (Table 2). Testicular Heavy-Biometal Concentrations The testicular levels of heavy metals in rats after exposure to OMLL for 7 days are shown in Fig. 1. Exposure to

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A 0.05

Control 12.5% OMLL 25% OMLL

*

* 0.8

*

* 0.02

ppm

0.03

Control 12.5% OMLL 25% OMLL

1.0

*

*

*

0.6

*

0.4

*

0.2

0.01

0.0

0.00 Cd

Fe Cu Biometals in the testes

Pb Ni Biometals in the testes

Control

Testicular Antioxidant Status

12.5% OMLL

24

9

After acute exposure to OMLL, GSH levels were not significantly affected in all of the treatment groups. However, levels of H2O2 and MDA were significantly increased in testes of OMLL-exposed rats compared with the control group (Fig. 2). Moreover, short-term exposure to OMLL significantly decreased testicular SOD, CAT, and GPx activities but markedly increased GST activity compared with control group (Table 3).

6

Marker Enzymes of Testicular Function

*

21

Units/mg protein

B

*

0.04

ppm

Fig. 1 Levels of Cd, Pb, Ni, Fe, and Cu in testes of rats after 7 days of exposure to OMLL. Each bar represents mean ± SD of 10 rats. *Values differ significantly from the control (p \ 0.05)

25% OMLL

*

18 15

* *

12

3 0 GSH

H2O2

MDA

End points in testes Fig. 2 Biomarkers of oxidative stress, namely, GSH, H2O2, and MDA levels, in testes of rats after 7 days of exposure to OMLL. Each bar represents mean ± SD of 10 rats. *Values differ significantly from the control (p \ 0.05)

OMLL resulted in a significant dose-dependent increase in levels of heavy-metal accumulation in testes of experimental rats compared with the control group. The trend of accumulation of the metals in the testes was Pb [ Cd [ Ni [ Fe [ Cu.

Figure 3 shows the activities of marker enzymes of testicular function after OMLL exposure for 7 days. Exposure to OMLL was associated with a significant increase in testicular activities of LDH-X, GGT, and G6PD compared with the control group (Fig. 3). Exposure to OMLL Decreased Plasma Hormonal Levels in Rats The influence of OMLL exposure on plasma concentrations of LH, FSH, prolactin, testosterone, TSH, T3, and T4 in the experimental rats are presented in Figs. 4 and 5. Exposure to OMLL elicited a dose-dependent effect on hormonal homeostasis in the experimental rats. Compared with the control group, acute OMLL exposure resulted in a marked

Table 3 Testicular antioxidant enzymes activities after OMLL exposure for 7 days in rats End points SOD (U/mg protein)

Control

12.5 % OMLL

25 % OMLL

1.44 ± 0.08

1.03 ± 0.10*

0.91 ± 0.07*

CAT (lmole H2O2 consumed/min/mg protein)

215.59 ± 15.69

194.07 ± 17.06*

168.01 ± 12.83*

GPX (U/mg protein)

157.82 ± 6.09

132.21 ± 7.02*

128.89 ± 6.97*

0.64 ± 0.08

0.83 ± 0.05*

0.88 ± 0.07*

GST (lmole CDNB–GSH complex formed/min/mg protein) Data are expressed as mean ± SD for 10 rats/group * p \ 0.05 against control

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79 Control

* 36

12.5% OMLL 25% OMLL

*

32

24

*

* *

*

20

Discussion

16 12 8 4 0 LDH

GGT

G6PD

Marker enzymes of testicular function Fig. 3 Marker enzymes of testicular function, namely, LDH, GGT, and G6PD, in testes of rats after 7 days of exposure to OMLL. Each bar represents mean ± SD of 10 rats. *Values differ significantly from the control (p \ 0.05)

decrease in the circulatory concentrations of LH, FSH, prolactin, testosterone, TSH, T3, and T4 levels. Histopathological Observations of Testes and Epididymides Figure 6 shows the representative histopathological findings in the testes and epididymides from all of the experimental groups. Control animals showed testicular architecture with normal interstitial cells, seminiferous epithelium, spermatogonia, spermatocytes, spermatids, and spermatozoa. Slight interstitial oedema, with focal area of sloughing of cellular debris into the lumen of some tubules, was observed in testes of rats exposed to 12.5 % OMLL. Vacuolation of the germinal epithelia cells, along with interstitial congestion and necrosis of tubules, was observed at 25 % OMLL. Moreover, epididymis of the control rats showed normal

Control 12.5% OMLL 25% OMLL

A 16

B

14 12

Control 12.5% OMLL 25% OMLL

6 5

* *

10

ng/ml

Fig. 4 Plasma concentrations of LH, FSH, prolactin, and testosterone in rats after 7 days of exposure to OMLL. Each bar represents mean ± SD of 10 rats. *Values differ significantly from control (p \ 0.05)

In the present study, heavy-metal analyses showed accumulation of Cu, Cd, Fe, Ni, and Pb in testes of OMLLexposed animals. Many redox- and nonredox-reactive metals are known to cause oxidative stress leading to hydrogen peroxide accumulation and LPO in the cells (Schutzendubel et al. 2001). It is well known that the accumulation of transition metals, such as Cu and Fe, favours reactive oxygen species (ROS) generation by way of Fenton-type chemistry. Cd exposure decreases male fertility by inducing oxidative stress, germ cell death, and inhibition of testicular steroidogenesis (Siu et al. 2009). Ni has also be reported to induce apoptosis by way of different pathways including generation of ROS and activation of caspase proteins (Au et al. 2006). Exposure to Pb induces oxidative stress and decreases sperm quality and steroidogenic enzymes activities in testes of rats (Sainath et al. 2011). Accumulation of these toxic heavy metals suggests their roles in testicular toxicity observed in OMLL-exposed animals in the present study. Furthermore, activities of some marker enzymes of testicular function were determined to show the influence of acute exposure to OMLL in the present study. GGT is a marker enzyme of Sertoli cell function. The activity of this enzyme varies inversely with sperm number and its maturation (Pant and Srivastava 2003). The increased GGT activity observed in the present study is characteristic of testicular atrophy associated with damaged Sertoli cells (Pant and Srivastava 2003). G6PD is a key marker enzyme

*

8 6

*

4

ng/ml

Units/mg protein

28

epithelial lining of the epididymal tubules with abundant sperm in the lumen. Epididymis of 12.5 % OMLL-treated rats showed mild erosion of the epithelial lining with oedema. Epididymis of rats exposed to 25 % OMLL showed severely disrupted epithelium with some parts of the epithelium sloughing off the epididymal tubules.

3

* *

*

*

2

4

1

2

0

0

LH

FSH

End points in plasma

Prolactin

Testosterone

End points in plasma

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Control 12.5% OMLL 25% OMLL

7.5

*

*

Units/dL

6.0

4.5

*

* 3.0

*

*

1.5

0.0 TSH

T3

T4

End points in plasma Fig. 5 Plasma concentrations of TSH, T3, and T4 in rats after 7 days of exposure to OMLL. Each bar represents mean ± SD of 10 rats. *Values differ significantly from control (p \ 0.05)

of testicular function that supplies reducing equivalents (NADPH) for the hydroxylation of steroids necessary for spermatogenesis. Although the increase in testicular G6PD activity observed in the OMLL-exposed rats probably suggests homeostatic adjustments in the GSH level by way of an increase in NADPH biosynthesis, excessive NADPH production could also trigger ROS generation by increasing NADPH oxidase activity, thus resulting in oxidative stress (Farombi et al. 2010). LDH-X is associated with survival

and maturation of germ cells as well as adenosine triphosphate production, which is required for spermatogenesis (Erkkila et al. 2002). The increase in testicular LDH-X activity observed in the present study may therefore indicates an adaptive mechanism by testes to mitigate germ cell death and azoospermia in OMLL-exposed rats. In the present study, the antioxidant status of testes was assessed by measuring the levels of hydrogen peroxide and MDA along with some endogenous antioxidants which have significant roles as suppressors or scavengers of free radicals. Our data clearly showed that SOD and CAT activities were significantly decreased in testes after acute exposure to OMLL. SOD is a family of metalloenzymes that is known to accelerate the conversion of endogenous cytotoxic superoxide radicals to H2O2. The elimination of H2O2 is either effected by CAT or GPx with the latter predominating in the case of testes (Farombi et al. 2010). The decreased activity of testicular SOD may suggest its inhibition by the increased H2O2 level resulting from decreased CAT activity. GSH, together with GPx, participates in the glutathione redox cycle by converting H2O2 and lipid peroxides to nontoxic products (Sanocka and Kurpisz 2004). GST is directly responsible for the elimination of electrophilic oxidants at the expense of GSH (Habig et al. 1974). In the present study, OMLL exposure significantly decreased GPx activity but markedly increased GST activity without affecting GSH level in testes of experimental rats. This observation possibly indicates that the detoxification process mediated by these enzymes was induced along with a

Control

12.5% OMLL

25% OMLL

Control

12.5% OMLL

25% OMLL

Testes

Epididymis

Fig. 6 Upper panel represents photomicrographs of testes from control and OMLL exposed rats. Control testes showing normal architecture. Slight interstitial oedema with focal area of sloughing of cellular debris (black arrow) into the lumen of some tubules at 12.5 % OMLL. Vacuolation of the germinal epithelia cells along with interstitial congestion and necrosis of tubules (chevron) at 25 %

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OMLL. Lower panel represents photomicrographs of epididymides from control and OMLL exposed rats. Control epididymides showing normal architecture. Mild erosion of the epithelial lining with oedema (arrowhead) at 12.5 % OMLL. Severe disrupted epithelium with some parts of the epithelium sloughing off the epididymal tubules (red arrow) at 25 % OMLL (Color figure online)

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compensatory synthesis of GSH by way of the GSH cycle. However, the increased levels of H2O2 and MDA in testes of OMLL-exposed rats showed that OMLL exposure increased the generation of ROS that exceeded the physiological capacity of the antioxidant system. ROS species attack cellular components involving polyunsaturated fatty acid residues of phospholipids to produce peroxyl radicals, which can be rearranged by way of a cyclization reaction to endoperoxides (precursors of MDA), with the final product of the peroxidation process being MDA (Marnett 1999). Testicular damage due to OMLL-induced oxidative stress was confirmed by light microscopy, which showed treatment-related testes histopathology in the treated animals. Excessive LPO is capable of disrupting testicular structure and function. The results of the present investigation showed that shortterm exposure to OMLL resulted in significant decrease in fluid intake without affecting weight gain in the experimental animals. The reason for the decrease in fluid intake could not be explained presently; however, a previous study from our laboratory indicated that the physicochemical characteristics of OMLL were significantly greater than acceptable limits by regulatory authorities (Farombi et al. 2012). An increase in the physicochemical characteristics above the normal values can cause severe degradation of groundwater quality and preclude its use for domestic water supply purposes (Lee and Jones-Lee 1996) including drinking. It is interesting to note that whereas exposure to OMLL did not significantly affect the relative weights of testes, seminal vesicles, and prostate glands, the relative weight of epididymis at 25 % OMLL exposure was significantly decreased compared with control animals. Moreover, OMLL exposure decreased ESN and sperm motility but increased sperm abnormalities without affecting sperm viability, TSN, and DSP in rats. Our findings suggest that although the observed testicular damage was not sufficient to impair spermatogenesis within the time of this investigation, OMLL exposure adversely affected the sperm store in epididymal compartment, which could have caused the decreased epididymal weight observed at 25 % OMLL exposure. The hypothalamic–pituitary–gonadal axis regulates development, reproduction, and aging in both animals and humans. Gonadal steroids act on the hypothalamus to regulate gonadotropin-releasing hormone pulses and at the pituitary level to regulate gonadotropin (LH and FSH) secretion. FSH, together with LH, regulates the process of spermatogenesis by suppressing proapoptotic signals and consequently promotes spermatogenic cell survival (O’Shaughnessy et al. 2010). LH is the primary tropic hormone required for the stimulation of Leydig cell to synthesize and secrete testosterone during spermatogenesis, whereas prolactin enhances the sensitivity of LH receptors in Leydig cells. In the present investigation, a

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significant decrease in plasma concentrations of testosterone, LH, and FSH was observed in OMLL-exposed rats. The diminution in the level of prolactin could lead to decreased sensitivity of LH receptors to circulating LH and consequently result in the decreased testosterone levels observed in OMLL-exposed rats. Thyroid hormones are vital in the physiological regulation of testicular basal metabolic activity (Maran 2003). Thyroid hormones reportedly influence the testicular twin functions of steroidogenesis and spermatogenesis (Wagner et al. 2008). Moreover, the present study showed that acute exposure to OMLL significantly decreased the plasma levels of TSH, T3, and T4 in the experimental animals. The hypothyroidism observed in the OMLL-exposed rats may have lead to a decrease in SHBG levels and consequently contribute to the decrease in the circulatory concentrations of plasma testosterone LH and FSH. Hypothyroidism adversely affects fertility by compromising semen volume and progressive sperm motility (Krajewska-Kulak and Sengupta 2013).

Conclusion Our data evidently showed that OMLL exposure elicits detrimental effects on testes functions in rats. Testicular toxicity of OMLL may be due to oxidative stress resulting from heavy-metal accumulation. Moreover, exposure to OMLL suppressed the gonadotropins and adversely affected the fine coordination between the hypothalamus–pituitary– gonad and hypothalamus–pituitary–thyroid axes. It should be noted, however, that other unidentified constituents, such as microbes, could be contributory factors to the observed effects in OMLL-exposed rats. Inappropriate methods employed in waste-management practices in Nigeria create the potential for a negative impact on the male reproductive health of the residents who may be exposed to leachatecontaminated substances such as water. In general, the public health impact of indiscriminate disposal of wastes into the environment needs attention. Acknowledgments This work was supported in part by Multidisciplinary Research Grants under the Staff Training and Research Capacity Building Programme of the John D. and Catherine T. MacArthur Foundation Grant (USA) endowment from the University of Ibadan, Nigeria, awarded to E. O. Farombi. The technical assistance of Omoko Ejiro of the Department of Veterinary Surgery and Reproduction, University of Ibadan, is gratefully appreciated by the authors.

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