http://informahealthcare.com/txm ISSN: 1537-6516 (print), 1537-6524 (electronic) Toxicol Mech Methods, 2014; 24(2): 136–141 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2013.869779
RESEARCH ARTICLE
Junling Wang1, Haojun Zhang1,2, Feixue Xu3, Feihua Xu1, Ke Zhang1, and Yingmei Zhang4 1
School of Public Health, Lanzhou University, Lanzhou, China, 2People’s Hospital of Gansu Province, Lanzhou, China, 3The First Affiliated Hospital of Lanzhou University, Lanzhou, China, and 4School of Life Sciences, Lanzhou University, Lanzhou, China
Abstract
Keywords
Sodium fluoride (NaF) has been found to interfere with the reproductive system of animals. However, the cellular mechanisms underlying the reproductive toxicity of fluoride are unclear. The present study aims to define a possible mechanism of NaF-induced reproductive toxicity with respect to mineral, oxidative stress and c-Fos expression and the role of aluminum (Al) in intervening the toxic effect of NaF on rat testes. Fifty-six male Wistar rats were treated with normal saline, 1.0, 2.0, and 3.0 mg NaF/kg body weight (bw)/day, and each NaF concentration plus Al ion (0.1 mg Al3þ/kg bw/day). After 90 days, no significant changes in the contents of Fe and Cu were observed in any of the NaF-treated groups compared with those of the control group. There were, however, significant decreases in the contents of Ca in the 1.0 mg NaF group, Zn in all NaF-treated groups and Mg in the 3.0 mg NaF group. The levels of malondialdehyde (MDA) in the 1.0 mg NaF group and hydrogen peroxide (H2O2) in the 2.0 mg NaF group significantly increased, whereas the activity of nitric oxide synthase (NOS) significantly decreased in the 1.0 mg NaF group. Meanwhile, the protein expression of c-Fos increased significantly in the 1.0 and 2.0 mg NaF groups compared with the control group. Conversely, these changes were partially attenuated in rats simultaneously administered Al. The present study suggested that NaF could decrease the contents of Ca, Fe and Mg and enhance oxidative stress leading to c-Fos overexpression, and some deleterious effects were more prominent at lower NaF intake. Furthermore, Al within the research concentration could minimize reproductive toxicity caused by fluoride.
Aluminum, c-Fos, fluoride, male rat reproduction, mineral, oxidative stress
Introduction Fluoride is a persistent and non-degradable poison that people may be exposed through drinking water, taking food and breathing air (Chouhan & Flora, 2008; Guney et al., 2007b). It is evidenced that prolonged exposure to high fluoride damages not only skeletal tissue and teeth but also other soft tissues, such as brain, liver, kidney, and spinal cord (Zhang et al., 2007). Furthermore, there is an evidence that mass fluoride exposure interferes with the reproductive system of animals (Huang et al., 2008). However, the cellular mechanisms of reproductive toxicity induced by fluoride are unclear. There are many evidences that oxidative stress is an important mediator of fluoride-induced toxicity, but the results of these studies have been inconsistent (Guney et al., 2007a; Zhang et al., 2007). Earlier researches have shown that fluoride induces increase in the amounts of reactive oxygen
Address for correspondence: Yingmei Zhang, School of Life Sciences, Lanzhou University, Lanzhou 730000, P. R. China. Tel: +86 13919123067. Fax: +86 931 8910864. E-mail: [email protected]
History Received 13 September 2013 Revised 19 November 2013 Accepted 24 November 2013 Published online 16 December 2013
species, malondialdehyde (MDA) and hydrogen peroxide (H2O2), and inhabitation of activity in certain antioxidant enzymes in testes of rats. However, Chouhan and Flora (2008) demonstrated that fluoride at a relatively low concentration creates a condition of oxidative stress whereas at higher concentration it acts as an inhibitor of free radical production. Therefore, studies on the role of oxidative stress in fluorideinduced reproductive damage in male rats are discrepant. In 1921, Betrand and Vladesco first found that the content of zinc (Zn) in seminal fluid is higher than that in other body fluid (Abou-Shakra et al., 1989). Then many researches began to focus on the effects of mineral on reproduction. Several studies reported that mineral [e.g. calcium (Ca), iron (Fe), Zn, copper (Cu) and magnesium (Mg)] have close links with the secretion of sex hormone, sexual function and reproductive diseases of men and male mice (Colagar et al., 2009; Croxford et al., 2011; Sarosiek et al., 2009). Therefore, the minerals mentioned above play an important role in the reproductive function. Activator protein-1 (AP-1) collectively describes a group of structurally and functionally related members of the Jun
20 14
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by Monash University on 12/08/14 For personal use only.
The antagonism of aluminum against fluoride-induced oxidative stress and c-Fos overexpression in rat testes
The antagonism of aluminum against fluoride
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by Monash University on 12/08/14 For personal use only.
DOI: 10.3109/15376516.2013.869779
protein family [Jun (originally described as c-Jun), JunB and JunD] and Fos protein family [Fos (originally described as c-Fos), FosB, Fra-1 and Fra-2]. It is a redox-sensitive transcription factors with important roles in the processes of proliferation, differentiation and apoptotic cell death and the stress response (Herbein et al., 2006). The expression of c-Fos after being activated is the most sensitive and characteristic during the process of the activation of AP-1 (Cobellis et al., 2002; Theas et al., 2006). There is an evidence that Zn deficiency can induce oxidative stress, activate or inhibit oxidant sensitive transcription factor (Herbein et al., 2006). Thus, we hypothesize that the changes in mineral contents, oxidative stress and the expression of c-Fos in the testicular tissue play important roles in fluoride-induced reproductive toxicity. Nitric oxide synthase (NOS) catalyzes the conversion of arginine and oxygen molecule to nitric oxide (NO). NO is a signal molecule and participator in oxidative stress and usually plays a diverse role at low concentrations (Lue et al., 2003). In addition, the effects of NO and NOS on reproduction are complex. They can both inhibit and enhance spermatogenesis, sperm mature, sperm motility and fertilization ability (Waalkes et al., 2000; Weissman et al., 2005). Thus, we attempt to better understand the roles of NO and NOS in the testes of rats treated with fluoride. Aluminum (Al) is the third most common element in the earth’s crust and its absorption/accumulation in humans can occur via the diet, drinking water. The lower concentration Al can inhibit the toxic effect of fluoride (Lubkowska et al., 2006). However, other research reported that Al can cause Alzheimer’s disease and excessive amount of Al leads to accumulation in target organs, which is associated with the damage of testicular tissue of both humans and animals (Yousef, 2004; Yousef et al., 2005). The combined effect of fluoride and Al herein may be somewhat mixed and need to be clarified. The present study therefore aimed to explore a possible mechanism of fluoride-induced reproductive toxicity with respect to testicular mineral, oxidative stress and protein expression of c-Fos. Furthermore, the antagonism of Al against fluoride-induced toxic effects on testicular tissue was also investigated.
Materials and methods Chemicals Sodium fluoride (NaF) and aluminum chloride (AlCl3), AR, was obtained from Beijing Reagents Corp. (Beijing, China). Primary antibodies to c-Fos and b-actin and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemical reagents were of analytical grade unless indicated otherwise. Animal treatment Fifty-six adult male Wistar-albino rats weighing 170–180 g were obtained from the Experimental Animal Center of Lanzhou University and were acclimated in the laboratory for 2 weeks before the experiment. The rats were housed in
137
groups in stainless steel cages on a 12-h/12-h light/dark cycle at 22 2 C and 50% relative humidity with a standard chow diet and ad libitum access to tap water containing50.3 mg F/l. All rats received humane care in compliance with the Institution Animal Care and Use Committee of China. The rats were divided randomly into seven equal groups as follows: the control group was administered normal saline as control (C) and the rats in the three experimental groups received 1.0, 2.0 and 3.0 mg NaF/kg body weight (bw)/day (group A1, A2, A3), respectively, and each NaF concentration plus Al ion (0.1 mg Al3þ/kg bw/day) (group B1, B2, B3) by intragastric gavage. Table 1 summarizes individual concentrations of fluoride and Al ions, to which each experimental group was exposed. After 90 days, all rats were sacrificed by cervical dislocation, and the testes were quickly excised, weighed, and the related index was calculated. Measurements of mineral A known weight (0.5 g) of the testes sample was put into 6 ml mixed acid (HNO3:HClO4 ¼ 4:1) for 24 h. The fluid was then heated on hot plate to produce smoke. After cooling, 5 ml distilled water was put into the fluid. The fluid was heated again to produce smoke and then diluted with deionized water to 10 ml. The contents of Ca, Fe, Zn, Cu and Mg were assayed using an atomic absorption spectrometry. Measurements of MDA, H2O2, NOS and NO Testis tissue was immediately weighed and then homogenized with 1:9 (w/v) saline solution at 4 C. MDA (Hussein et al., 2013; Lan et al., 2013; Sohn et al., 2013), H2O2 and NOS activity and NO content in the testis tissue were determined with the reagent kit provided by the Nanjing Jianchen Biological Institute (China). Western blot analysis A portion of the testes was homogenized in 1.0 ml RIPA containing 10 ml phenylmethyl sulfonyl fluoride (PMSF) and centrifuged for 20 min at 13 000 rpm at 4 C. Protein concentrations were then measured using the Coomassie brilliant blue protein kit (Jianchen, China). c-Fos were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 4% SDS-PAGE, respectively, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA). Membranes were then blocked for 12 h at room temperature with 5% non-fat milk in Trisbuffered saline (TBS) and then rinsed with TBS-Tween 20 (TBST) buffer. The membranes were incubated with rabbit anti-rat c-Fos Table 1. Summary of NaF and aluminum ion concentration administered to individual experimental groups. Group C A1 A2 A3 B1 B2 B3
NaF (mg/kg bw/day)
Al3þ (mg/kg bw/day)
0 1.0 2.0 3.0 1.0 2.0 3.0
0 0 0 0 0.1 0.1 0.1
138
J. Wang et al.
Toxicol Mech Methods, 2014; 24(2): 136–141
antibodies at a dilution of 1/200. Blots were washed with 0.5% TBST and incubated overnight at 4 C in HRPconjugated goat anti-rabbit secondary antibody at a dilution of 1/2000. Detection was done using DAB as substrate. c-Fos-specific signals were quantified using Image Processing Plus (Image-Pro Plus) software, version 6.0, the relative expression levels of c-Fos was analyzed by the band intensities of c-Fos-/b-actin.
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by Monash University on 12/08/14 For personal use only.
Statistical analysis Data were expressed as mean standard deviation (SD) and analyzed with analysis of variance using Statistical Product and Service Solutions (SPSS, Version 13.0, Chicago, IL) followed by Tukey’s least significant difference post-hoc test. p50.05 was considered statistically significant.
Results Effects of NaF or combined with Al ion on body weight and relative testes weights BW and relative testes weights in the control group and all experimental groups are summarized in Table 2. During the 90-day period of treatment and observation, there were no significant changes in body and relative testes weights between the control and all experimental groups. No deaths occurred in any of the groups. Influence of NaF or combined with Al ion on mineral in the testis As seen in Table 3, no significant changes in Fe and Cu were observed in any of the NaF-treated groups. There were, however, significant decreases in the contents of Ca in group A1, Zn in all groups and Mg in group A3 compared with that
Table 2. The effects of NaF and combined with Al ion on body weight and relative testes weights of male rats (mean SD, n ¼ 8).
Group
Initial weight (g)
90 day (g)
Relative testes weights (g/100 g)
175.75 20.80 177.00 13.24 179.33 16.45 170.44 10.83 170.78 19.35 173.56 12.42 168.00 19.31
266.63 49.32 299.50 39.74 300.71 35.34 306.22 22.21 288.00 35.61 291.44 33.19 265.78 29.67
1.03 0.21 1.01 0.16 0.87 0.20 1.00 0.04 1.02 0.12 1.06 0.18 1.05 0.11
C A1 A2 A3 B1 B2 B3
of group C. Meanwhile, Ca in group B1 was significantly increased compared with group A1. Effects of NaF or combined with Al ion on testicular oxidative stress As shown in Figure 1, there were significant increases in MDA content in group A1 and H2O2 content in group A2 compared with group C. On the other hand, H2O2 content in group B2 was significantly decreased compared with group A2. Effects of NaF or combined with Al ion on NO content and NOS activity in testes The analysis of enzyme activity in testicular tissue isolated from control and experimental animals revealed a significant decrease in NOS activity in group A1. Significant increases in NO content in groups B1 and B3 and NOS activity in all NaF combined with Al ion groups were also found compared with the each parallel NaF-treated group (Figure 2). Effect of NaF or combined with Al ion on c-Fos expression in the testis The expression levels of c-Fos in testicular tissue in the different groups were detected by Western blot analysis. The results revealed that c-Fos expression (Figure 3) was low in normal testicular tissues, whereas significantly increased in groups A1 and A2. At the same time, there were significant decreases in all NaF combined with Al ion groups compared with the each parallel NaF-treated group.
Discussion In recent years, the influence of fluoride on reproductive system arouses researchers’ attention. Previous studies reported that the minerals like Ca, Fe, Zn, Cu and Mg could play very different roles in the reproductive system. The moderate contents of these minerals can regulate sperm activity, spermatogenesis and activity of sperm enzymes and influence the growth of younger generation (Bench et al., 2000; Glogowski et al., 1996; Liu et al., 2013; Meizel, 1997; Sarosiek et al., 2009; Sorensen et al., 1999; Yamaguchi et al., 2009; Zheng et al., 2001). In the present study, the decreases of Ca in group A1, Zn in all NaF-treated groups and Mg in group A3 were detected. Therefore, it is plausible that excess fluoride can influence male reproduction by changing the contents of Ca, Zn and Mg in the testes, whereas Fe and Cu
Table 3. Influence of NaF and combined with Al ion on mineral in the testes (mean SD, n ¼ 8). Group C A1 A2 A3 B1 B2 B3
Ca (mg/g)
Fe (mg/g)
Zn (mg/g)
Cu (mg/g)
Mg (mg/g)
77.57 6.66 56.15 4.21** 76.54 5.63 76.06 8.75 72.15 5.25aa 68.84 3.48 74.48 2.33
12.93 5.62 8.66 1.99 9.44 1.03 9.07 0.88 9.46 2.53 7.50 2.10 9.32 1.79
7.16 0.28 4.80 0.55** 4.56 0.33** 5.46 0.79** 5.85 0.99 5.03 0.54 5.58 0.27
5.81 0.59 5.55 0.32 5.48 0.96 5.87 0.21 5.62 0.65 5.63 0.47 5.69 0.49
42.54 8.07 38.89 1.68 35.97 5.79 32.44 1.53** 36.71 6.89 35.83 4.15 36.63 4.35
**p50.01 compared with the control group. p50.01 compared with the each parallel NaF-treated group.
aa
The antagonism of aluminum against fluoride
DOI: 10.3109/15376516.2013.869779
(B) 1000
(A) 12 10
**
139
*
800
8 600
a nmol/L
nmol/mg protein
a
6
400 4 200
2
0 C
A1
A2
A3
B1
B2
B3
C
A1
A2
A3
B1
B2
B3
Figure 1. Testicular content of MDA (A) and H2O2 (B) in control, NaF and combination of NaF and Al ion groups. Mean SD of eight independent experiments were indicated. *p50.05 as compared with the control group. **p50.01 as compared with the control group. ap50.05 compared with the each parallel NaF-treated group.
14 NO NOS
12
aa
10 mmol/g protein
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by Monash University on 12/08/14 For personal use only.
0
aa 8 6 aa
4
aa
aa
**
2 0 C
A1
A2
A3
B1
B2
B3
Figure 2. Testicular enzyme activities of NO and NOS content in control, NaF and combination of NaF and Al ion groups. Mean SD of eight independent experiments were indicated. **p50.01 as compared with the control group. aap50.05 compared with the each parallel NaF-treated group.
may not have effects on reproductive damage within the research concentrations. Moreover, numerous studies have indicated that Zn is close related with many dehydrogenase activities and plays an important role in regulating the oxidative stress of reproductive system (Herbein et al., 2006; Prasad et al., 2004). Zn can prevent oxidative stress by scavenging free radicals (e.g. H2O2) and lipid peroxidation (LPO) generated (e.g. decreased the MDA content) and maintain the activities of antioxidant enzymes [e.g. superoxide dismutase (SOD) and catalase (CAT)] (Taysi et al., 2012). Modification in Ca concentration has been associated with the production of reactive oxygen species (ROS) (Ciarcia et al., 2010). Mg deficiency has been shown to be associated with increased oxidative stress. Different mechanisms including systemic reactions and cellular changes are supposed to be involved in development and maintenance of the oxidative stress due to Mg deficiency (Chandra et al., 2013; Spasov et al., 2012). In this study, the
increases of H2O2 and MDA at lower dosages of NaF were also observed, which is consistent with earlier reports of oxidative stress induced by fluoride (Chlubek et al., 1999; Chouhan & Flora, 2008; Wang et al., 1997, 2009) and showed that the oxidative stress may have the relationship with the changes of minerals. On the other hand, a large proportion of NO in the testes is produced by NOS derived from testicular macrophages and the changes of NO and NOS can induce oxidative stress leading to functional damage of germ cell (Waalkes et al., 2000; Wang et al., 2002; Weissman et al., 2005). In the present study, a remarkable decrease was found in NOS activity in the testicular tissue of rats in the group A1. Therefore, the results also showed that NOS was a critical factor responsible for the oxidative stress at lower dosages of NaF. Fos family proteins are constituted by several members, such as c-Fos, FosB, Fra-1, and Fra-2, which have been proved to have different transcriptional activities (Karin et al., 1997). Although c-Fos activity has been detected in mammalian testes, particularly in premeiotic germ cells during spermatogenesis in the mouse (Cobellis et al., 2002), only a few studies appear to have addressed c-Fos expression induced by fluoride in the testes. The result from this study showed an increase of c-Fos expression in the testes induced by fluoride, suggesting that fluoride may influence the male reproductive function through inducing the c-Fos abnormal expression. In addition, previous studies showed that Zn and some other mineral deficiency can not only induce oxidative stress but also subsequently activate/inhibit oxidant-sensitive transcription factors such as c-Fos that can affect cell function, proliferation and survival (Harman, 2003; Herbein et al., 2006; Ho et al., 2004; Prasad et al., 2004). Our result showed that excess fluoride can decrease the contents of Ca, Zn and Mg and NOS activity and induce oxidative stress by changing the contents of MDA and H2O2 at lower dosages of fluoride. These findings suggested that the changes of Ca, Zn and Mg and NOS activity may influence oxidative stress leading to the c-Fos overexpression. At the same time, the changes in Ca, oxidative stress and expression of c-Fos followed a similar trend that all changes are more prominent at lower dosages of fluoride. Of course, further studies are
J. Wang et al.
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by Monash University on 12/08/14 For personal use only.
Figure 3. Western blot analysis for c-Fos in testes sample of rats treated with NaF. (A) Lane 1 control group, Lane 2 A1 group, Lane 3 A2 group, Lane 4 A3 group, Lane 5 B1 group, Lane 6 B2 group, Lane 7 B3 group. (B) Densitometric analysis showed significant increase in the c-Fos level in A1 and A2 groups compared with controls. Values are Mean SD of 4 observations. **p50.01 as compared with the control group. aap50.01 as compared with the each parallel NaF-treated group.
Toxicol Mech Methods, 2014; 24(2): 136–141
(A) 1
2
3
4
5
6
7 Fos (55KD) β-actin (39KD)
(B) 1.4 Protrin expression (ratio of optical densities)
140
**
1.2
**
1.0 .8 .6 aa .4
aa aa
.2 0.0 C
needed to determine the possible molecular mechanism involved in this process. The recommended tolerable daily intake of Al is 1 mg per kg (WHO Joint Expert Committee on Food Additives, JECFA, 1998). In our study, the experimental dose of Al by 0.1 mg Al3þ/kg bw/day is assured to be a safe dose to rats. Previous studies in testes of rats showed that fluoride induced increased production of reactive oxygen species, causing decreased activity in certain antioxidant enzymes (Chinoy et al., 2005; Huang et al., 2007). The present study found that fluoride can influence oxidative stress by increasing the contents of MDA and H2O2 and the activity of NOS. According to the reports by Lubkowska et al. (2006), Al could inhibit the fluoride enteric ingestion and increase the fluoride excretion at lower concentration. Thus, the absorption and storage of fluoride are decreased. The present study also demonstrated the efficacy of Al in alleviating the deleterious effect by increasing mineral and decreasing oxidative stress in testicular tissue. This has in turn led to minimal the c-Fos overexpression. In conclusion, excess fluoride exposure may possible influence the reproductive function of male rats by causing Ca, Zn and Mg deficiency and inducing oxidative stress leading to the increase of c-Fos expression, and some deleterious effects were more prominent at lower fluoride intake. Furthermore, the results suggest that Al within research concentration is effective in alleviating the toxic effects of NaF on testicular tissue.
Declaration of interest This research was sponsored by the Fundamental Research Funds for the Central Universities (Grant No. lzujbky-2012156). The authors declare no conflicts of interests.
A1
A2
A3
B1
B2
B3
References Abou-Shakra FR, Ward NI, Everard DM. (1989). The role of trace elements in male infertility. Fertil Steril 52:307–10. Bench G, Corzett MH, Kramer CE, et al. (2000). Zinc is sufficiently abundant within mammalian sperm nuclei to bind stoichiometrically with protamine 2. Mol Reprod Dev 56:512–19. Chandra AK, Sengupta P, Goswami H, Sarkar M. (2013). Effects of dietary magnesium on testicular histology, steroidogenesis, spermatogenesis and oxidative stress markers in adult rats. Indian J Exp Biol 51:37–47. Chinoy NJ, Mehta D, Jhala DD. (2005). Effects of different protein diets on fluoride induced oxidative stress in mice testis. Fluoride 38: 269–75. Chlubek D, Zawierta J, Kazmierczyk A, et al. (1999). Effect of different fluoride ion concentrations on malondialdehyde (MDA) formation in the mitochondrial fraction of human placental cells. Bromatol Chem Toksykol 32:119–22. Chouhan S, Flora SJ. (2008). Effects of fluoride on the tissue oxidative stress and apoptosis in rats: biochemical assays supported by IR spectroscopy data. Toxicology 254:61–7. Ciarcia R, d’Angelo D, Pacilio C, et al. (2010). Dysregulated calcium homeostasis and oxidative stress in chronic myeloid leukemia (CML) cells. J Cell Physiol 224:443–53. Cobellis G, Meccariello R, Fienga G, et al. (2002). Cytoplasmic and nuclear Fos protein forms regulate resumption of spermatogenesis in the frog, Rana esculenta. Endocrinology 143:163–70. Colagar AH, Marzony ET, Chaichi MJ. (2009). Zinc levels in seminal plasma are associated with sperm quality in fertile and infertile men. Nutr Res 29:82–8. Croxford TP, McCormick NH, Kelleher SL. (2011). Moderate zinc deficiency reduces testicular Zip6 and Zip10 abundance and impairs spermatogenesis in mice. J Nutr 141:359–65. Glogowski J, Babiak I, Goryczko K, Dobosz S. (1996). Activity of aspartate aminotransferase and acid phosphatase in cryopreserved trout sperm. Reprod Fertil Dev 8:1179–84. Guney M, Oral B, Demirin H, et al. (2007a). Protective effects of vitamins C and E against endometrial damage and oxidative stress in fluoride intoxication. Clin Exp Pharmacol Physiol 34: 467–74. Guney M, Oral B, Take G, et al. (2007b). Effect of fluoride intoxication on endometrial apoptosis and lipid peroxidation in rats: role of vitamins E and C. Toxicology 231:215–23.
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by Monash University on 12/08/14 For personal use only.
DOI: 10.3109/15376516.2013.869779
Harman D. (2003). The free radical theory of aging. Antioxid Redox Signal 5:557–61. Herbein G, Varin A, Fulop T. (2006). NF-kappaB, AP-1, Zinc-deficiency and aging. Biogerontology 7:409–19. Ho LH, Ruffin RE, Murgia C, et al. (2004). Labile zinc and zinc transporter ZnT4 in mast cell granules: role in regulation of caspase activation and NF-kappaB translocation. J Immunol 172:7750–60. Huang C, Yang HB, Niu RY, et al. (2008). Effects of sodium fluoride on androgen receptor expression in male mice. Fluoride 41:10–17. Huang CH, Niu RY, Wang JD. (2007). Toxic effects of sodium fluoride on reproductive function in male mice. Fluoride 40:162–8. Hussein AE, Abd-Elkhabir A, Abozahra A, et al. (2013). Pancreatic injury secondary to renal ischemia/reperfusion (I/R) injury: possible role of oxidative stress. Physiol Res. [Epub ahead of print]. Karin M, Liu Z, Zandi E. (1997). AP-1 function and regulation. Curr Opin Cell Biol 9:240–6. Lan Z, Bi KS, Chen XH. (2013). Ligustrazine attenuates elevated levels of indoxyl sulfate, kidney injury molecule-1 and clusterin in rats exposed to cadmium. Food Chem Toxicol. [Epub ahead of print]. Liu XF, Zhang LM, Zhang Z, et al. (2013). Manganese-induced effects on testicular trace element levels and crucial hormonal parameters of Hyline cocks. Biol Trace Elem Res 151:217–24. Lubkowska A, Chlubek D, Machoy-Mokrzyniska A. (2006). [The effect of alternating administration of aluminum chloride and sodium fluoride in drinking water on the concentration of fluoride in serum and its content in bones of rats]. Ann Acad Med Stetin 52:67–71. Lue Y, Sinha Hikim AP, Wang C, et al. (2003). Functional role of inducible nitric oxide synthase in the induction of male germ cell apoptosis, regulation of sperm number, and determination of testes size: evidence from null mutant mice. Endocrinology 144: 3092–100. Meizel S. (1997). Amino acid neurotransmitter receptor/chloride channels of mammalian sperm and the acrosome reaction. Biol Reprod 56: 569–74. Prasad AS, Bao B, Beck FW, et al. (2004). Antioxidant effect of zinc in humans. Free Radic Biol Med 37:1182–90. Sarosiek B, Pietrusewicz M, Radziwoniuk J, Glogowski J. (2009). The effect of copper, zinc, mercury and cadmium on some sperm enzyme activities in the common carp (Cyprinus carpio L.). Reprod Biol 9: 295–301. Sohn SH, Kim SK, Kim YO, et al. (2013). A comparison of antioxidant activity of Korean White and Red Ginsengs on H2O2-induced oxidative stress in HepG2 hepatoma cells. J Ginseng Res 37:442–50.
The antagonism of aluminum against fluoride
141
Sorensen MB, Bergdahl IA, Hjollund NH, et al. (1999). Zinc, magnesium and calcium in human seminal fluid: relations to other semen parameters and fertility. Mol Hum Reprod 5:331–7. Spasov AA, Zheltova AA, Kharitonov MV. (2012). Magnesium and the oxidative stress. Ross Fiziol Zh Im I M Sechenova 98:915–23. Taysi S, Okumus S, Akyuz M, et al. (2012). Zinc administration modulates radiation-induced oxidative injury in lens of rat. Pharmacogn Mag 8:245–9. Theas MS, Rival C, Dietrich SJ, et al. (2006). Death receptor and mitochondrial pathways are involved in germ cell apoptosis in an experimental model of autoimmune orchitis. Hum Reprod 21: 1734–42. Waalkes MP, Keefer LK, Diwan BA. (2000). Induction of proliferative lesions of the uterus, testes, and liver in swiss mice given repeated injections of sodium arsenate: possible estrogenic mode of action. Toxicol Appl Pharmacol 166:24–35. Wang JL, Zhang YM, Zhang HJ, et al. (2009). Toxic effects of fluoride on reproductive ability in male rats: sperm motility, oxidative stress, cell cycle, and testicular apoptosis. Fluoride 42:174–8. Wang Y, Newton DC, Miller TL, et al. (2002). An alternative promoter of the human neuronal nitric oxide synthase gene is expressed specifically in Leydig cells. Am J Pathol 160:369–80. Wang YY, Zhao BL, Li XJ. (1997). Spin trapping technique studies on active oxygen radicals from human polymorphonuclear leukocytes during fluoride-stimulated respiratory burst. Fluoride 30:5–15. Weissman BA, Niu E, Ge R, et al. (2005). Paracrine modulation of androgen synthesis in rat leydig cells by nitric oxide. J Androl 26: 369–78. Yamaguchi S, Miura C, Kikuchi K, et al. (2009). Zinc is an essential trace element for spermatogenesis. Proc Natl Acad Sci USA 106: 10859–64. Yousef MI. (2004). Aluminium-induced changes in hemato-biochemical parameters, lipid peroxidation and enzyme activities of male rabbits: protective role of ascorbic acid. Toxicology 199:47–57. Yousef MI, El-Morsy AM, Hassan MS. (2005). Aluminium-induced deterioration in reproductive performance and seminal plasma biochemistry of male rabbits: protective role of ascorbic acid. Toxicology 215:97–107. Zhang M, Wang A, He W, et al. (2007). Effects of fluoride on the expression of NCAM, oxidative stress, and apoptosis in primary cultured hippocampal neurons. Toxicology 236:208–16. Zheng YY, Li XY, Yuan XL, et al. (2001). Effect of iron deficiency anemia on the reproduction of female rats and the development of their offspring. J Hyg Res 30:163–5.
RESEARCH ARTICLE
Junling Wang1, Haojun Zhang1,2, Feixue Xu3, Feihua Xu1, Ke Zhang1, and Yingmei Zhang4 1
School of Public Health, Lanzhou University, Lanzhou, China, 2People’s Hospital of Gansu Province, Lanzhou, China, 3The First Affiliated Hospital of Lanzhou University, Lanzhou, China, and 4School of Life Sciences, Lanzhou University, Lanzhou, China
Abstract
Keywords
Sodium fluoride (NaF) has been found to interfere with the reproductive system of animals. However, the cellular mechanisms underlying the reproductive toxicity of fluoride are unclear. The present study aims to define a possible mechanism of NaF-induced reproductive toxicity with respect to mineral, oxidative stress and c-Fos expression and the role of aluminum (Al) in intervening the toxic effect of NaF on rat testes. Fifty-six male Wistar rats were treated with normal saline, 1.0, 2.0, and 3.0 mg NaF/kg body weight (bw)/day, and each NaF concentration plus Al ion (0.1 mg Al3þ/kg bw/day). After 90 days, no significant changes in the contents of Fe and Cu were observed in any of the NaF-treated groups compared with those of the control group. There were, however, significant decreases in the contents of Ca in the 1.0 mg NaF group, Zn in all NaF-treated groups and Mg in the 3.0 mg NaF group. The levels of malondialdehyde (MDA) in the 1.0 mg NaF group and hydrogen peroxide (H2O2) in the 2.0 mg NaF group significantly increased, whereas the activity of nitric oxide synthase (NOS) significantly decreased in the 1.0 mg NaF group. Meanwhile, the protein expression of c-Fos increased significantly in the 1.0 and 2.0 mg NaF groups compared with the control group. Conversely, these changes were partially attenuated in rats simultaneously administered Al. The present study suggested that NaF could decrease the contents of Ca, Fe and Mg and enhance oxidative stress leading to c-Fos overexpression, and some deleterious effects were more prominent at lower NaF intake. Furthermore, Al within the research concentration could minimize reproductive toxicity caused by fluoride.
Aluminum, c-Fos, fluoride, male rat reproduction, mineral, oxidative stress
Introduction Fluoride is a persistent and non-degradable poison that people may be exposed through drinking water, taking food and breathing air (Chouhan & Flora, 2008; Guney et al., 2007b). It is evidenced that prolonged exposure to high fluoride damages not only skeletal tissue and teeth but also other soft tissues, such as brain, liver, kidney, and spinal cord (Zhang et al., 2007). Furthermore, there is an evidence that mass fluoride exposure interferes with the reproductive system of animals (Huang et al., 2008). However, the cellular mechanisms of reproductive toxicity induced by fluoride are unclear. There are many evidences that oxidative stress is an important mediator of fluoride-induced toxicity, but the results of these studies have been inconsistent (Guney et al., 2007a; Zhang et al., 2007). Earlier researches have shown that fluoride induces increase in the amounts of reactive oxygen
Address for correspondence: Yingmei Zhang, School of Life Sciences, Lanzhou University, Lanzhou 730000, P. R. China. Tel: +86 13919123067. Fax: +86 931 8910864. E-mail: [email protected]
History Received 13 September 2013 Revised 19 November 2013 Accepted 24 November 2013 Published online 16 December 2013
species, malondialdehyde (MDA) and hydrogen peroxide (H2O2), and inhabitation of activity in certain antioxidant enzymes in testes of rats. However, Chouhan and Flora (2008) demonstrated that fluoride at a relatively low concentration creates a condition of oxidative stress whereas at higher concentration it acts as an inhibitor of free radical production. Therefore, studies on the role of oxidative stress in fluorideinduced reproductive damage in male rats are discrepant. In 1921, Betrand and Vladesco first found that the content of zinc (Zn) in seminal fluid is higher than that in other body fluid (Abou-Shakra et al., 1989). Then many researches began to focus on the effects of mineral on reproduction. Several studies reported that mineral [e.g. calcium (Ca), iron (Fe), Zn, copper (Cu) and magnesium (Mg)] have close links with the secretion of sex hormone, sexual function and reproductive diseases of men and male mice (Colagar et al., 2009; Croxford et al., 2011; Sarosiek et al., 2009). Therefore, the minerals mentioned above play an important role in the reproductive function. Activator protein-1 (AP-1) collectively describes a group of structurally and functionally related members of the Jun
20 14
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by Monash University on 12/08/14 For personal use only.
The antagonism of aluminum against fluoride-induced oxidative stress and c-Fos overexpression in rat testes
The antagonism of aluminum against fluoride
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by Monash University on 12/08/14 For personal use only.
DOI: 10.3109/15376516.2013.869779
protein family [Jun (originally described as c-Jun), JunB and JunD] and Fos protein family [Fos (originally described as c-Fos), FosB, Fra-1 and Fra-2]. It is a redox-sensitive transcription factors with important roles in the processes of proliferation, differentiation and apoptotic cell death and the stress response (Herbein et al., 2006). The expression of c-Fos after being activated is the most sensitive and characteristic during the process of the activation of AP-1 (Cobellis et al., 2002; Theas et al., 2006). There is an evidence that Zn deficiency can induce oxidative stress, activate or inhibit oxidant sensitive transcription factor (Herbein et al., 2006). Thus, we hypothesize that the changes in mineral contents, oxidative stress and the expression of c-Fos in the testicular tissue play important roles in fluoride-induced reproductive toxicity. Nitric oxide synthase (NOS) catalyzes the conversion of arginine and oxygen molecule to nitric oxide (NO). NO is a signal molecule and participator in oxidative stress and usually plays a diverse role at low concentrations (Lue et al., 2003). In addition, the effects of NO and NOS on reproduction are complex. They can both inhibit and enhance spermatogenesis, sperm mature, sperm motility and fertilization ability (Waalkes et al., 2000; Weissman et al., 2005). Thus, we attempt to better understand the roles of NO and NOS in the testes of rats treated with fluoride. Aluminum (Al) is the third most common element in the earth’s crust and its absorption/accumulation in humans can occur via the diet, drinking water. The lower concentration Al can inhibit the toxic effect of fluoride (Lubkowska et al., 2006). However, other research reported that Al can cause Alzheimer’s disease and excessive amount of Al leads to accumulation in target organs, which is associated with the damage of testicular tissue of both humans and animals (Yousef, 2004; Yousef et al., 2005). The combined effect of fluoride and Al herein may be somewhat mixed and need to be clarified. The present study therefore aimed to explore a possible mechanism of fluoride-induced reproductive toxicity with respect to testicular mineral, oxidative stress and protein expression of c-Fos. Furthermore, the antagonism of Al against fluoride-induced toxic effects on testicular tissue was also investigated.
Materials and methods Chemicals Sodium fluoride (NaF) and aluminum chloride (AlCl3), AR, was obtained from Beijing Reagents Corp. (Beijing, China). Primary antibodies to c-Fos and b-actin and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemical reagents were of analytical grade unless indicated otherwise. Animal treatment Fifty-six adult male Wistar-albino rats weighing 170–180 g were obtained from the Experimental Animal Center of Lanzhou University and were acclimated in the laboratory for 2 weeks before the experiment. The rats were housed in
137
groups in stainless steel cages on a 12-h/12-h light/dark cycle at 22 2 C and 50% relative humidity with a standard chow diet and ad libitum access to tap water containing50.3 mg F/l. All rats received humane care in compliance with the Institution Animal Care and Use Committee of China. The rats were divided randomly into seven equal groups as follows: the control group was administered normal saline as control (C) and the rats in the three experimental groups received 1.0, 2.0 and 3.0 mg NaF/kg body weight (bw)/day (group A1, A2, A3), respectively, and each NaF concentration plus Al ion (0.1 mg Al3þ/kg bw/day) (group B1, B2, B3) by intragastric gavage. Table 1 summarizes individual concentrations of fluoride and Al ions, to which each experimental group was exposed. After 90 days, all rats were sacrificed by cervical dislocation, and the testes were quickly excised, weighed, and the related index was calculated. Measurements of mineral A known weight (0.5 g) of the testes sample was put into 6 ml mixed acid (HNO3:HClO4 ¼ 4:1) for 24 h. The fluid was then heated on hot plate to produce smoke. After cooling, 5 ml distilled water was put into the fluid. The fluid was heated again to produce smoke and then diluted with deionized water to 10 ml. The contents of Ca, Fe, Zn, Cu and Mg were assayed using an atomic absorption spectrometry. Measurements of MDA, H2O2, NOS and NO Testis tissue was immediately weighed and then homogenized with 1:9 (w/v) saline solution at 4 C. MDA (Hussein et al., 2013; Lan et al., 2013; Sohn et al., 2013), H2O2 and NOS activity and NO content in the testis tissue were determined with the reagent kit provided by the Nanjing Jianchen Biological Institute (China). Western blot analysis A portion of the testes was homogenized in 1.0 ml RIPA containing 10 ml phenylmethyl sulfonyl fluoride (PMSF) and centrifuged for 20 min at 13 000 rpm at 4 C. Protein concentrations were then measured using the Coomassie brilliant blue protein kit (Jianchen, China). c-Fos were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 4% SDS-PAGE, respectively, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA). Membranes were then blocked for 12 h at room temperature with 5% non-fat milk in Trisbuffered saline (TBS) and then rinsed with TBS-Tween 20 (TBST) buffer. The membranes were incubated with rabbit anti-rat c-Fos Table 1. Summary of NaF and aluminum ion concentration administered to individual experimental groups. Group C A1 A2 A3 B1 B2 B3
NaF (mg/kg bw/day)
Al3þ (mg/kg bw/day)
0 1.0 2.0 3.0 1.0 2.0 3.0
0 0 0 0 0.1 0.1 0.1
138
J. Wang et al.
Toxicol Mech Methods, 2014; 24(2): 136–141
antibodies at a dilution of 1/200. Blots were washed with 0.5% TBST and incubated overnight at 4 C in HRPconjugated goat anti-rabbit secondary antibody at a dilution of 1/2000. Detection was done using DAB as substrate. c-Fos-specific signals were quantified using Image Processing Plus (Image-Pro Plus) software, version 6.0, the relative expression levels of c-Fos was analyzed by the band intensities of c-Fos-/b-actin.
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by Monash University on 12/08/14 For personal use only.
Statistical analysis Data were expressed as mean standard deviation (SD) and analyzed with analysis of variance using Statistical Product and Service Solutions (SPSS, Version 13.0, Chicago, IL) followed by Tukey’s least significant difference post-hoc test. p50.05 was considered statistically significant.
Results Effects of NaF or combined with Al ion on body weight and relative testes weights BW and relative testes weights in the control group and all experimental groups are summarized in Table 2. During the 90-day period of treatment and observation, there were no significant changes in body and relative testes weights between the control and all experimental groups. No deaths occurred in any of the groups. Influence of NaF or combined with Al ion on mineral in the testis As seen in Table 3, no significant changes in Fe and Cu were observed in any of the NaF-treated groups. There were, however, significant decreases in the contents of Ca in group A1, Zn in all groups and Mg in group A3 compared with that
Table 2. The effects of NaF and combined with Al ion on body weight and relative testes weights of male rats (mean SD, n ¼ 8).
Group
Initial weight (g)
90 day (g)
Relative testes weights (g/100 g)
175.75 20.80 177.00 13.24 179.33 16.45 170.44 10.83 170.78 19.35 173.56 12.42 168.00 19.31
266.63 49.32 299.50 39.74 300.71 35.34 306.22 22.21 288.00 35.61 291.44 33.19 265.78 29.67
1.03 0.21 1.01 0.16 0.87 0.20 1.00 0.04 1.02 0.12 1.06 0.18 1.05 0.11
C A1 A2 A3 B1 B2 B3
of group C. Meanwhile, Ca in group B1 was significantly increased compared with group A1. Effects of NaF or combined with Al ion on testicular oxidative stress As shown in Figure 1, there were significant increases in MDA content in group A1 and H2O2 content in group A2 compared with group C. On the other hand, H2O2 content in group B2 was significantly decreased compared with group A2. Effects of NaF or combined with Al ion on NO content and NOS activity in testes The analysis of enzyme activity in testicular tissue isolated from control and experimental animals revealed a significant decrease in NOS activity in group A1. Significant increases in NO content in groups B1 and B3 and NOS activity in all NaF combined with Al ion groups were also found compared with the each parallel NaF-treated group (Figure 2). Effect of NaF or combined with Al ion on c-Fos expression in the testis The expression levels of c-Fos in testicular tissue in the different groups were detected by Western blot analysis. The results revealed that c-Fos expression (Figure 3) was low in normal testicular tissues, whereas significantly increased in groups A1 and A2. At the same time, there were significant decreases in all NaF combined with Al ion groups compared with the each parallel NaF-treated group.
Discussion In recent years, the influence of fluoride on reproductive system arouses researchers’ attention. Previous studies reported that the minerals like Ca, Fe, Zn, Cu and Mg could play very different roles in the reproductive system. The moderate contents of these minerals can regulate sperm activity, spermatogenesis and activity of sperm enzymes and influence the growth of younger generation (Bench et al., 2000; Glogowski et al., 1996; Liu et al., 2013; Meizel, 1997; Sarosiek et al., 2009; Sorensen et al., 1999; Yamaguchi et al., 2009; Zheng et al., 2001). In the present study, the decreases of Ca in group A1, Zn in all NaF-treated groups and Mg in group A3 were detected. Therefore, it is plausible that excess fluoride can influence male reproduction by changing the contents of Ca, Zn and Mg in the testes, whereas Fe and Cu
Table 3. Influence of NaF and combined with Al ion on mineral in the testes (mean SD, n ¼ 8). Group C A1 A2 A3 B1 B2 B3
Ca (mg/g)
Fe (mg/g)
Zn (mg/g)
Cu (mg/g)
Mg (mg/g)
77.57 6.66 56.15 4.21** 76.54 5.63 76.06 8.75 72.15 5.25aa 68.84 3.48 74.48 2.33
12.93 5.62 8.66 1.99 9.44 1.03 9.07 0.88 9.46 2.53 7.50 2.10 9.32 1.79
7.16 0.28 4.80 0.55** 4.56 0.33** 5.46 0.79** 5.85 0.99 5.03 0.54 5.58 0.27
5.81 0.59 5.55 0.32 5.48 0.96 5.87 0.21 5.62 0.65 5.63 0.47 5.69 0.49
42.54 8.07 38.89 1.68 35.97 5.79 32.44 1.53** 36.71 6.89 35.83 4.15 36.63 4.35
**p50.01 compared with the control group. p50.01 compared with the each parallel NaF-treated group.
aa
The antagonism of aluminum against fluoride
DOI: 10.3109/15376516.2013.869779
(B) 1000
(A) 12 10
**
139
*
800
8 600
a nmol/L
nmol/mg protein
a
6
400 4 200
2
0 C
A1
A2
A3
B1
B2
B3
C
A1
A2
A3
B1
B2
B3
Figure 1. Testicular content of MDA (A) and H2O2 (B) in control, NaF and combination of NaF and Al ion groups. Mean SD of eight independent experiments were indicated. *p50.05 as compared with the control group. **p50.01 as compared with the control group. ap50.05 compared with the each parallel NaF-treated group.
14 NO NOS
12
aa
10 mmol/g protein
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by Monash University on 12/08/14 For personal use only.
0
aa 8 6 aa
4
aa
aa
**
2 0 C
A1
A2
A3
B1
B2
B3
Figure 2. Testicular enzyme activities of NO and NOS content in control, NaF and combination of NaF and Al ion groups. Mean SD of eight independent experiments were indicated. **p50.01 as compared with the control group. aap50.05 compared with the each parallel NaF-treated group.
may not have effects on reproductive damage within the research concentrations. Moreover, numerous studies have indicated that Zn is close related with many dehydrogenase activities and plays an important role in regulating the oxidative stress of reproductive system (Herbein et al., 2006; Prasad et al., 2004). Zn can prevent oxidative stress by scavenging free radicals (e.g. H2O2) and lipid peroxidation (LPO) generated (e.g. decreased the MDA content) and maintain the activities of antioxidant enzymes [e.g. superoxide dismutase (SOD) and catalase (CAT)] (Taysi et al., 2012). Modification in Ca concentration has been associated with the production of reactive oxygen species (ROS) (Ciarcia et al., 2010). Mg deficiency has been shown to be associated with increased oxidative stress. Different mechanisms including systemic reactions and cellular changes are supposed to be involved in development and maintenance of the oxidative stress due to Mg deficiency (Chandra et al., 2013; Spasov et al., 2012). In this study, the
increases of H2O2 and MDA at lower dosages of NaF were also observed, which is consistent with earlier reports of oxidative stress induced by fluoride (Chlubek et al., 1999; Chouhan & Flora, 2008; Wang et al., 1997, 2009) and showed that the oxidative stress may have the relationship with the changes of minerals. On the other hand, a large proportion of NO in the testes is produced by NOS derived from testicular macrophages and the changes of NO and NOS can induce oxidative stress leading to functional damage of germ cell (Waalkes et al., 2000; Wang et al., 2002; Weissman et al., 2005). In the present study, a remarkable decrease was found in NOS activity in the testicular tissue of rats in the group A1. Therefore, the results also showed that NOS was a critical factor responsible for the oxidative stress at lower dosages of NaF. Fos family proteins are constituted by several members, such as c-Fos, FosB, Fra-1, and Fra-2, which have been proved to have different transcriptional activities (Karin et al., 1997). Although c-Fos activity has been detected in mammalian testes, particularly in premeiotic germ cells during spermatogenesis in the mouse (Cobellis et al., 2002), only a few studies appear to have addressed c-Fos expression induced by fluoride in the testes. The result from this study showed an increase of c-Fos expression in the testes induced by fluoride, suggesting that fluoride may influence the male reproductive function through inducing the c-Fos abnormal expression. In addition, previous studies showed that Zn and some other mineral deficiency can not only induce oxidative stress but also subsequently activate/inhibit oxidant-sensitive transcription factors such as c-Fos that can affect cell function, proliferation and survival (Harman, 2003; Herbein et al., 2006; Ho et al., 2004; Prasad et al., 2004). Our result showed that excess fluoride can decrease the contents of Ca, Zn and Mg and NOS activity and induce oxidative stress by changing the contents of MDA and H2O2 at lower dosages of fluoride. These findings suggested that the changes of Ca, Zn and Mg and NOS activity may influence oxidative stress leading to the c-Fos overexpression. At the same time, the changes in Ca, oxidative stress and expression of c-Fos followed a similar trend that all changes are more prominent at lower dosages of fluoride. Of course, further studies are
J. Wang et al.
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by Monash University on 12/08/14 For personal use only.
Figure 3. Western blot analysis for c-Fos in testes sample of rats treated with NaF. (A) Lane 1 control group, Lane 2 A1 group, Lane 3 A2 group, Lane 4 A3 group, Lane 5 B1 group, Lane 6 B2 group, Lane 7 B3 group. (B) Densitometric analysis showed significant increase in the c-Fos level in A1 and A2 groups compared with controls. Values are Mean SD of 4 observations. **p50.01 as compared with the control group. aap50.01 as compared with the each parallel NaF-treated group.
Toxicol Mech Methods, 2014; 24(2): 136–141
(A) 1
2
3
4
5
6
7 Fos (55KD) β-actin (39KD)
(B) 1.4 Protrin expression (ratio of optical densities)
140
**
1.2
**
1.0 .8 .6 aa .4
aa aa
.2 0.0 C
needed to determine the possible molecular mechanism involved in this process. The recommended tolerable daily intake of Al is 1 mg per kg (WHO Joint Expert Committee on Food Additives, JECFA, 1998). In our study, the experimental dose of Al by 0.1 mg Al3þ/kg bw/day is assured to be a safe dose to rats. Previous studies in testes of rats showed that fluoride induced increased production of reactive oxygen species, causing decreased activity in certain antioxidant enzymes (Chinoy et al., 2005; Huang et al., 2007). The present study found that fluoride can influence oxidative stress by increasing the contents of MDA and H2O2 and the activity of NOS. According to the reports by Lubkowska et al. (2006), Al could inhibit the fluoride enteric ingestion and increase the fluoride excretion at lower concentration. Thus, the absorption and storage of fluoride are decreased. The present study also demonstrated the efficacy of Al in alleviating the deleterious effect by increasing mineral and decreasing oxidative stress in testicular tissue. This has in turn led to minimal the c-Fos overexpression. In conclusion, excess fluoride exposure may possible influence the reproductive function of male rats by causing Ca, Zn and Mg deficiency and inducing oxidative stress leading to the increase of c-Fos expression, and some deleterious effects were more prominent at lower fluoride intake. Furthermore, the results suggest that Al within research concentration is effective in alleviating the toxic effects of NaF on testicular tissue.
Declaration of interest This research was sponsored by the Fundamental Research Funds for the Central Universities (Grant No. lzujbky-2012156). The authors declare no conflicts of interests.
A1
A2
A3
B1
B2
B3
References Abou-Shakra FR, Ward NI, Everard DM. (1989). The role of trace elements in male infertility. Fertil Steril 52:307–10. Bench G, Corzett MH, Kramer CE, et al. (2000). Zinc is sufficiently abundant within mammalian sperm nuclei to bind stoichiometrically with protamine 2. Mol Reprod Dev 56:512–19. Chandra AK, Sengupta P, Goswami H, Sarkar M. (2013). Effects of dietary magnesium on testicular histology, steroidogenesis, spermatogenesis and oxidative stress markers in adult rats. Indian J Exp Biol 51:37–47. Chinoy NJ, Mehta D, Jhala DD. (2005). Effects of different protein diets on fluoride induced oxidative stress in mice testis. Fluoride 38: 269–75. Chlubek D, Zawierta J, Kazmierczyk A, et al. (1999). Effect of different fluoride ion concentrations on malondialdehyde (MDA) formation in the mitochondrial fraction of human placental cells. Bromatol Chem Toksykol 32:119–22. Chouhan S, Flora SJ. (2008). Effects of fluoride on the tissue oxidative stress and apoptosis in rats: biochemical assays supported by IR spectroscopy data. Toxicology 254:61–7. Ciarcia R, d’Angelo D, Pacilio C, et al. (2010). Dysregulated calcium homeostasis and oxidative stress in chronic myeloid leukemia (CML) cells. J Cell Physiol 224:443–53. Cobellis G, Meccariello R, Fienga G, et al. (2002). Cytoplasmic and nuclear Fos protein forms regulate resumption of spermatogenesis in the frog, Rana esculenta. Endocrinology 143:163–70. Colagar AH, Marzony ET, Chaichi MJ. (2009). Zinc levels in seminal plasma are associated with sperm quality in fertile and infertile men. Nutr Res 29:82–8. Croxford TP, McCormick NH, Kelleher SL. (2011). Moderate zinc deficiency reduces testicular Zip6 and Zip10 abundance and impairs spermatogenesis in mice. J Nutr 141:359–65. Glogowski J, Babiak I, Goryczko K, Dobosz S. (1996). Activity of aspartate aminotransferase and acid phosphatase in cryopreserved trout sperm. Reprod Fertil Dev 8:1179–84. Guney M, Oral B, Demirin H, et al. (2007a). Protective effects of vitamins C and E against endometrial damage and oxidative stress in fluoride intoxication. Clin Exp Pharmacol Physiol 34: 467–74. Guney M, Oral B, Take G, et al. (2007b). Effect of fluoride intoxication on endometrial apoptosis and lipid peroxidation in rats: role of vitamins E and C. Toxicology 231:215–23.
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by Monash University on 12/08/14 For personal use only.
DOI: 10.3109/15376516.2013.869779
Harman D. (2003). The free radical theory of aging. Antioxid Redox Signal 5:557–61. Herbein G, Varin A, Fulop T. (2006). NF-kappaB, AP-1, Zinc-deficiency and aging. Biogerontology 7:409–19. Ho LH, Ruffin RE, Murgia C, et al. (2004). Labile zinc and zinc transporter ZnT4 in mast cell granules: role in regulation of caspase activation and NF-kappaB translocation. J Immunol 172:7750–60. Huang C, Yang HB, Niu RY, et al. (2008). Effects of sodium fluoride on androgen receptor expression in male mice. Fluoride 41:10–17. Huang CH, Niu RY, Wang JD. (2007). Toxic effects of sodium fluoride on reproductive function in male mice. Fluoride 40:162–8. Hussein AE, Abd-Elkhabir A, Abozahra A, et al. (2013). Pancreatic injury secondary to renal ischemia/reperfusion (I/R) injury: possible role of oxidative stress. Physiol Res. [Epub ahead of print]. Karin M, Liu Z, Zandi E. (1997). AP-1 function and regulation. Curr Opin Cell Biol 9:240–6. Lan Z, Bi KS, Chen XH. (2013). Ligustrazine attenuates elevated levels of indoxyl sulfate, kidney injury molecule-1 and clusterin in rats exposed to cadmium. Food Chem Toxicol. [Epub ahead of print]. Liu XF, Zhang LM, Zhang Z, et al. (2013). Manganese-induced effects on testicular trace element levels and crucial hormonal parameters of Hyline cocks. Biol Trace Elem Res 151:217–24. Lubkowska A, Chlubek D, Machoy-Mokrzyniska A. (2006). [The effect of alternating administration of aluminum chloride and sodium fluoride in drinking water on the concentration of fluoride in serum and its content in bones of rats]. Ann Acad Med Stetin 52:67–71. Lue Y, Sinha Hikim AP, Wang C, et al. (2003). Functional role of inducible nitric oxide synthase in the induction of male germ cell apoptosis, regulation of sperm number, and determination of testes size: evidence from null mutant mice. Endocrinology 144: 3092–100. Meizel S. (1997). Amino acid neurotransmitter receptor/chloride channels of mammalian sperm and the acrosome reaction. Biol Reprod 56: 569–74. Prasad AS, Bao B, Beck FW, et al. (2004). Antioxidant effect of zinc in humans. Free Radic Biol Med 37:1182–90. Sarosiek B, Pietrusewicz M, Radziwoniuk J, Glogowski J. (2009). The effect of copper, zinc, mercury and cadmium on some sperm enzyme activities in the common carp (Cyprinus carpio L.). Reprod Biol 9: 295–301. Sohn SH, Kim SK, Kim YO, et al. (2013). A comparison of antioxidant activity of Korean White and Red Ginsengs on H2O2-induced oxidative stress in HepG2 hepatoma cells. J Ginseng Res 37:442–50.
The antagonism of aluminum against fluoride
141
Sorensen MB, Bergdahl IA, Hjollund NH, et al. (1999). Zinc, magnesium and calcium in human seminal fluid: relations to other semen parameters and fertility. Mol Hum Reprod 5:331–7. Spasov AA, Zheltova AA, Kharitonov MV. (2012). Magnesium and the oxidative stress. Ross Fiziol Zh Im I M Sechenova 98:915–23. Taysi S, Okumus S, Akyuz M, et al. (2012). Zinc administration modulates radiation-induced oxidative injury in lens of rat. Pharmacogn Mag 8:245–9. Theas MS, Rival C, Dietrich SJ, et al. (2006). Death receptor and mitochondrial pathways are involved in germ cell apoptosis in an experimental model of autoimmune orchitis. Hum Reprod 21: 1734–42. Waalkes MP, Keefer LK, Diwan BA. (2000). Induction of proliferative lesions of the uterus, testes, and liver in swiss mice given repeated injections of sodium arsenate: possible estrogenic mode of action. Toxicol Appl Pharmacol 166:24–35. Wang JL, Zhang YM, Zhang HJ, et al. (2009). Toxic effects of fluoride on reproductive ability in male rats: sperm motility, oxidative stress, cell cycle, and testicular apoptosis. Fluoride 42:174–8. Wang Y, Newton DC, Miller TL, et al. (2002). An alternative promoter of the human neuronal nitric oxide synthase gene is expressed specifically in Leydig cells. Am J Pathol 160:369–80. Wang YY, Zhao BL, Li XJ. (1997). Spin trapping technique studies on active oxygen radicals from human polymorphonuclear leukocytes during fluoride-stimulated respiratory burst. Fluoride 30:5–15. Weissman BA, Niu E, Ge R, et al. (2005). Paracrine modulation of androgen synthesis in rat leydig cells by nitric oxide. J Androl 26: 369–78. Yamaguchi S, Miura C, Kikuchi K, et al. (2009). Zinc is an essential trace element for spermatogenesis. Proc Natl Acad Sci USA 106: 10859–64. Yousef MI. (2004). Aluminium-induced changes in hemato-biochemical parameters, lipid peroxidation and enzyme activities of male rabbits: protective role of ascorbic acid. Toxicology 199:47–57. Yousef MI, El-Morsy AM, Hassan MS. (2005). Aluminium-induced deterioration in reproductive performance and seminal plasma biochemistry of male rabbits: protective role of ascorbic acid. Toxicology 215:97–107. Zhang M, Wang A, He W, et al. (2007). Effects of fluoride on the expression of NCAM, oxidative stress, and apoptosis in primary cultured hippocampal neurons. Toxicology 236:208–16. Zheng YY, Li XY, Yuan XL, et al. (2001). Effect of iron deficiency anemia on the reproduction of female rats and the development of their offspring. J Hyg Res 30:163–5.
