Indium Acetate Toxicity in Male Reproductive System in Rats Kuo-Hsin Lee,1,2 Hsiu-Ling Chen,3 Chung-Man Leung,1,4 Hsin-Pao Chen,1,5 Ping-Chi Hsu1,6 1
Department of Safety, Health and Environmental Engineering, National Kaohsiung First University of Science and Technology, Kaohsiung 811, Taiwan
2
Department of Emergency Medicine, E-Da Hospital, I-Shou University, Kaohsiung 824, Taiwan
3
Institute of Occupational Safety and Hazard Prevention, Hung Kuang University, Taichung 43302, Taiwan
4
Department of Radiation Oncology, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan
5
Department of Surgery, E-DA Hospital, I-Shou University, Kaohsiung 824, Taiwan
6
School of Health Sciences, Faculty of Health and Medicine, The University of Newcastle, Callaghan NSW 2308, Australia
Received 22 December 2013; revised 18 June 2014; accepted 21 June 2014 ABSTRACT: Indium, a rare earth metal characterized by high plasticity, corrosion resistance, and a low melting point, is widely used in the electronics industry, but has been reported to be an environmental pollutant and a health hazard. We designed a study to investigate the effects of subacute exposure of indium compounds on male reproductive function. Twelve-week old male Sprague-Dawley rats were randomly divided into test and control groups, and received weekly intraperitoneal injections of indium acetate (1.5 mg/kg body weight) and normal saline, respectively, for 8 weeks. Serum indium levels, cauda epididymal sperm count, motility, morphology, chromatin DNA structure, mitochondrial membrane potential, oxidative stress, and testis DNA content were investigated. The indium acetate-treated group showed significant reproductive toxicity, as well as an increased percentage of sperm morphology abnormality, chromatin integrity damage, and superoxide anion generation. Furthermore, positive correlations among sperm morphology abnormalities, chromatin DNA damage, and superoxide anion generation were also noted. The results of this study demonstrated the toxic effect of subacute low-dose indium exposure during the period of sexual maturation on male reproductive function in adulthood, through an increase in C 2014 oxidative stress and sperm chromatin DNA damage during spermiogenesis, in a rodent model. V Wiley Periodicals, Inc. Environ Toxicol 31: 68–76, 2016.
Keywords: indium acetate; serum indium level; male reproductive toxicity; sperm function; sperm DNA damage
INTRODUCTION Correspondence to: P.-C. Hsu; e-mail: [email protected] Contract grant sponsor: National Science Council (NSC) of Taiwan. Contract grant number: 101–2221-E-327-014-MY3 Published online 5 July 2014 in Wiley (wileyonlinelibrary.com). DOI: 10.1002/tox.22022
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Indium, a metallic rare earth element belonging to Group IIIA, is a soft, silvery, white metal with superior ductility, malleability, and plasticity. Indium has been used for surface protection of metals and alloys. It shows a high optical transmittance in the visible light spectrum, and high electrical
C 2014 Wiley Periodicals, Inc. V
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TOXICITY OF INDIUM EXPOSURE ON MALE REPRODUCTION
conductivity. Indium tin oxide (indium oxide doped with tin; ITO) is a transparent, conductive oxide. In thin film form, ITO is extensively used to make transparent electrodes, with a wide range of optoelectronic applications. Production of ITO continues to be the leading end-use of indium and accounts for most global indium consumption. Indium compounds such as indium acetate are used to produce transparent, conductive ITO films by the dip-coating and vacuum-sputtering method. This method is primarily used for producing electrical conductors in a variety of flat-panel devices, most commonly liquid crystal displays (LCDs). Indium is thus an indispensable raw material for flat panel displays (FPDs) including LCDs, plasma display panels, and micro-display projections. Taiwan has become a major FPD manufacturer in Asia, with FPD-related industries also covering a wide range of other areas. In response to Taiwan’s industrial development and technical advancements, the consumption of indium and its compounds continues to grow annually to provide the raw materials for various commercial products. Until the beginning of the 1990s, because of a lack of information on the adverse effects of exposure to indium compounds on the health of humans or animals, indium compounds were considered ineffective as compared to other heavy metal compounds (Tanaka 2004). However, because of their increasing industrial use, occupational exposure to indium compounds has attracted increased attention. In 2003, the first case of interstitial pneumonia caused by occupational exposure to ITO was reported (Homma et al., 2003). Chronic pulmonary toxicity of indium compounds, especially indium arsenide (InAs), indium phosphide (InP), and ITO, has been demonstrated clinically (Tanaka et al., 2010). Furthermore, adverse effects on other organ systems have been investigated (Castronovo and Wagner, 1973; Conner et al., 1995; Asakura et al., 2009; Nagano et al., 2011). However, the data on reproductive toxicity arising from exposure to indium compounds are limited. Another study found that testicular toxicity resulted from repetitive intratracheal instillation of InAs and InP in hamsters for 2 years (Omura et al., 2000), whereas indium-induced testicular toxicity in rats is controversial (Omura et al., 1996). In light of the ever-increasing consumption of indium and its compounds, assessment of the related health hazards would provide an important reference for environmental and occupational health agencies when implementing the necessary precautionary measures and legislation governing its industrial use. The aims of this study, therefore, are to investigate the adverse effects of subacute indium exposure on the male reproductive system, and the mechanisms involved, using a rodent model.
MATERIALS AND METHODS Animals, Grouping, and Experimental Protocol Thirteen male Sprague Dawley rats were obtained at the age of postnatal day (PND) 42 from the National Laboratory
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Animal Center (Taipei, Taiwan). The animals were housed in a rodent vivarium in the Laboratory of Industrial Hygiene, National Kaohsiung First University of Science and Technology, and were acclimated until PND 84 under a 12-h light:12-h dark cycle and at controlled temperature. Laboratory Rodent Diet 5001 (LabDiet, Richmond, IN) and water were made available ad libitum. Before the experiment, the rats were randomized into two groups (seven rats in the indium acetate group and six rats in the control group). Each rat received one weekly dose of either indium acetate or normal saline for 8 weeks, to evaluate the effects of subacute exposure. On the first day of the experiment, the seven rats in the indium acetate group received an intraperitoneal injection of indium acetate solution at a dosage of 1.5 mg/kg body weight, whereas the control group received an equivalent volume (i.e. 1 mL/kg body weight) of intraperitoneal physiological saline. The animals were sacrificed on PND 150, which was the 16th day after the last intraperitoneal injection. In order to get a more stable internal dose of indium, the intraperitoneal injection route of exposure was used in a subcute rodent model. The administered dose in this study was based on the effective dose of indium compound (Conner et al., 1995). The rationale of choosing the timing for treatment is to simulate the effects of indium exposure during the period of sexual maturation on later reproductive system in adulthood.
Chemicals and Preparation Indium acetate was obtained from Alfa Aesare (A Johnson Matthey Company, Lancashire, United Kingdom) with a purity of more than 99.99% in powder form and slight solubility in water. Indium acetate solution was obtained by totally dissolving 30 mg indium acetate in 20 mL physiological saline.
Blood Collection Each rat provided 10 mL of blood drawn by needle aspiration from the heart into two vacuum blood-drawing tubes without any anticoagulant on PND 150. The blood samples were aimed to analyze serum indium (S-In) concentration and centrifuged at 3000 3 g for 15 min to separate blood cells and serum. Serum was stored at 285 C until the analysis of S-In.
Body and Reproductive Organ Weight The body weights of rats were recorded every day during the experiment from PND 84 to PND 150. The animals were sacrificed by CO2 and the paired testes, paired epididymis, and seminal vesicles were removed and weighed on PND 150. Relative organ weight was measured by calculating the ratio between organ weight and body weight. The left testis was used for DNA content analysis by flow cytometry
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(FCM). The left cauda epididymis was used to perform sperm chromatin structure analysis (SCSA). The sample of sperm suspension was measured for sperm count, motility, morphology, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) generation.
Sperm Motility Analysis The left cauda epididymis of each control and treated rat was removed and placed in a medium of 1 mL HTF, maintained at 34 C in an environment saturated with 5% CO2. After 5 min, the cauda epididymis was minced with curved scissors and the sperm was dispersed. After 20 min, the sperm was collected and transferred to a fresh tube. A volume of 10 lL aliquots of sperm suspension was placed in a pre-warmed Makler chamber (10 lm depth; Sefi-Medical Instruments, Haifa, Israel). Sperm motility was expressed as the ratio between the number of motile sperm and the total number of sperm.
Sperm Morphology Analysis The sperm suspension was diluted with phosphate-buffered saline before mixing with 1% aqueous eosin Y (10:1). The resulting mixture was set aside for 30 min. A drop of the mixture was placed on a clear slide and a uniform smear was made. Two samples were made for each rat. After air drying, the slides were briefly rinsed in methanol to remove excess stain, air dried again, and cover-slipped with mounting medium. Two hundred cells from each rat were examined for morphological abnormalities under a light microscope (4003) (Zeiss, Axioskop2, Germany). Abnormalities were classified as abnormal head (which included flattened head and pin head), abnormal neck, abnormal tail, or multiple abnormalities (which included tailless sperm) (Linder et al., 1990; Brown et al., 1994).
Sperm Chromatin Structure Assay (SCSA) The procedure of FCM SCSA has been described in detail (Evenson and Jost, 1994). Briefly, the left cauda epididymis was removed from each rat and placed in TNE buffer solution (0.15 M NaCl, 0.01 M Tris-HCL, 1 mM disodium EDTA, pH 7.4) in a Petri dish and minced. After allowing the tissue fragments to settle, the sperm suspension was filtered through a 0.2 mm nylon mesh into 2 mL cryogenic vials and mixed with glycerol to form a final solution of 10% (v/v) concentration. The solution was then frozen at 280 C. For SCSA analysis, the frozen samples were diluted to a concentration of 1 to 2 3 106 sperm/mL with TNE buffer solution; 50 lL of the diluted sample was put into a Falcon tube (Becton Dickinson Immunocytometry Systems, San Jose, CA) and 100 lL low-pH detergent solution was added (0.1% Triton X-100, 0.15 M NaCl, and 0.08 N HCl, pH 1.2). After 30 seconds, the cells were stained with 0.3 mL of acridine orange (AO) staining solution containing
Environmental Toxicology DOI 10.1002/tox
6 mg/L of AO in staining buffer (0.1 M citric acid, 0.2 M Na2HPO4, 1 mM EDTA, 0.15 M NaCl, pH 6.0). After 3 min of staining, the sample was analyzed using a FACScan FCM (Becton Dickinson Immunocytometry Systems, San Jose, CA). A total of 10,000 spermatozoa were collected and analyzed at a flow rate of 100 to 200 cells/s. AO is a metachromatic fluorochrome used to differentially stain double- and singlestranded nucleic acids. When excited by blue light at 488 nm, AO intercalates into double-stranded DNA to give a green fluorescence (FL1, native DNA). If, however, DNA is damaged, AO attaches to single-stranded nucleic acids and exhibits a red fluorescence (FL3, denatured, single stranded DNA). In SCSA analysis, the metachromatic shift from green to red fluorescence is expressed as a ratio between red and total fluorescence (red and green), and referred to as the DNA fragmentation index (DFI). SCSA variables in the present study were mean DFI, standard deviation (SD) DFI, and the percentage of cells outside the main population (% DFI). Data calculations were further performed using FCS Express software version 2 (De Novo Software, Thornhill, Ontario, Canada).
Sperm Mitochondrial Membrane Potential (MMP) The lipophilic cation with dual emission fluorophore JC-1 was used to measure the MMP (Garner et al., 1997; Gravance et al., 2001).When excited by blue light at 488 nm, the JC-1 molecule remains in its monomeric form. After passing through the mitochondrial membrane, it exhibits green fluorescence at 530 nm, representing low potential (inactivity or death). If the JC-1 molecule transforms into the J-aggregate form, it exhibits an orange fluorescence at 590 nm, indicating high membrane potential (high activity). Concentrations of 1.53 mM JC-1 stock solution were prepared in DMSO. The sperm suspension was diluted to 1 to 2 3 106 sperm/mL and stained with JC-1 to a final stain concentration of 3.0 lM. The samples were set aside for 10 min at 34 C, and then analyzed using a FACScan FCM. A total of 10,000 spermatozoa were collected and analyzed at a flow rate of 100 to 200 cells/s. The percentage of orange stained (mitochondrial-active sperm) cells was recorded.
Sperm Reactive Oxygen Species (ROS) Generation The sperm hydrogen peroxide (H2O2) level in most cells can be measured using a fluorescent probe, 2,7-dichlorofluorescin (DCFH-DA). To do this, H2O2 is incorporated into hydrophobic lipid regions of the cell (Bass et al., 1983). The acetate moieties are cleaved off leaving the non-fluorescent DCFH-DA. DCFH-DA is a stable compound that passively diffuses into cells and is hydrolyzed by intracellular esterase to yield DCHF, which is trapped inside cells. H2O2 produced by cells oxidizes DCFH to the highly fluorescent compound 2,7-dichlorofluorescein, which is fluorescent at 530 nm. The
TOXICITY OF INDIUM EXPOSURE ON MALE REPRODUCTION
DCFH-DA stock dissolves in absolute alcohol at 2.5 mM (1.2 mg/mL). We modified the method used by Fisher et al. (2005) to evaluate the H2O2 level in sperm. Sperm cells at a concentration of 1 to 2 3 106 sperm/mL were added into 2.5 mM DCFH-DA to a concentration of 12.5 mM. The mixture was set aside and maintained at 34 C for 30 min. The level of sperm superoxide anion (O22 •) was measured using a modification of a previously described method (Marchetti et al., 2002). The dihydroethidium (DHE) stock dissolves in DMSO at 0.33 mM, and can be directly oxidized into ethidium bromide by O22 • produced by sperm. Sperm cells at a concentration of 1 to 2 3 106 sperm/mL were added into 0.33 mM DHE to a concentration of 1.65 lM. The mixture was set aside and maintained at 34 C for 30 min. Intracellular H2O2 and O22 • levels were measured using a FACScan FCM with excitation and emission settings at 488 and 530 nm, respectively. We measured the percentage of cells with positive DHE and DCFH (which indicated positive ROS cells), and the data were expressed as the percentage of fluorescent spermatozoa (Mahfouz et al., 2009).
FCM DNA Content Analysis of Testis Cell Testis monocellular suspensions were prepared according to the procedure described by Spano et al. (1996). Briefly, the left testis was minced and treated with 1 mL 0.1% pepsinHCl solution, mixed by gentle magnetic stirring at room temperature for 10 min. These monocellular suspensions were then filtered through a 37 mm nylon mesh to separate the cells from the tissue. The resulting monocellular suspensions were stabilized with 4 mL of 95% ethanol and stored at 220 C for several weeks. For the FCM analysis, fixed samples were added to 0.5% pepsin-HCl solution to a 1:1 volume. After 10 min of gentle magnetic stirring, cells were immediately added to 50 lg/mL propidium iodide (PI) in 0.1 M Tris-HCl buffer solution (pH 7.5) containing 0.04 mg/mL DNAse-free RNAse. The fluorescence intensity of the DNA content of the testicular cells was measured using a FACScan FCM. PI fluorescent emissions were monitored using a 620 nm band-pass filter. A total of 2 3 104 cells were collected for each sample. Based on the DNA content, main germ cell peaks could be classified into four categories: (1) mature haploid (elongated spermatids; stages XXIV), (2) immature haploid (round and elongated spermatids; stages I-IX), (3) diploid (spermatogonia, secondary spermatocytes, tissue somatic cells), and (4) tetraploid (mostly primary spermatocytes). The region between the diploid and tetraploid peaks, called the S-phase, is composed of cells that actively synthesize DNA (Spano et al., 1996). The relative proportions of haploid, diploid, Sphase, and tetraploid cell types were calculated.
Analysis of S-In The S-In analysis was modified from methods of Hamaguchi et al. (2008). For pre-treatment, 0.25 mL of serum was
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diluted by 2.5 mL of 68 to 70% ultra-pure nitric acid (HNO3, GR for analysis, Merck) and 0.25 mL of 30% ultrapure hydrogen peroxide (H2O2, TAMAPURE-AA-100). After digestion by a 100 W/vessel microwave oven (Mars, microwave digestion system, CEM) (Liu et al., 2012), 0.1 mL 10 lg/L was added in each sample for external calibration. Finally, inductively-coupled plasma mass spectroscopy (ICP-MS, Perkin Elmer Sciex ELAN DRC, San Jose, CA) was used to analyze the serum indium concentration on PND 150.
Statistical Analysis Average values are expressed as mean 6 SD. All statistical comparisons were performed using the JMP statistical package (SAS Institute Inc., Gary, NC) for the unexposed control and the exposed groups. The body weights of the animals exposed to indium acetate and those of the control group were compared with Student’s t test, with P < 0.05 considered statistically significant. Welch’s test was adopted instead of Student’s t test if the variances of the two groups were not equal. The reproductive organ weights, sperm count, motility, morphology, SCSA, MMP, generation of H2O2 and O22 • as well as DNA content of testicular cells of the indium and the control groups were compared using a nonparametric Wilcoxon rank sums test, with P < 0.05 considered statistically significant. The relationships between sperm morphology, DFI, and O22 • were evaluated using general linear regression analysis.
RESULTS Body and Reproductive Organ Weights A significant reduction in mean body weight was noted in the indium-exposed animals compared to those in the control group from PND 113 to PND 143 (Fig. 1). There was a significant decrease of absolute seminal vesicle weight in indium exposed group at PND 150 (Table I). However, there was no significant change in the seminal vesicle weight to body weight ratio between the two groups. Similarly, there were no significant differences in the absolute and relative weights of the body, paired testis, paired epididymis, and paired cauda epididymis between the two groups (Table I).
Serum Indium Levels, Sperm Count, Motility, and Morphology The mean S-In level in the exposed group (143.95 lg/L) was significantly increased as compared to the control group (0.30 lg/L) (Table II). There were also no significant differences in sperm count and motility between the two groups, as shown in Table II. There was a significant increase in the number of sperm with abnormal heads, abnormal necks, abnormal tails, and multiple abnormalities, and total
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was not present in the generation of H2O2 between the two groups (Table II).
DNA Content Analysis of Testis Cells There were no significant differences between the two groups in the testis DNA content among the four cell types (mature haploid, immature haploid, diploid, and tetraploid) and that of the S-phase cell (data not shown).
Relationships Among Abnormal Sperm Morphology, Mean DFI, % DFI, and ROS Generation Fig. 1. Body weight of each rat recorded daily after exposure to either indium acetate or normal saline and presented as means 6 standard deviation between the indium acetate and the control group. *Significant differences as compared with control group (P < 0.05).
morphological abnormalities in the indium-exposed group as compared to the control group (Fig. 2).
Regression analysis of the incidence of total morphologically abnormal sperm, mean DFI, % DFI, and percentage of positive DHE cell showed that the incidence of morphologically total abnormal sperm was positively and significantly associated with the generation of sperm O22 • (r 5 0.67, P 5 0.012), the % DFI (r 5 0.68, P 5 0.011) and the mean DFI (r 5 0.77, P 5 0.022) [Figs. 3 and 4(A,B)].
DISCUSSION FCM Analysis of SCSA, MMP, and ROS Generation Sperm samples from the cauda epididymis were analyzed by FCM SCSA to investigate possible indium acetate-induced damages in chromatin integrity. There were significant increases in % DFI and the DFI mean in the indium-exposed group, whereas there were no significant differences in the SD DFI and MMP between the two groups (Table II). To evaluate sperm ROS generation, we measured H2O2 and O22 • levels and found a significant increase in the percentage of positive DHE cells in the indium-exposed group compared to that of the control group. However, this difference
In this study we demonstrated the impact of subacute indium treatment on the reproductive functions of male rats and investigated the underlying mechanism of the effect of the indium. The results showed that prolonged subacute administration of indium acetate into male rats reduced body weight and absolute seminal vesicle weight, increased the percentage of sperm with abnormal morphology, augmented impairment of sperm chromatin integrity, and induced the generation of O22 •. We further found significant and positive correlations among abnormal sperm morphology, chromatin integrity damage, and O22 • generation. A study by Omura et al., on testicular toxicities of two intratracheally administered semiconductor materials,
TABLE I. Body and tissue weight between rats exposed to indium acetate and unexposed control at postnatal day 150 Parameters Body weight (kg) Absolute tissue weight Testis (g) Epididymis (g) Cauda epididymis (g) Seminal vesicles (g) Relative tissue weight Testis (g/kg B.W.) Epididymis (g/kg B.W.) Cauda epididymis (g/kg B.W.) Seminal vesicles (g/kg B.W.) All data was expressed mean 6 standard deviation. *P < 0.05 as compared with control group.
Environmental Toxicology DOI 10.1002/tox
Control (n 5 6)
Indium Acetate (n 5 7)
P
0.57 6 0.05
0.52 6 0.05
0.1004
3.25 6 0.28 1.10 6 0.05 0.48 6 0.64 2.04 6 0.22
3.06 6 0.96 1.11 6 0.08 0.51 6 0.29 1.57 6 0.21*
0.8303 0.9431 0.3531 0.0124
5.74 6 0.64 1.95 6 0.21 0.86 6 0.19 3.62 6 0.54
5.94 6 1.94 2.15 6 0.18 0.98 6 0.08 3.04 6 0.50
0.2246 0.1336 0.2840 0.1004
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TOXICITY OF INDIUM EXPOSURE ON MALE REPRODUCTION
TABLE II. Serum indium level, sperm quality, epididymal sperm chromatin structure assay (SCSA), mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation in rats exposed to indium acetate and unexposed control group Parameters Serum indium level (lg/L) Mean DFI (AU) % DFI SD DFI (AU) MMP (%) Sperm motility (%) Sperm count (106/ml) DHE, % (O22) DCFH-DA, % (H2O2)
Control (n 5 6)
Indium Acetate (n 5 7)
P
0.30 206.9 6 7.0 2.3 6 1 21.9 6 10.4 18.3 6 17.6 51.2 6 9.2 16.5 6 4.9 1.1 6 1.1 0.7 6 0.5
143.95* 227.7 6 15.1** 14.6 6 12.7** 29.3 6 11.9 9.8 6 10.6 43.6 6 3.8 17.3 6 5.2 3.5 6 1.5** 0.5 6 0.3
27% is associated with pregnancy failure in assisted reproductive technology (Larson et al., 2000; Larson-Cook et al., 2003). Our data demonstrated positive correlations among abnormal sperm morphology, chromatin integrity damage, and O22 • generation. Several studies have supported the hypothesis that abnormal sperm morphology is statistically associated with an increase in the incidence of chromosomal abnormalities (Calogero et al., 2001; Sun et al., 2006), and chromatin integrity damage (Fischer et al., 2003; Kubo-Irie et al., 2005). A great deal of evidence has indicated that most sperm chromatin integrity damage is associated with ROS generation. In 2010, Aitken and De Iuliis put forward a two-step hypothesis to explain the etiology of oxidative DNA damage in germ cells. Step 1 of the hypothesis suggests the generation of a state of oxidative stress in the testes that impairs spermiogenesis, leading to the generation of poorly remodeled chromatin. In Step 2, this vulnerable DNA is oxidatively attacked, possibly as a result of the generation of mitochondrial ROS as these vulnerable cells succumb to apoptosis (Aitken and De Iuliis, 2010). Nagano et al., (2011) found that the average concentrations of indium in testis tissues were less lower than lung tissues contents in male rats exposed to ITO at 0.1 mg/m3 for 26 weeks (0.006 mg/g for testis vs. 20.2 mg/g for lung). Another study showed that the presence of electron-dense deposits characteristic of indium in the lysosomes of testicular Leydig and Sertoli cells as well as sufferance in mitochondria of indium-treated rats with total dose of 28 mg/kg (Samira et al., 2011). The non-specific toxic effects might due to relatively high toxic dose of indium. In this study, the mean S-In level in the exposed group was 143.95 lg/L and it was approximately up to 48-fold higher than the biological exposure index value of 3 mg/L set by Japan Society for Occupational Health (JSOH, 2007).
TOXICITY OF INDIUM EXPOSURE ON MALE REPRODUCTION
Our study also had several limitations for human health risk evaluation: the different routes of exposure, absorption, metabolism, distribution, and excretion of indium between human beings and experimental animals. Although the limitations of the rodent model must be kept in mind when interpreting results, the findings herein may be used in assessing human and ecological risk associated with its production, use and disposal. In conclusion, this study showed that subacute indium treatment increased the percentage of sperm with abnormal morphology, enhanced damage of chromatin integrity, and augmented ROS generation. Moreover, the positive correlations among abnormal sperm morphology, chromatin integrity damage, and ROS generation were demonstrated. Base on the findings of the present study, which have not been previously reported, we hypothesize that indium acetate treatment may generate oxidative stress during spermiogenesis and cause sperm chromatin remodeling, thereby leading to abnormal sperm morphology and subsequent male infertility. Given the current popular use of indium compounds in electronics industries, the clinical implications warrant further elucidation.
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Mahfouz R, Sharma R, Lackner J, Aziz N, Agarwal A. 2009. Evaluation of chemiluminescence and flow cytometry as tools in assessing production of hydrogen peroxide and superoxide anion in human spermatozoa. Fertil Steril 92:819–827. Marchetti C, Obert G, Deffosez A, Formstecher P, Marchetti P. 2002. Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm. Hum Reprod 17:1257–1265. Massanyi P, Trandzik J, Nad P, Korenekova B, Skalicka M, Toman R, Lukac N, Halo M, Strapak P. 2004. Concentration of copper, iron, zinc, cadmium, lead, and nickel in bull and ram semen and relation to the occurrence of pathological spermatozoa. J Environ Sci Health A Tox Hazard Subst Environ Eng 39: 3005–3014. Nagano K, Nishizawa T, Umeda Y, Kasai T, Noguchi T, Gotoh K, Ikawa N, Eitaki Y, Kawasumi Y, Yamauchi T, Arito H, Fukushima S. 2011. Inhalation carcinogenicity and chronic tox-
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Spano M, Bartoleschi C, Cordelli E, Leter G, Segre L, Mantovani A, Fazzi P, Pacchierotti F. 1996. Flow cytometric and histological assessment of 1,2:3,4-diepoxybutane toxicity on mouse spermatogenesis. J Toxicol Environ Health 47:423–441. Sun F, Ko E, Martin RH. 2006. Is there a relationship between sperm chromosome abnormalities and sperm morphology? Reprod Biol Endocrinol 4:1. Tanaka A. 2004. Toxicity of indium arsenide, gallium arsenide, and aluminium gallium arsenide. Toxicol Appl Pharmacol 198: 405–11. Tanaka A, Hirata M, Kiyohara Y, Nakano M, Omae K, Shiratani M, Koga K. 2010. Review of pulmonary toxicity of indium compounds to animals and humans. Thin solid Films 518: 2934–2936. Xu DX, Shen HM, Zhu QX, Chua L, Wang QN, Chia SE, Ong CN. 2003. The associations among semen quality, oxidative DNA damage in human spermatozoa and concentrations of cadmium, lead and selenium in seminal plasma. Mutat Res 534:155–163.
Department of Safety, Health and Environmental Engineering, National Kaohsiung First University of Science and Technology, Kaohsiung 811, Taiwan
2
Department of Emergency Medicine, E-Da Hospital, I-Shou University, Kaohsiung 824, Taiwan
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Institute of Occupational Safety and Hazard Prevention, Hung Kuang University, Taichung 43302, Taiwan
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Department of Radiation Oncology, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan
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Department of Surgery, E-DA Hospital, I-Shou University, Kaohsiung 824, Taiwan
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School of Health Sciences, Faculty of Health and Medicine, The University of Newcastle, Callaghan NSW 2308, Australia
Received 22 December 2013; revised 18 June 2014; accepted 21 June 2014 ABSTRACT: Indium, a rare earth metal characterized by high plasticity, corrosion resistance, and a low melting point, is widely used in the electronics industry, but has been reported to be an environmental pollutant and a health hazard. We designed a study to investigate the effects of subacute exposure of indium compounds on male reproductive function. Twelve-week old male Sprague-Dawley rats were randomly divided into test and control groups, and received weekly intraperitoneal injections of indium acetate (1.5 mg/kg body weight) and normal saline, respectively, for 8 weeks. Serum indium levels, cauda epididymal sperm count, motility, morphology, chromatin DNA structure, mitochondrial membrane potential, oxidative stress, and testis DNA content were investigated. The indium acetate-treated group showed significant reproductive toxicity, as well as an increased percentage of sperm morphology abnormality, chromatin integrity damage, and superoxide anion generation. Furthermore, positive correlations among sperm morphology abnormalities, chromatin DNA damage, and superoxide anion generation were also noted. The results of this study demonstrated the toxic effect of subacute low-dose indium exposure during the period of sexual maturation on male reproductive function in adulthood, through an increase in C 2014 oxidative stress and sperm chromatin DNA damage during spermiogenesis, in a rodent model. V Wiley Periodicals, Inc. Environ Toxicol 31: 68–76, 2016.
Keywords: indium acetate; serum indium level; male reproductive toxicity; sperm function; sperm DNA damage
INTRODUCTION Correspondence to: P.-C. Hsu; e-mail: [email protected] Contract grant sponsor: National Science Council (NSC) of Taiwan. Contract grant number: 101–2221-E-327-014-MY3 Published online 5 July 2014 in Wiley (wileyonlinelibrary.com). DOI: 10.1002/tox.22022
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Indium, a metallic rare earth element belonging to Group IIIA, is a soft, silvery, white metal with superior ductility, malleability, and plasticity. Indium has been used for surface protection of metals and alloys. It shows a high optical transmittance in the visible light spectrum, and high electrical
C 2014 Wiley Periodicals, Inc. V
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TOXICITY OF INDIUM EXPOSURE ON MALE REPRODUCTION
conductivity. Indium tin oxide (indium oxide doped with tin; ITO) is a transparent, conductive oxide. In thin film form, ITO is extensively used to make transparent electrodes, with a wide range of optoelectronic applications. Production of ITO continues to be the leading end-use of indium and accounts for most global indium consumption. Indium compounds such as indium acetate are used to produce transparent, conductive ITO films by the dip-coating and vacuum-sputtering method. This method is primarily used for producing electrical conductors in a variety of flat-panel devices, most commonly liquid crystal displays (LCDs). Indium is thus an indispensable raw material for flat panel displays (FPDs) including LCDs, plasma display panels, and micro-display projections. Taiwan has become a major FPD manufacturer in Asia, with FPD-related industries also covering a wide range of other areas. In response to Taiwan’s industrial development and technical advancements, the consumption of indium and its compounds continues to grow annually to provide the raw materials for various commercial products. Until the beginning of the 1990s, because of a lack of information on the adverse effects of exposure to indium compounds on the health of humans or animals, indium compounds were considered ineffective as compared to other heavy metal compounds (Tanaka 2004). However, because of their increasing industrial use, occupational exposure to indium compounds has attracted increased attention. In 2003, the first case of interstitial pneumonia caused by occupational exposure to ITO was reported (Homma et al., 2003). Chronic pulmonary toxicity of indium compounds, especially indium arsenide (InAs), indium phosphide (InP), and ITO, has been demonstrated clinically (Tanaka et al., 2010). Furthermore, adverse effects on other organ systems have been investigated (Castronovo and Wagner, 1973; Conner et al., 1995; Asakura et al., 2009; Nagano et al., 2011). However, the data on reproductive toxicity arising from exposure to indium compounds are limited. Another study found that testicular toxicity resulted from repetitive intratracheal instillation of InAs and InP in hamsters for 2 years (Omura et al., 2000), whereas indium-induced testicular toxicity in rats is controversial (Omura et al., 1996). In light of the ever-increasing consumption of indium and its compounds, assessment of the related health hazards would provide an important reference for environmental and occupational health agencies when implementing the necessary precautionary measures and legislation governing its industrial use. The aims of this study, therefore, are to investigate the adverse effects of subacute indium exposure on the male reproductive system, and the mechanisms involved, using a rodent model.
MATERIALS AND METHODS Animals, Grouping, and Experimental Protocol Thirteen male Sprague Dawley rats were obtained at the age of postnatal day (PND) 42 from the National Laboratory
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Animal Center (Taipei, Taiwan). The animals were housed in a rodent vivarium in the Laboratory of Industrial Hygiene, National Kaohsiung First University of Science and Technology, and were acclimated until PND 84 under a 12-h light:12-h dark cycle and at controlled temperature. Laboratory Rodent Diet 5001 (LabDiet, Richmond, IN) and water were made available ad libitum. Before the experiment, the rats were randomized into two groups (seven rats in the indium acetate group and six rats in the control group). Each rat received one weekly dose of either indium acetate or normal saline for 8 weeks, to evaluate the effects of subacute exposure. On the first day of the experiment, the seven rats in the indium acetate group received an intraperitoneal injection of indium acetate solution at a dosage of 1.5 mg/kg body weight, whereas the control group received an equivalent volume (i.e. 1 mL/kg body weight) of intraperitoneal physiological saline. The animals were sacrificed on PND 150, which was the 16th day after the last intraperitoneal injection. In order to get a more stable internal dose of indium, the intraperitoneal injection route of exposure was used in a subcute rodent model. The administered dose in this study was based on the effective dose of indium compound (Conner et al., 1995). The rationale of choosing the timing for treatment is to simulate the effects of indium exposure during the period of sexual maturation on later reproductive system in adulthood.
Chemicals and Preparation Indium acetate was obtained from Alfa Aesare (A Johnson Matthey Company, Lancashire, United Kingdom) with a purity of more than 99.99% in powder form and slight solubility in water. Indium acetate solution was obtained by totally dissolving 30 mg indium acetate in 20 mL physiological saline.
Blood Collection Each rat provided 10 mL of blood drawn by needle aspiration from the heart into two vacuum blood-drawing tubes without any anticoagulant on PND 150. The blood samples were aimed to analyze serum indium (S-In) concentration and centrifuged at 3000 3 g for 15 min to separate blood cells and serum. Serum was stored at 285 C until the analysis of S-In.
Body and Reproductive Organ Weight The body weights of rats were recorded every day during the experiment from PND 84 to PND 150. The animals were sacrificed by CO2 and the paired testes, paired epididymis, and seminal vesicles were removed and weighed on PND 150. Relative organ weight was measured by calculating the ratio between organ weight and body weight. The left testis was used for DNA content analysis by flow cytometry
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(FCM). The left cauda epididymis was used to perform sperm chromatin structure analysis (SCSA). The sample of sperm suspension was measured for sperm count, motility, morphology, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) generation.
Sperm Motility Analysis The left cauda epididymis of each control and treated rat was removed and placed in a medium of 1 mL HTF, maintained at 34 C in an environment saturated with 5% CO2. After 5 min, the cauda epididymis was minced with curved scissors and the sperm was dispersed. After 20 min, the sperm was collected and transferred to a fresh tube. A volume of 10 lL aliquots of sperm suspension was placed in a pre-warmed Makler chamber (10 lm depth; Sefi-Medical Instruments, Haifa, Israel). Sperm motility was expressed as the ratio between the number of motile sperm and the total number of sperm.
Sperm Morphology Analysis The sperm suspension was diluted with phosphate-buffered saline before mixing with 1% aqueous eosin Y (10:1). The resulting mixture was set aside for 30 min. A drop of the mixture was placed on a clear slide and a uniform smear was made. Two samples were made for each rat. After air drying, the slides were briefly rinsed in methanol to remove excess stain, air dried again, and cover-slipped with mounting medium. Two hundred cells from each rat were examined for morphological abnormalities under a light microscope (4003) (Zeiss, Axioskop2, Germany). Abnormalities were classified as abnormal head (which included flattened head and pin head), abnormal neck, abnormal tail, or multiple abnormalities (which included tailless sperm) (Linder et al., 1990; Brown et al., 1994).
Sperm Chromatin Structure Assay (SCSA) The procedure of FCM SCSA has been described in detail (Evenson and Jost, 1994). Briefly, the left cauda epididymis was removed from each rat and placed in TNE buffer solution (0.15 M NaCl, 0.01 M Tris-HCL, 1 mM disodium EDTA, pH 7.4) in a Petri dish and minced. After allowing the tissue fragments to settle, the sperm suspension was filtered through a 0.2 mm nylon mesh into 2 mL cryogenic vials and mixed with glycerol to form a final solution of 10% (v/v) concentration. The solution was then frozen at 280 C. For SCSA analysis, the frozen samples were diluted to a concentration of 1 to 2 3 106 sperm/mL with TNE buffer solution; 50 lL of the diluted sample was put into a Falcon tube (Becton Dickinson Immunocytometry Systems, San Jose, CA) and 100 lL low-pH detergent solution was added (0.1% Triton X-100, 0.15 M NaCl, and 0.08 N HCl, pH 1.2). After 30 seconds, the cells were stained with 0.3 mL of acridine orange (AO) staining solution containing
Environmental Toxicology DOI 10.1002/tox
6 mg/L of AO in staining buffer (0.1 M citric acid, 0.2 M Na2HPO4, 1 mM EDTA, 0.15 M NaCl, pH 6.0). After 3 min of staining, the sample was analyzed using a FACScan FCM (Becton Dickinson Immunocytometry Systems, San Jose, CA). A total of 10,000 spermatozoa were collected and analyzed at a flow rate of 100 to 200 cells/s. AO is a metachromatic fluorochrome used to differentially stain double- and singlestranded nucleic acids. When excited by blue light at 488 nm, AO intercalates into double-stranded DNA to give a green fluorescence (FL1, native DNA). If, however, DNA is damaged, AO attaches to single-stranded nucleic acids and exhibits a red fluorescence (FL3, denatured, single stranded DNA). In SCSA analysis, the metachromatic shift from green to red fluorescence is expressed as a ratio between red and total fluorescence (red and green), and referred to as the DNA fragmentation index (DFI). SCSA variables in the present study were mean DFI, standard deviation (SD) DFI, and the percentage of cells outside the main population (% DFI). Data calculations were further performed using FCS Express software version 2 (De Novo Software, Thornhill, Ontario, Canada).
Sperm Mitochondrial Membrane Potential (MMP) The lipophilic cation with dual emission fluorophore JC-1 was used to measure the MMP (Garner et al., 1997; Gravance et al., 2001).When excited by blue light at 488 nm, the JC-1 molecule remains in its monomeric form. After passing through the mitochondrial membrane, it exhibits green fluorescence at 530 nm, representing low potential (inactivity or death). If the JC-1 molecule transforms into the J-aggregate form, it exhibits an orange fluorescence at 590 nm, indicating high membrane potential (high activity). Concentrations of 1.53 mM JC-1 stock solution were prepared in DMSO. The sperm suspension was diluted to 1 to 2 3 106 sperm/mL and stained with JC-1 to a final stain concentration of 3.0 lM. The samples were set aside for 10 min at 34 C, and then analyzed using a FACScan FCM. A total of 10,000 spermatozoa were collected and analyzed at a flow rate of 100 to 200 cells/s. The percentage of orange stained (mitochondrial-active sperm) cells was recorded.
Sperm Reactive Oxygen Species (ROS) Generation The sperm hydrogen peroxide (H2O2) level in most cells can be measured using a fluorescent probe, 2,7-dichlorofluorescin (DCFH-DA). To do this, H2O2 is incorporated into hydrophobic lipid regions of the cell (Bass et al., 1983). The acetate moieties are cleaved off leaving the non-fluorescent DCFH-DA. DCFH-DA is a stable compound that passively diffuses into cells and is hydrolyzed by intracellular esterase to yield DCHF, which is trapped inside cells. H2O2 produced by cells oxidizes DCFH to the highly fluorescent compound 2,7-dichlorofluorescein, which is fluorescent at 530 nm. The
TOXICITY OF INDIUM EXPOSURE ON MALE REPRODUCTION
DCFH-DA stock dissolves in absolute alcohol at 2.5 mM (1.2 mg/mL). We modified the method used by Fisher et al. (2005) to evaluate the H2O2 level in sperm. Sperm cells at a concentration of 1 to 2 3 106 sperm/mL were added into 2.5 mM DCFH-DA to a concentration of 12.5 mM. The mixture was set aside and maintained at 34 C for 30 min. The level of sperm superoxide anion (O22 •) was measured using a modification of a previously described method (Marchetti et al., 2002). The dihydroethidium (DHE) stock dissolves in DMSO at 0.33 mM, and can be directly oxidized into ethidium bromide by O22 • produced by sperm. Sperm cells at a concentration of 1 to 2 3 106 sperm/mL were added into 0.33 mM DHE to a concentration of 1.65 lM. The mixture was set aside and maintained at 34 C for 30 min. Intracellular H2O2 and O22 • levels were measured using a FACScan FCM with excitation and emission settings at 488 and 530 nm, respectively. We measured the percentage of cells with positive DHE and DCFH (which indicated positive ROS cells), and the data were expressed as the percentage of fluorescent spermatozoa (Mahfouz et al., 2009).
FCM DNA Content Analysis of Testis Cell Testis monocellular suspensions were prepared according to the procedure described by Spano et al. (1996). Briefly, the left testis was minced and treated with 1 mL 0.1% pepsinHCl solution, mixed by gentle magnetic stirring at room temperature for 10 min. These monocellular suspensions were then filtered through a 37 mm nylon mesh to separate the cells from the tissue. The resulting monocellular suspensions were stabilized with 4 mL of 95% ethanol and stored at 220 C for several weeks. For the FCM analysis, fixed samples were added to 0.5% pepsin-HCl solution to a 1:1 volume. After 10 min of gentle magnetic stirring, cells were immediately added to 50 lg/mL propidium iodide (PI) in 0.1 M Tris-HCl buffer solution (pH 7.5) containing 0.04 mg/mL DNAse-free RNAse. The fluorescence intensity of the DNA content of the testicular cells was measured using a FACScan FCM. PI fluorescent emissions were monitored using a 620 nm band-pass filter. A total of 2 3 104 cells were collected for each sample. Based on the DNA content, main germ cell peaks could be classified into four categories: (1) mature haploid (elongated spermatids; stages XXIV), (2) immature haploid (round and elongated spermatids; stages I-IX), (3) diploid (spermatogonia, secondary spermatocytes, tissue somatic cells), and (4) tetraploid (mostly primary spermatocytes). The region between the diploid and tetraploid peaks, called the S-phase, is composed of cells that actively synthesize DNA (Spano et al., 1996). The relative proportions of haploid, diploid, Sphase, and tetraploid cell types were calculated.
Analysis of S-In The S-In analysis was modified from methods of Hamaguchi et al. (2008). For pre-treatment, 0.25 mL of serum was
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diluted by 2.5 mL of 68 to 70% ultra-pure nitric acid (HNO3, GR for analysis, Merck) and 0.25 mL of 30% ultrapure hydrogen peroxide (H2O2, TAMAPURE-AA-100). After digestion by a 100 W/vessel microwave oven (Mars, microwave digestion system, CEM) (Liu et al., 2012), 0.1 mL 10 lg/L was added in each sample for external calibration. Finally, inductively-coupled plasma mass spectroscopy (ICP-MS, Perkin Elmer Sciex ELAN DRC, San Jose, CA) was used to analyze the serum indium concentration on PND 150.
Statistical Analysis Average values are expressed as mean 6 SD. All statistical comparisons were performed using the JMP statistical package (SAS Institute Inc., Gary, NC) for the unexposed control and the exposed groups. The body weights of the animals exposed to indium acetate and those of the control group were compared with Student’s t test, with P < 0.05 considered statistically significant. Welch’s test was adopted instead of Student’s t test if the variances of the two groups were not equal. The reproductive organ weights, sperm count, motility, morphology, SCSA, MMP, generation of H2O2 and O22 • as well as DNA content of testicular cells of the indium and the control groups were compared using a nonparametric Wilcoxon rank sums test, with P < 0.05 considered statistically significant. The relationships between sperm morphology, DFI, and O22 • were evaluated using general linear regression analysis.
RESULTS Body and Reproductive Organ Weights A significant reduction in mean body weight was noted in the indium-exposed animals compared to those in the control group from PND 113 to PND 143 (Fig. 1). There was a significant decrease of absolute seminal vesicle weight in indium exposed group at PND 150 (Table I). However, there was no significant change in the seminal vesicle weight to body weight ratio between the two groups. Similarly, there were no significant differences in the absolute and relative weights of the body, paired testis, paired epididymis, and paired cauda epididymis between the two groups (Table I).
Serum Indium Levels, Sperm Count, Motility, and Morphology The mean S-In level in the exposed group (143.95 lg/L) was significantly increased as compared to the control group (0.30 lg/L) (Table II). There were also no significant differences in sperm count and motility between the two groups, as shown in Table II. There was a significant increase in the number of sperm with abnormal heads, abnormal necks, abnormal tails, and multiple abnormalities, and total
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was not present in the generation of H2O2 between the two groups (Table II).
DNA Content Analysis of Testis Cells There were no significant differences between the two groups in the testis DNA content among the four cell types (mature haploid, immature haploid, diploid, and tetraploid) and that of the S-phase cell (data not shown).
Relationships Among Abnormal Sperm Morphology, Mean DFI, % DFI, and ROS Generation Fig. 1. Body weight of each rat recorded daily after exposure to either indium acetate or normal saline and presented as means 6 standard deviation between the indium acetate and the control group. *Significant differences as compared with control group (P < 0.05).
morphological abnormalities in the indium-exposed group as compared to the control group (Fig. 2).
Regression analysis of the incidence of total morphologically abnormal sperm, mean DFI, % DFI, and percentage of positive DHE cell showed that the incidence of morphologically total abnormal sperm was positively and significantly associated with the generation of sperm O22 • (r 5 0.67, P 5 0.012), the % DFI (r 5 0.68, P 5 0.011) and the mean DFI (r 5 0.77, P 5 0.022) [Figs. 3 and 4(A,B)].
DISCUSSION FCM Analysis of SCSA, MMP, and ROS Generation Sperm samples from the cauda epididymis were analyzed by FCM SCSA to investigate possible indium acetate-induced damages in chromatin integrity. There were significant increases in % DFI and the DFI mean in the indium-exposed group, whereas there were no significant differences in the SD DFI and MMP between the two groups (Table II). To evaluate sperm ROS generation, we measured H2O2 and O22 • levels and found a significant increase in the percentage of positive DHE cells in the indium-exposed group compared to that of the control group. However, this difference
In this study we demonstrated the impact of subacute indium treatment on the reproductive functions of male rats and investigated the underlying mechanism of the effect of the indium. The results showed that prolonged subacute administration of indium acetate into male rats reduced body weight and absolute seminal vesicle weight, increased the percentage of sperm with abnormal morphology, augmented impairment of sperm chromatin integrity, and induced the generation of O22 •. We further found significant and positive correlations among abnormal sperm morphology, chromatin integrity damage, and O22 • generation. A study by Omura et al., on testicular toxicities of two intratracheally administered semiconductor materials,
TABLE I. Body and tissue weight between rats exposed to indium acetate and unexposed control at postnatal day 150 Parameters Body weight (kg) Absolute tissue weight Testis (g) Epididymis (g) Cauda epididymis (g) Seminal vesicles (g) Relative tissue weight Testis (g/kg B.W.) Epididymis (g/kg B.W.) Cauda epididymis (g/kg B.W.) Seminal vesicles (g/kg B.W.) All data was expressed mean 6 standard deviation. *P < 0.05 as compared with control group.
Environmental Toxicology DOI 10.1002/tox
Control (n 5 6)
Indium Acetate (n 5 7)
P
0.57 6 0.05
0.52 6 0.05
0.1004
3.25 6 0.28 1.10 6 0.05 0.48 6 0.64 2.04 6 0.22
3.06 6 0.96 1.11 6 0.08 0.51 6 0.29 1.57 6 0.21*
0.8303 0.9431 0.3531 0.0124
5.74 6 0.64 1.95 6 0.21 0.86 6 0.19 3.62 6 0.54
5.94 6 1.94 2.15 6 0.18 0.98 6 0.08 3.04 6 0.50
0.2246 0.1336 0.2840 0.1004
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TABLE II. Serum indium level, sperm quality, epididymal sperm chromatin structure assay (SCSA), mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation in rats exposed to indium acetate and unexposed control group Parameters Serum indium level (lg/L) Mean DFI (AU) % DFI SD DFI (AU) MMP (%) Sperm motility (%) Sperm count (106/ml) DHE, % (O22) DCFH-DA, % (H2O2)
Control (n 5 6)
Indium Acetate (n 5 7)
P
0.30 206.9 6 7.0 2.3 6 1 21.9 6 10.4 18.3 6 17.6 51.2 6 9.2 16.5 6 4.9 1.1 6 1.1 0.7 6 0.5
143.95* 227.7 6 15.1** 14.6 6 12.7** 29.3 6 11.9 9.8 6 10.6 43.6 6 3.8 17.3 6 5.2 3.5 6 1.5** 0.5 6 0.3
27% is associated with pregnancy failure in assisted reproductive technology (Larson et al., 2000; Larson-Cook et al., 2003). Our data demonstrated positive correlations among abnormal sperm morphology, chromatin integrity damage, and O22 • generation. Several studies have supported the hypothesis that abnormal sperm morphology is statistically associated with an increase in the incidence of chromosomal abnormalities (Calogero et al., 2001; Sun et al., 2006), and chromatin integrity damage (Fischer et al., 2003; Kubo-Irie et al., 2005). A great deal of evidence has indicated that most sperm chromatin integrity damage is associated with ROS generation. In 2010, Aitken and De Iuliis put forward a two-step hypothesis to explain the etiology of oxidative DNA damage in germ cells. Step 1 of the hypothesis suggests the generation of a state of oxidative stress in the testes that impairs spermiogenesis, leading to the generation of poorly remodeled chromatin. In Step 2, this vulnerable DNA is oxidatively attacked, possibly as a result of the generation of mitochondrial ROS as these vulnerable cells succumb to apoptosis (Aitken and De Iuliis, 2010). Nagano et al., (2011) found that the average concentrations of indium in testis tissues were less lower than lung tissues contents in male rats exposed to ITO at 0.1 mg/m3 for 26 weeks (0.006 mg/g for testis vs. 20.2 mg/g for lung). Another study showed that the presence of electron-dense deposits characteristic of indium in the lysosomes of testicular Leydig and Sertoli cells as well as sufferance in mitochondria of indium-treated rats with total dose of 28 mg/kg (Samira et al., 2011). The non-specific toxic effects might due to relatively high toxic dose of indium. In this study, the mean S-In level in the exposed group was 143.95 lg/L and it was approximately up to 48-fold higher than the biological exposure index value of 3 mg/L set by Japan Society for Occupational Health (JSOH, 2007).
TOXICITY OF INDIUM EXPOSURE ON MALE REPRODUCTION
Our study also had several limitations for human health risk evaluation: the different routes of exposure, absorption, metabolism, distribution, and excretion of indium between human beings and experimental animals. Although the limitations of the rodent model must be kept in mind when interpreting results, the findings herein may be used in assessing human and ecological risk associated with its production, use and disposal. In conclusion, this study showed that subacute indium treatment increased the percentage of sperm with abnormal morphology, enhanced damage of chromatin integrity, and augmented ROS generation. Moreover, the positive correlations among abnormal sperm morphology, chromatin integrity damage, and ROS generation were demonstrated. Base on the findings of the present study, which have not been previously reported, we hypothesize that indium acetate treatment may generate oxidative stress during spermiogenesis and cause sperm chromatin remodeling, thereby leading to abnormal sperm morphology and subsequent male infertility. Given the current popular use of indium compounds in electronics industries, the clinical implications warrant further elucidation.
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