Protective Effects of Melatonin Against Formaldehyde-Induced Oxidative Damage and Apoptosis in Rat Testes: An Immunohistochemical and Biochemical Study

Abstract

This study investigated the protective effects of melatonin against formaldehyde-induced oxidative damage and apoptosis in rat testes. A total of 21 male Wistar rats were divided into three groups. Group I was used as a control, Group II was injected every other day with formaldehyde for 1 month, whereas Group III was injected every other day with formaldehyde and melatonin for 1 month. At the end of the experimental period animals were sacrificed and the testes removed and dissected from the surrounding tissues for immunohistochemical evaluation. In addition, the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) were determined. The levels of SOD and GSH-Px decreased significantly, whereas the level of MDA significantly increased in animals treated with formaldehyde compared with the controls. Apoptosis of spermatogenetic and Leydig cells of testicular tissues was observed. In contrast, rats with melatonin SOD and GSH-Px enzyme activity increased whereas MDA levels decreased with formaldehyde exposure along with apoptosis. In view of the present findings, it is suggested that melatonin treatment may prevent formaldehyde-induced oxidative damage and apoptosis in rat testes.

Keywords:

• apoptosis, formaldehyde, melatonin, oxidative damage, testes

Abbreviations
FA	=	formaldehyde
SOD	=	superoxide dismutase
GSH-Px	=	glutathione peroxidase
MDA	=	malondialdehyde
MEL	=	melatonin

Introduction

Formaldehyde (FA), a member of the aldehyde family and one of the simplest organic molecules, is a colorless, pungent, and irritant gas. It is commonly used in hospitals, laboratories, and industrial settings. FA is frequently found in the domestic environment (e.g. paint, plywood, fabrics, cosmetics, heating and cooking emissions, etc.) [Simith [1992]; Franklin et al. [2000]; Zhou et al. [2006]; Usanmaz et al. [2002]]. Everyone may be exposed to it and exposure levels can vary with occupations such as anatomists and medical students, where levels of exposure to FA vapor during dissection sessions can be significant [Chohen et al. [1998]].

FA is considered toxic over certain doses and the risk of harmful effects is increased at room temperature because of its volatility [Ozen et al. [2003]; Songur et al. [2003]]. FA can react with different macromolecules such as nucleic acid and proteins, or with substances of low molecular weight such as amino acids [Cheng et al. [2003]; Metz et al. [2004]]. It is a highly water soluble compound which can rapidly penetrate into various tissues [Gurel et al. [2005]].

FA exerts an acutely irritating and allergic effect, primarily on the eyes, the upper and lower airways, and the skin. It has been shown that FA is mutagenic and carcinogenic in experimental animals [Kilburn et al. [1987]; Monteiro Riviere and Popp, [1986]; Chang et al. [1983]; Feron et al. [1988]]. Studies have also implicated detrimental effects of FA on germinal cells [Pradip and Vijay [1995]; Thrasher and Kilburn [2001]]. It has also been reported to cause primary and secondary infertility in both sexes [Pradip and Vijay [1995]; Collins et al. [2001]]. It disrupts the menstrual functions in women, and because of its teratogenic potential, it endangers gestation and embryonic development [Halperin et al. [1983]]. In experimental studies, FA exposure has been reported to cause histopathological changes in the testicular tissue and reduce sperm counts as well as blood testosterone levels [Chowdhury et al. [1992]; Majumder and Kumar [1995]; Tang et al. [2003]; Ozen et al. [2005]; Zhou et al. [2006]]. Reports of congenital and non-congenital pathologies such as spontaneous abortus, anemia, and babies with low-birth rate have been made [Taskinen et al. [1994]]. However, immunohistochemical studies on the apoptotic changes in testicular tissue associated with FA toxicity have remained largely unexplored.

Melatonin (MEL) is an endogenous neurohormone released by the pineal gland. This hormone possesses lipophilic and hydrophilic properties, and easily penetrates all biological membranes including both the placenta and the blood-brain barrier [Menendez-Pelaez et al. [1993]; Shida et al. [1994]; Costa et al. [1995]; Okatani et al. [1998]]. MEL is known to be involved in a variety of physiological processes including the regulation of endocrine rhythms [Forsling et al. [1993]], neuroprotective effects [Zararsiz et al. [2007]], and stimulation of the immune function [Guerrero and Reiter [1992]]. There is also evidence that MEL may regulate smooth muscle tone [Ayar et al. [2001]]. In addition to these functions, many recent studies have shown that MEL functions effectively as an antioxidant, i.e. a hydroxyl radical and a peroxyl radical scavenger [Kus et al. [2005]; Ogeturk et al. [2005]]. It has also been shown that when animals and tissues are subjected to lipid peroxidation, MEL affords substantial protection against the oxidative destruction of lipids [Longoni et al. [1998]]. However, to the best of our knowledge, there are no experimental studies that ascertain the protective effects of MEL against apoptosis and oxidative damage in testicular tissue induced by FA. To this end, this experimental study immunohistochemically evaluated apoptosis developing in the testicular tissue as a function of FA toxicity. The potential protective effects of MEL against apoptosis and oxidative damage associated with FA toxicity were investigated.

Results and discussion

Biochemical Analysis

For this study, 21 male Wistar rats were divided into three groups. Group I was used as a control, Group II was injected every other day with formaldehyde for 1 month, and Group III was injected every other day with formaldehyde and melatonin for 1 month. At the end of the experimental period animals were sacrificed and the testes removed for evaluation. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) levels were determined using a spectrophotometric assay. The levels of SOD and GSH-Px, which are antioxidant enzymes, were significantly reduced in the animals treated with FA as compared to the control group (p < 0.001). In comparison, the levels of MDA, an important parameter in determining tissue damage and an indicator of lipid peroxidation, were significantly increased in the rats that were given FA compared to the control group (p < 0.001) (Table 1). The levels of SOD and GSH-Px in animals exposed to FA and MEL were significantly increased compared to those treated with FA alone, while the level of MDA of the same group significantly decreased (p < 0.001) (Table 1).

Testicular MDA, SOD and GSH-Px Values in All Groups

Parameters	Control	FA	FA+MEL
MDA (nmol/g)	6.1±0.6	13.7±0.8*	7.3±0.5
SOD (U/g)	263.8±2.7	148.4±3.2*	242.5±3.9
GSH-Px (U/g)	237.4±3.1	77.5±5.3*	198.2±5.6

Values are expressed as means±SD. n=7 per group. FA, formaldehyde; MEL, melatonin.

*p < 0.001 vs. other groups.

Immunohistochemical Analysis

The findings are summarized in Table 2 and shown in Figure 1. The testicular tissue sections of the rats in all three groups were evaluated for apoptosis using Bax antibody staining. The evaluation was performed considering the severity of the reactions detected by this method, which is based on staining in order to render the Bax protein visible in the testicular tissue. Immunolocalization of Bax in spermogenetic cells was evaluated in both spermatogonia which line the basal lamina and spermatides in the adluminal compartment of the seminiferous epithelium. Bax staining (0.00±0.00) was absent in both the spermogenetic cells in the seminiferous tubules and the Leydig cells in the interstitial areas (Table 2 and Fig. 1A, B) of testicular tissue specimens in Group I, the control group. In Group II, heavy Bax staining was detected in the cytoplasm of the spermogenetic (2.87±0.41) and Leydig cells (2.95±0.52) (Table 2), (Fig. 1C, D). Whereas minimal Bax staining was observed in the cytoplasm of the spermogenetic (0.65±0.53) and Leydig cells (0.75±0.55) in Group III (Table 2), (Fig. 1E).

Grading Immunoreactive Bax in Cytoplasm of Spermatogenetic and Leydig Cells. Density of Immunohistochemical Staining was Graded from 0 to 3+ for Each Group

Treatment	Groups	Spermatogenetic cells	Leydig cells
Control	I	0.00±0.00	0.00±0.00
FA	II	2.87±0.41	2.95±0.52
FA+MEL	III	0.65±0.53	0.75±0.55
p values	I–II	0.001	0.001
	I–III	0.019	0.021
	II–III	0.002	0.002

Differences between two groups were determined with Mann–Whitney U test. Values are expressed as means±SD. N=7 per group. FA, formaldehyde; MEL, melatonin.

FIGURE 1 Bax localization in the rat testes. Immunohistochemically, Bax-stained cells were not observed in the seminiferous tubules of control rats. Section of testicular tissue from control group (A). No Bax staining was in the cytoplasm of Leydig cells (B). In rats exposed to FA, heavy Bax staining was detected in the cytoplasm of spermatogenetic cells (C) (arrows). Due to FA exposure, heavy Bax staining was observed in the cytoplasm of Leydig cells (D) (arrows). The density of immunohistochemical Bax staining was minimal in the cytoplasm of spermatogenetic (thin arrow) and Leydig cells (large arrow) in rats treated with FA and MEL (E). A preimmune control for Bax (F). Magnification×40.

Living organisms are protected against oxidative damage by enzymatic or non-enzymatic antioxidant systems and molecules. SOD and GSH-Px are among the enzymatic antioxidant systems effective at the cellular level [Tang et al. [1997]]. Both SOD and GSH-Px enzyme activities were significantly reduced in the testicular tissue samples of the animals that were given FA. This is suggestive of the damage inflicted by FA on the antioxidant defense mechanism.

MDA is one of the products formed after lipid peroxidation and is commonly used as a marker of oxidative damage [Kamal et al. [1989]]. In the present study, the level of MDA in animals given FA significantly increased. The increase in MDA is consistent with the view that FA induced lipid peroxidation increases oxidative damage in the testes. Zhou et al. [[2006]], have shown that FA inhalation leads to oxidative damage in the testicular tissue and exposure to FA lowers the level of both SOD and GSH-Px, while increasing MDA. Similarly, Tang et al. [[2003]] showed that the level of MDA increases in testicular tissues of the rats that were administered FA intraperitoneally for 5 days. The biochemical findings presented in this study are in agreement with the results of Zhou et al. [[2006]] and Tang et al. [[2003]] with respect to oxidative damage inflicted by FA exposure. This is similar to the oxidative damage observed in various tissues due to FA exposure [Zararsiz et al. [2006a],[b]]. This parallels other studies that associated changes in the morphological structure of the testes with FA exposure [Chowdhury et al. [1992]; Tang et al. [2003]; Ozen et al. [2005]; Zhou et al. [2006]]. In our previous study, we determined that testicular weights, diameters of seminiferous tubules and blood testosterone levels decreased following FA inhalation in rats [Ozen et al. [2005]]. Similarly, Zhou et al. [[2006]] showed seminiferous tubular atrophy in the rats that inhaled FA for 2 weeks. In contrast, Majumder and Kumar [[1995]] reported lower testicular DNA levels in the rats that were administered FA at a dose of 10 mg/kg for one month. In other experimental studies, FA has been reported to lower sperm counts [Chowdhury et al. [1992]; Majumder and Kumar [1995]; Tang et al. [2003]; Zhou et al. [2006]].

Apoptosis is the genetically regulated form of cell death (programmed cell death) that permits the safe disposal of cells when they are damaged or have fulfilled their intended biological function. Mitochondria play an important role in the apoptotic process. Death signals cause an increase in the permeability of the outer mitochondrial membrane, which in turn causes apoptosis. Some proteins regulate the permeability of the outer membrane of mitochondria. The most important of them are the proteins of the Bcl-2 family. Some of the proteins in this family are pro-apoptotic, whereas some are anti-apoptotic. Bax is a pro-apoptotic protein and it causes cytochrome c release into cytoplasm from the mitochondrial membrane. Upon release, cytochrome c starts the apoptotic process by activating caspase in the cytoplasm. Bcl-2 is an anti-apoptotic protein that inhibits the release of cytochrome c by preventing the insertion of Bax into the mitochondrial membrane.

In summary, the observed immunohistochemical Bax staining in cytoplasm of cells of the spermatogenic lineage and Leydig cells in response to FA treatment is indicative of apoptosis [Walker et al. [1988]; Nagata [1997]; Lu et al. [2000]; Debatin [2004]]. This is consistent with a negative impact on sperm production following decreased testosterone levels. Thus, the immunohistochemical findings of our study are parallel with the results of previous histological studies. MEL hormone, which is expressed by the pineal gland, acts in regulation of many physiological functions. In addition, it has been shown to be a very potent antioxidant [Stastica et al. [1998]; Zang et al. [1998]] and prevent oxidative damage due to lipid peroxidation [Sewerynek et al. [1995]; Longoni et al. [1998]]. In addition to its strong antioxidant effect, it stimulates antioxidant enzymes such as SOD, GSH-Px, and glutathione reductase [Reiter et al. [1997]].

Recent studies on various tissues have emphasized the antioxidant property of MEL and the protection provided by the MEL hormone against tissue damage due to oxidative damage [Kabuto et al. [1998]; Tan et al. [1998]; Arslan et al. [2002]; Sener et al. [2003]; Erol et al. [2004]; Topal et al. [2004]]. The MEL hormone has been reported to prevent apoptosis development in various tissues [Mayo et al. [1998]; Yoo et al., [2002]; Pedreanez et al. [2004]; Juknat et al. [2005]]. For example, in vitro [Kabuto et al. [1998]], MEL prevented oxidative damage induced by iron in the brain cortex. Similarly, Tan et al. [[1998]] have shown protection provided by MEL against neuronal damage induced by cyanic acid in rat hippocampus. Erol et al. [[2004]] observed the increased level of brain cortex MDA following exposure to gamma-radiation was reduced and lipid peroxidation was prevented by MEL administration. In addition, experimental pulmonary tissue damage was prevented by MEL administration [Arslan et al. [2002]; Sener et al. [2003]; Topal et al. [2004]].

FA exposure leads to apoptosis in the spermogenetic cells on the walls of the seminiferous tubules and in the Leydig cells responsible for testosterone production. The levels of both SOD and GSH-Px in rats exposed to FA along with MEL increased, while their MDA levels had decreased. The biochemical and immunohistochemical evaluation of the effect of FA exposure on testicular tissue revealed that MEL affords some protection against oxidative damage and apoptosis in the testicular tissue.

Materials and methods

Animals and Treatments

Adult male Wistar rats (weighing 230–250 g, n=21) were used in this study. They were kept at a constant temp (21±1°C) and under controlled light conditions (light, 07.00–19.00 h). Food (standard pellet diet) and tap water were supplied ad libitum. All of the protocols were approved by the local ethics committee of the Medical School.

The animals were divided into three equal groups. The rats in Group I (n=7) were designated as control. The rats in Group II (n=7) were injected with FA (10 mg/kg, body weight, intraperitoneally) every other day (at 04:00 p.m.). The rats in Group III (n=7) received MEL every other day (at 05:00 p.m.) intraperitoneally (25 mg/kg, body weight) (Sigma, St. Louis, MO, USA) one hour later than FA. At the end of one-month experimental period, all rats were sacrificed by decapitation. The testes of rats were removed and dissected from the surroundings tissues. Some of the testicular tissue specimens were washed twice with cold saline solution, placed into glass bottles, labeled, and stored frozen (−30°C) for subsequent analysis. The remaining specimens were used for immunohistochemical evaluation.

Biochemical Analysis of Testicular Tissues

For biochemical analysis, the tissues were weighed and homogenized in four volumes of ice-cold 50 mM Tris-HCl, pH 7.4, buffer containing 0.05% Triton X-100 with a homogenizer (IKA Ultra-Turrax T 25 Basic) for 2 min at 13,000 rpm. All the procedures were performed at +4°C. The level of MDA was determined in the homogenate. Tissue homogenates were centrifuged at 5,000 × g for 60 min at +4°C to remove debris, and the clear supernatant fluids were separated and kept at −40°C until the enzyme activity was assessed.

Determination of SOD Activity

Total (Cu–Zn and Mn) SOD (EC 1.15.1.1) activity was determined based on the method of Sun et al. [[1988]] using the nitro blue Tetrazolium (NBT) reduction by the xanthine-xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the supernatant after 1 ml of ethanol-chloroform mixture (5:3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the amount of enzyme causing 50% inhibition in the NBT reduction rate. The SOD activity was expressed as U/g protein.

Determination of Glutathione Peroxidase Activity

Glutathione peroxidase (GSH-Px, EC 1.6.4.2) activity was measured by the method of Paglia and Valentine [[1967]]. The reaction contained NADPH, reduced glutathione (GSH), sodium azide and glutathione reductase and was initiated by the addition of H₂O₂, and the change in absorbance at 340 nm was monitored by a spectrophotometer. Activity was expressed as U/g protein.

Determination of MDA Level

The tissue MDA level was determined using a method of Esterbauer and Cheeseman [[1990]] based on reaction with thiobarbituric acid (TBA) at 90–100°C. In the TBA test reaction, MDA and TBA react to produce a pink pigment with an absorption maximum at 532 nm. The reaction was performed at pH 2–3 and 90°C for 15 min. The sample was mixed with two volumes of cold 10% (w/v) trichloroacetic acid to precipitate the protein. The precipitate was centrifuged and an aliquot of the supernatant was reacted with an equal volume of 0.67% (w/v) TBA in a boiling water-bath for 10 min. After cooling, the absorbance at 532 nm was determined. Results were expressed as nmol/g wet tissue, by reference to a standard curve prepared from measurements made with a standard solution (1,1,3,3-tetramethoxypropane).

Immunohistochemical Analysis

Specimens were fixed in neutral formalin solution (10%) then embedded in paraffin wax and cut into 5 μm sections. Paraffin sections were cleaned in xylene, hydrated and then placed in phosphate buffered saline (PBS; pH 7.6). Antigen retrieval was performed by boiling for 15 min in 10 mM citrate buffer. Sections were treated with 3% hydrogen peroxide for 5 min to quench endogenous peroxidase activity, rinsed with deionized water and then washed with PBS. Sections were incubated first with 1% pre-immune rabbit serum to decrease non-specific staining and then with a monoclonal antibody against Bax protein (Dako, Carpinteria CA, USA) at 23°C in a moist chamber for 1 h. Detection of the antibody was performed using a biotin-streptavidin detection system (Bio-Genex, San Ramon CA, USA) with 3-amino 9-ethyl carbazole (AEC) as chromogen (Dako, Carpinteria CA, USA). Sections were counterstained with Mayer's hematoxylin, dehydrated and then cover-slipped with Permount. The level of Bax (a marker protein of apoptosis) was based on staining in order to render Bax protein visible in spermatogonia and spermatids in the adluminal compartment of the seminiferous epithelium. Intensity of immunostaining was graded as follows: no staining (0), minimal (1+), moderate (2+), heavy (3+). Cytoplasmic intensity of immunostaining of spermatogenetic and Leydig cells was the primary criterion for semi-quantitative evaluation.

Statistical Analysis

The biochemical data (SOD, GSH-Px and MDA values) are expressed as means±standard deviations (SD). Statistical analyses employed SPSS, version 11.0 (SPSS, Chicago, IL, USA). The data were tested for normal distribution using the Kolmogorov-Smirnov test. Since the data was distributed normally, within group comparisons were carried out using one-way ANOVA followed by an LSD post-hoc test. Since the immunoreactive grading of Bax showed an abnormal distribution, immunohistochemical data were considered to be non-parametric. They were analyzed using Kruskal–Wallis H-test. Differences between two groups were determined with Mann–Whitney U test. The level of significance was set at p < 0.05.