Effects of vitamin E on reproductive hormones and testis structure in chronic dioxin-treated mice

Hai-Ping Yin1,2, Jian-Ping Xu1,3, Xian-Qing Zhou1,4 and Ying Wang1

Toxicology and Industrial Health 28(2) 152161 The Author(s) 2012 Reprints and permission: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233711408381 tih.sagepub.com

Abstract The purpose of this study was to investigate the effects of vitamin E on reproductive hormones and testis structure in mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Five experimental groups of a combination of TCDD and vitamin E were designed as follows: 0 ng/kg/d and 0 mg/kg/d (control group), 100 ng/kg/d and 0 mg/kg/d (Group I), 100 ng/kg/d and 20 mg/kg/d (Group II), 100 ng/kg/d and 100 mg/kg/d (Group III), and 100 ng/kg/d and 500 mg/kg/d (Group IV) respectively. Vitamin E and TCDD were given by oral gavage for 7 weeks. The results demonstrated that TCDD decreased the levels of brain gonadotropin releasing hormone (GnRH), testis luteinizing hormone (LH) and follicle stimulating hormone (FSH), serum testosterone and testis spermatozoa number, and damaged testis structure. Vitamin E at 20 mg/kg alleviated the decrease of GnRH; vitamin E at 20, 100, and 500 mg/kg antagonized the decline of LH and FSH; vitamin E at 20 and 100 mg/kg reversed the decrease of testosterone and spermatozoa number; and vitamin E at 100 mg/kg decreased the damage of the testis structure caused by TCDD. The results indicate that vitamin E antagonizes the reproductive endocrine toxicity and alleviates the changes in testicular structure caused by TCDD. Keywords Vitamin E, mouse, reproductive hormone, spermatozoa number, testis structure, 2, 3, 7, 8-tetrachlorodibenzop-dioxin

Introduction
2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), which is both ubiquitous and persistent, is one of the most potent environmental contaminants. TCDD is lipophilic, has a low rate of metabolism and excretion, and is prone to bioaccumulation. TCDD has potential adverse effects on humans and animals. It has been shown to lower the level of serum testosterone and decrease fertility, and it delayed the growth and maturation of spermatozoa in a rat model (Latchoumycandane et al., 2002a). It can also destroy the antioxidant defense systems of testicular chondrosomes and cytomicrosomes, cause oxidative stress (Latchoumycandane and Mathur 2002b), and lead to the feminization and sterility of male offspring (Mably et al., 1992). Vitamin E is an essential lipid-soluble vitamin in the human body, and it plays multiple important physiological roles. Vitamin E is not only an effective antioxidant and immunomodulator (Moriguchi and Muraga 2000), but it can also antagonize the

neurotoxic effects caused by TCDD (Kim and Yang, 2005). However, little information is available about the effects of vitamin E on reproductive endocrine function and testis structure in TCDD-treated mice. The present study was designed to evaluate the effects
1

Department of Laboratory Animal Science, School of Basic Medical Sciences, Capital Medical University, Beijing, China 2 Gansu International Traval Healthcare Centre, Entry-Exit Inspection and Quarantine Bureau, Gansu, Peoples Republic of China 3 Yanjing Medical College, Capital Medical University, Beijing, Peoples Republic of China 4 Department of Health Toxicology and Health Chemistry, School of Public Health and Family Medicine, Capital Medical University, Beijing, Peoples Republic of China Corresponding author: Xian-Qing Zhou, Department of Health Toxicology and Health Chemistry, School of Public Health and Family Medicine, Capital Medical University, Beijing, 100069, Peoples Republic of China E-mail: xianqingzhou@yahoo.com.cn

Yin et al.

153

of different levels of vitamin E on reproductive performance in TCDD-treated mice, which is expected to provide an approach for preventing or alleviating the toxicity elicited by TCDD.

approximately 8:00 a.m.9:00 a.m., and blood, testes, hypothalamus and appendix suprasphenoidalis were collected from each animal for analysis. Serum was separated by centrifugation at 3500 rpm for 15 min at 4 C, and then stored at 80 C until analysis.

Materials and methods Animals and husbandry

Five-week-old mice used in this study were obtained from Beijing Vital River Laboratory Animal Technology Limited Corporation, China. Mice were housed individually in cages that measured 26 15 15 cm3. They were maintained at a constant temperature of 22 + 2 C and a humidity of 50 + 5% with a 12:12 light/dark cycle, and given a standard commercial pelleted diet without vitamin E and tap water ad libitum. The nutritional makeup of the diet consisted of 20.6% protein, 4.16% fat, 53.17% carbohydrate, 4.92% crude fiber, 7.5% ash content, and 9.65% moisture content. The experiment was initiated after the mice were acclimatized to laboratory conditions for 1 week.

Determination of gonadotropin releasing hormone

Gonadotropin releasing hormone (GnRH) was measured as described by Wu et al. (2006). Brain tissue (1 g) was placed in a tube for homogenization, 0.2 ml 1 mol/L HCl was added and the sample was homogenized thoroughly. Tubes were maintained at room temperature for 100 min, 0.2 ml 1 mol/L NaOH was added, and then the tubes were centrifuged at 4000 rpm for 10 min at 4 C. The supernatant was used in an Elisa kit to measure GnRH purchased from the Animal Diagnostic Laboratory (ADL) of Penn State University (University Park, PA, USA).

Experimental design and methods

Forty healthy male mice with an initial body weight ranging 19.033.3 g were selected. They were randomly assigned to five groups, each containing eight male mice. The levels of both TCDD and vitamin E were 0 ng/kg/d and 0 mg/kg/d (control group), 100 ng/kg/d and 0 mg/kg/d (experimental group I), 100 ng/kg/d and 20 mg/kg/d (experimental group II), 100 ng/kg/d and 100 mg/kg/d (experimental group III), and 100 ng/kg/d and 500 mg/kg/d (experimental group IV). The levels of vitamin E and TCDD were established based on a previous work by Fujimaki et al. (2002) and Zhou et al. (2004), respectively. Both TCDD and vitamin E were dissolved in corn oil, and were given by oral gavage. TCDD was given at approximately 8 a.m. daily, and after 2 h, vitamin E was administered. The mice in the control group received corn oil of equal volume. The other experimental conditions were the same as the acclimatization. The experiments were conducted in accordance with institutional guidelines for animal welfare, and the protocols have been reviewed by the animal experimentation committee. TCDD (97% purity) was provided by the Research Center for Eco-Environmental Sciences, the Chinese Academy Sciences, Beijing, China. Vitamin E (95% purity) was obtained from Sigma (St Louis, MO, USA). After 7 weeks of treatment, all mice were killed by decapitation at

Determination of follicle stimulating hormone and luteinizing hormone

Follicle stimulating hormone (FSH) and luteinizing hormone (LH) were measured as described by Taketoh et al. (2007). Testicular tissue (1 g) was placed in a tube for homogenization and 0.4 ml deionized water was added. Samples were homogenized thoroughly and centrifuged at 7500 rpm for 10 min at 4 C. The supernatant was used for measuring FSH and LH using Elisa kits obtained from ADL.

Determination of serum testosterone

Serum testosterone was determined in 50-ml samples by 125I-radioimmunoassay kits (Beijing Furui Bio-engineering Company, Peoples Republic of China). Samples were mixed with 100 ml of antiserum and incubated at 37 C for 1 h. Following the incubation, 500 ml of separation reagent was added. The mixture was maintained at room temperature for 15 min and then centrifuged at 3500 rpm for 15 min at 4 C. The radioactivity of the supernatant was measured on a gamma counter (FJ-2008PS g-counter).

Histological assessment of testes and sperm count

Testes were rapidly removed from mice and fixed in 10% buffered formalin for 24 h. Testicular tissue was then dehydrated and embedded in paraffin by

154

Toxicology and Industrial Health 28(2)

Gonadotropin releasing hormone (ng/g)

250 200 a,* 150 100 50 0 Control group Group I

b a a

t test, and differences among experimental groups IIV were analyzed by one-way analysis of variance (ANOVA) followed by LSDs (least significant difference procedure) multiple range test. All values are expressed as means + standard error (SE). The results were considered significant at p < 0.05.

Group II

Group III

Group IV

Results
The effects of vitamin E on brain GnRH in male mice treated with TCDD The t tests indicated that TCDD had significant effects on brain GnRH in male mice (t 2.793, p 0.017). The level of GnRH in group I was significantly lower than that of the control group (p < 0.05) (Figure 1). ANOVA tests showed that vitamin E had significant effects on brain GnRH in TCDD-treated male mice (F 3.017, p 0.029). The level of GnRH in group II was notably higher than those of group I, group III, and group IV (p < 0.05), while there were no significant differences among group I, group III, and group IV (Figure 1).

Figure 1. Effects of vitamin E on the level of brain gonadotropin releasing hormone in TCDD-treated male mice (n 8, mean + SE). T test, *represents a significant difference between the control group and experimental group I. LSD multiple range test, different superscript letters represent significant differences among experimental groups (p < 0.05).

300 Luteinizing hormone (ng/g) 250 200 150 100 50 0 Control group Group I a,*

Group II

Group III

Group IV

The effects of vitamin E on testis LH in male mice treated with TCDD

TCDD had significant effects on testicular LH levels in male mice (t 3.162, p 0.01). The level of LH in group I was clearly lower than that of the control group (p < 0.05) (Figure 2). Vitamin E had significant effects on the levels of testicular LH in TCDD-treated male mice (F 3.017, p 0.029). The levels of LH in groups II, III, and IV increased significantly compared to group I (p < 0.05), while there were no significant differences among group II, III, and IV. The levels of testicular LH had a decrease tendency with the increase of vitamin E dosage range group IIIV (Figure 2).

Figure 2. Effects of vitamin E on the level of testicular luteinizing hormone in TCDD-treated male mice (n 8, mean + SE). T test, *represents a significant difference between the control group and experimental group I. LSD multiple range test, different superscript letters represent significant differences among experimental groups (p < 0.05).

standard procedures. Sections (4-mm thick) were deparaffinized and rehydrated. After hematoxylineosin (HE) staining, the histopathology of the testes were observed, and the number of spermatozoa were counted under a light microscope (10 40). Six samples of each group were used for HE staining. Twenty fields for every sample were chosen to count spermatozoa. The average number of spermatozoa for the 20 fields was used as the sperm count for each sample.

The effects of vitamin E on testicular FSH in male mice treated with TCDD
TCDD had significant effects on the level of testicular FSH in male mice (t 2.550, p 0.029). The level of FSH in group I clearly decreased compared to the control group (p < 0.05) (Figure 3). Vitamin E had major effects on the level of testicular FSH in TCDD-treated male mice (F 3.521, p 0.034). The levels of FSH in group II, group III, and group IV were significantly higher than that of group I

Statistical analysis
All data were analyzed by the statistical software package SPSS 11.5. Differences between the control group and experimental group I was assessed by the

Yin et al.

155

Follicle-stimulating hormone (ng/g)

350 300 250 200 150 100 50 0 Control group Group I Group II a,* b

Testiclesperms (number /10 40 visual field)

80 b 60 40 20 0 Control group Group I Group II Group III Group IV a,* a b

Group III

Group IV

Figure 3. Effects of vitamin E on the level of testicular folliclestimulating hormone in TCDD-treated male mice (n 8, mean + SE). T test, *represents a significant difference between the control group and experimental group I. LSD multiple range test, different superscript letters represent significant differences among experimental groups (p < 0.05).

Figure 5. Effects of vitamin E on the number of spermatozoa produced by testes of mice treated with TCDD (n 8, mean + SE). T test, *represents a significant difference between the control group and experimental group I. LSD multiple range test, different superscript letters represent significant differences among experimental groups (p < 0.05).

and group IV levels (p > 0.05), and also between group II and group III (p > 0.05) (Figure 4).
0.3 Serum testosterone (ng/ml) b b

0.2

0.1 a,* 0 Control group Group I Group II

The effects of vitamin E on the number of spermatozoa produced by testes of male mice treated with TCDD
a

Group III

Group IV

Figure 4. Effects of vitamin E on the level of serum testosterone in male mice treated with TCDD (n 8, mean + SE). T test, *represents a significant difference between the control group and experimental group I. LSD multiple range test, different superscript letters represent significant differences among experimental groups (p < 0.05).

(p < 0.05), while there were no significant differences among group II, III, and IV (Figure 3).

TCDD had significant effects on the numbers of spermatozoa produced by testes of male mice (t 6.894, p < 0.001). The number of spermatozoa in group I decreased compared to the control group (Figure 5). Vitamin E had significant effects on the number of spermatozoa in the TCDD-treated male mice (F 20.45, p < 0.001). The spermatozoa numbers in both group II and group III were notably higher than those of group I and group IV, while there was no significant difference between group I and group IV, and also between group II and group III (p > 0.05) (Figure 5).

The effects of vitamin E on testosterone in male mice treated with TCDD

TCDD had significant effects on the level of serum testosterone in male mice (t 7.716, p 0.001). The serum testosterone level in group I was notably lower than that of the control group (p < 0.01) (Figure 4). Vitamin E had significant effects on the levels of serum testosterone in TCDD-treated male mice (F 57.56, p < 0.01). The levels of serum testosterone in both group II and group III were notably higher than those of group I and group IV (p < 0.001), while there was no significant difference between group I

The effects of vitamin E on the structure of testis in male mice treated with TCDD
TCDD damaged the testicular structure. The contortion observed in normal seminiferous tubules in testes was intact (Figure 6A), and the layers of male spermatogonia were clearly visible in the control group. The sperm, spermatocytes, and spermatogonia were identified in turn from the center to the bottom of the contorted seminiferous tubules, and are indicated by arrows in Figure 6C. The seminiferous tubules in the testes of group I displayed obvious swelling and increased areas of open space, and the arrows showed the rarefaction and edema of tubules (Figuer 6B).

156

Toxicology and Industrial Health 28(2)

Figure 6. The effects of TCDD on testis structure of male mice. (A) Testis slice from the control group (HE staining, magnification, 10 10). (B) Testis slice from group I (HE staining, magnification, 10 10). (C) Testis slice from the control group (HE staining, magnification, 10 40). (D) Testis slice from group I (HE staining, magnification, 10 40).

Necrosis of the spermatocytes and spermatogonia was also observed (Figue 6D). Vitamin E had a protective effect on the damage to the testicular structure elicited by TCDD. The layer of contorted seminiferous tubules in the testes was clear in group III, and the spermatogenic cells at different developmental stages are shown in turn from the bottom to the center of the tubules by arrows (spermatogonia, spermatocytes, and sperm). Necrosis of spermatogenic cells and edema was observed (Figure 7C), while necrosis of spermatocytes and edema in group II was found (the arrows showed necrosis of the spermatocytes) (Figure 7B). In addition, there was a greater amount of necrosis of spermatocytes in group IV compared to group II (the arrows showed necrosis of the spermatocytes and edema) (Figure 7D).

Discussion The effects of vitamin E on reproductive hormones in male mice treated with TCDD
GnRH is a key signaling molecule and one of the most important hormones controlling animal reproduction.

It is synthesized and secreted by the arcuate nucleus of the hypothalamus (Moore and Wray, 2000) and is typically associated with the medial preoptic area in mice (Saitoh et al., 1992). It can regulate the production of gonadal steroid hormones indirectly by enhancing the synthesis of LH and FSH. In addition, it can affect the accessory glands directly (Kovacs and Schally, 2001). FSH and LH secreted by the anterior pituitary can affect Sertoli cells and Leydig cells and, in turn, increase the secretion of testosterone from Leydig cells. The secretions of GnRH, FSH, and LH are regulated by hormones secreted from the testes. This feedback loop maintains a balance in the level of androgenic hormone secretion (Yao, 2005). The present study demonstrated that TCDD inhibited the levels of GnRH, LH, FSH, and testosterone. This finding is consistent with the results obtained from previous studies in male rats (Bookstaff et al., 1990a). However, Chaffin et al. (1997) found that TCDD induced the secretion of GnRH, LH, and FSH in female rats. These findings indicate that TCDD could have sex-specific effects on reproductive

Yin et al.

157

Figure 7. The effects of vitamin E on testis structure in TCDD-treated male mice. (A) Testis slice from group I, which was the same as panel D in figure 6 (HE staining, magnification, 10 40). (B) Testis slice from group II (HE staining, magnification, 10 40). (C) Testis slice from group III (HE staining, magnification, 10 40). (D) Testis slice from group IV (HE staining, magnification, 10 40).

hormones that differ between male and female animals. The mechanism of toxicity of TCDD in male mice is not clear. TCDD could have caused the decrease in testosterone by damaging the smooth endoplasmic reticulum or mitochondria of the Leydig cells, thereby interfering with enzymes involved in androgen synthesis (Roman et al., 1995). TCDD might reduce endocrine toxicity by changing the responsiveness of testicular granule cells to regulatory hormones or growth factors (Bookstaff et al., 1990b). Vitamin E is essential for reproduction in animals. It is an antioxidant present in all mammalian cells. Vitamin E stimulated both LH (Karanth et al., 2003) and FSH (Li et al., 2006) release. The results from the current study demonstrate that vitamin E at 20 mg/kg significantly reversed the decrease of GnRH, LH, and FSH levels caused by chronic TCDD exposure. Vitamin E at concentrations of 100 and 500 mg/kg antagonized the depression of LH and FSH levels, but there were no significant effects on the observed decrease of the GnRH level caused by TCDD, which might be due to feedback of LH and FSH to GnRH. Vitamin E at 100 and 500 mg/kg could boost the secretion of LH and FSH by the anterior pituitary,

which could then inhibit the secretion of GnRH. It is possible that the levels of LH and FSH in groups administered vitamin E at 20 mg/kg could not reach the threshold levels needed to stimulate the negative feedback to GnRH. Vitamin E at concentrations of 20 and 100 mg/kg alleviated the decrease in the serum testosterone level elicited by TCDD, while vitamin E at 500 mg/kg had no significant effect on this decrease. One potential explanation for this observation is that the accumulated overabundance of vitamin E in testis tissue in the 500 mg/kg group could have directly inhibited the secretion of testosterone. Vitamin E is positively associated with fetal growth in humans (Scholl et al., 2006), and it is a potent antioxidative agent both in vitro (Farris, 1990; Hassoun et al., 1995b) and in vivo (Bagchi et al., 1993; Hassoun et al., 1995a). Vitamin E reacts with hydroxy radicals in membrane phospholipids and effectively attenuates testicular DNA damage and the production of lipid peroxidation induced by Aspergillus flavus (Verma and Nair, 2002). However, vitamin E dosages above 2 mg/100 g body weight were not protective or therapeutic in the thymus and spleen of burned rats (Gao et al., 1992). Vitamin E can both act as an antioxidant and

158

Toxicology and Industrial Health 28(2)

promote oxidative action, and an overabundance of vitamin E could tip the balance towards the promotion of oxidative action. The mechanism of the antagonistic effects of vitamin E on TCDD-induced endocrine toxicity could result from one of the following mechanisms. (1) Vitamin E suppressed the aryl hydrocarbon receptor (AhR)mediated target gene expression induction by TCDD. It is well known that TCDD produces toxic effects through activating AhR, and the cytochrome P450 (CYP 450) gene is the target gene of AhR. Vitamin E could inhibit the transcription and expression of CYP450 and steroid dehydrogenase genes by inhibiting the TCDD-induced activation of AhR, and in turn, decrease the competitive binding of TCDD to the androgen receptor, thereby antagonizing TCDD toxicity. TCDD was found to increase the levels of CYP1A1, CYP1A2, and CYP1B1 mRNA in the human thymus cell line MCF10A (Ronald et al., 2006) and improve the expressions of osteoblast aryl hydrocarbon receptor (AhR) and CYP1A1 (Wejheden et al., 2006). Palaniappan et al. (2007) demonstrated that vitamin E could alleviate the increases of mRNA levels of CYP450, 3b-hydroxysteroid dehydrogenase and 17b-hydroxysteroid dehydrogenase. (2) Vitamin E inhibited oxidative stress caused by TCDD. Vitamin E antagonized the decrease in antioxidant activity and alleviated the increase of hydrogen dioxide elicited by TCDD (Latchoumycandane and Mathur, 2002b). Additionally, vitamin E reversed the increase of malondialdehyde (MDA) in testis tissue of mice (Tengerdy et al., 1984) and decreased lipid peroxidation caused by aflatoxins (Verma and Nair, 2001). Vitamin E succinate alone or in combination with ellagic acid suppressed brain oxidative stress caused by TCDD in a rat model (Hassoun et al., 2004, 2006). Previously, we found that vitamin E reversed the decrease in liver antioxidant gene transcription elicited in acute TCDD-treated mice (Yin et al., 2008)

The effects of vitamin E on sperm number and testis pathology in mice treated with TCDD
TCDD has antiandrogenic effects on both humans and animals, and it could therefore affect the form and function of testes. TCDD inhibited the growth and differentiation of seminal vesicle epithelium during postnatal development (Hamm et al., 2000), and caused atrophy and decreases in the diameter of the seminiferous tubules and in the spermatogonial population (el-Sabeawy et al., 1998). It also caused

the feminization of male mice and as well as their sexual dysfunction (Gray et al., 1997). An epidemiology study found that the testosterone level had a negative correlation with serum TCDD, and the number of spermatozoa decreased to half that of the normal levels in men exposed chronically to TCDD (Egeland et al., 1994). In the present study, we found that TCDD resulted in a decrease in the spermatozoa number and damage of the testis structure in mice. Most toxic effects induced by TCDD were mediated by binding to the AhR, which binds together with a second protein, aryl hydrocarbon receptor nuclear translocator, to the response elements of a number of target genes and thus modulates gene expression (Nau, 2006). The activated AhR induces the expression of various genes with xenobiotic response elements in their enhancer regions, such as the gene for cytochrome P450 1A1 (CYP1A1). Esser et al. (2005) found that TCDD interfered with the physiological signaling of the AhR, led to cell-specific changes in gene transcription and cell differentiation, and thereby producing toxic effects. TCDD combined with AhR, and thereby induced a change in the expression of monoamine oxidase related to CYP450 enzymes activated by AhR (Poland and Kimbrough, 1984). In the present study, the changes in the spermatozoa number and structure of the testis in mice could be related to oxidative stress caused by TCDD. TCDD decreased the antioxidant activity and increased the levels of hydrogen dioxide (Latchoumycandane and Mathur, 2002b) and MDA in the testis tissue in mice (Tengerdy et al., 1984) .Vitamin E could reduce the proportion of abnormal and dead spermatozoa of semen significantly and the quantities of alkaline phosphatase (ALP), glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) (Biswas et al., 2009). In the present study, we found that 100 mg/kg vitamin E could antagonize the structural changes elicited by TCDD, while 20 and 100 mg/kg vitamin E could alleviate the decrease in sperm number observed in TCDD-treated mice. The results indicated that vitamin E alleviated the reproductive toxicity of TCDD. In previous work, it was found that vitamin E reversed the decrease in the weight of the testis, epididymis, seminal vesicle and prostate induced by TCDD (Latchoumycandane and Mathur, 2002b), and it protected the spermatozoa and alleviated the biochemical changes in testis of mice exposed to Aspergillus flavus (Awad et al., 1994). Vitamin E also reversed the decrease of the number and vigor of

Yin et al.

159 vitamin E deficient rats using F2 isoprostanes. Journal of Nutrition 124(6): 810816. Bagchi D, Hassoun EA, Bagchi M, et al. (1993) Protective effects of antioxidants against endrin-induced hepatic lipid peroxidation, DNA damage and excretion of urinary lipid metabolises. Free Radical Biology and Medicine 15: 217222. Biswas, Mohan J, and Sastry KV (2009) Effect of higher dietary vitamin E concentrations on physical and biochemical characteristics of semen in Kadaknath cockerels. British Poultry Science 50(6): 733738. Bookstaff RC, Kamel F, Moore RW, et al. (1990b) Altered regulation of pituitary gonadotropin releasing homone (GnRH) receptor number and pituitary responsiveness to GnRH in 2, 3, 7, 8-tetra-chlorodibenzo-p-dioxintreated male rats. Toxicology and Applied Pharmacology 105(1): 7892. Bookstaff RC, Moore RW, and Peterson RE (1990a) 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin increases the potency of androgens and estrogens as feedback inhibitors of luteinizing hormone secretion in male rats. Toxicology and Applied Pharmacology 104(2): 212224. Chaffin CL, Trewin AL, Watanabe G, et al. (1997) Alterations to the pituitary gonadal axis in the peripubertal female rat exposed in utero and through lactation to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin. Biology of Reproduction 56(6): 14981502. Egeland GM, Sweeney MH, Fingerhu MA, et al. (1994) Total serum testosterone and gonadotropinsin workers exposed to dioxin. American Journal of Epidemiology 139: 272281. el-Sabeawy F, Wang S, Overstreet J, et al. (1998) Treatment of rats during pubertal development with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin alters both signaling kinase activities and epidermal growth factor receptor binding in the testis and the motility and acrosomal reation of sperm. Toxicology and Applied Pharmacology 150(2): 427442. Esser C, Steinwachs S, Herder C, et al. (2005) Effects of a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin, given at post-puberty, in senescent mice. Toxicology Letters 157(2): 8998. Farris MW (1990) Oxygen toxicity: unique cytoprotective properties of vitamin E succinate in hepatocytes. Free Radical Biology and Medicine 9: 333343. Fujimaki H, Nohara K., Kobayashi T, et al. (2002) Effect of a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin on immune function in male NC/Nga mice. Toxicology Science 66(1): 117124. Gao LX, Xu DY, Jin H, et al. (1992) A study on the adequate dosage of vitamin E in prevention and treatment

spermatozoa in mice exposed to mercuric chloride (Rao and Sharma, 2001), and it increased the ejaculate amount and the number of spermatozoa and decreased the number of malformed and necrotic sperm in male rabbits (Yousef et al., 2003). The mechanism of the antagonistic effects of vitamin E to TCDD could result from the ability of vitamin E to suppress the activated target gene expression of AhR and oxidative stress caused by TCDD. TCDD upregulated the transcription and expression of CYP1A1, CYP1A2, and CYP1B1 genes via AhR (Yang et al., 2001), while, vitamin E alleviated the increase of P450 1A2 mRNA caused by chronic TCDD treatment (Yin et al., 2008). The study reported by Latchoumycandane et al. (2002c) showed that vitamin E reversed the decrease of antioxidant activity and the increases of lipid peroxidation in testis of TCDD-treated rats. Hamm et al. (2000) found that vitamin E decreased the oxidative stress observed in the epididymis and epididymis spermatozoa and prevented the structural abnormalities of testis caused exposed to methoxychlor.

Conclusions
Our results suggested that vitamin E antagonized the reproductive endocrine toxicity, and alleviated the changes in testis structure and spermatoza number caused by TCDD. In the present study, the optimal dose of vitamin E was found to be 100 mg/kg/day. The present findings provided a potential approach for preventing or alleviating the endocrine toxicity associated with exposure to TCDD. Authors note
Hai-PingYin and Jian-Ping Xu are the first co-authors for this article.

Acknowledgements
The authors thank Dr Yakun Wan (Institute for System Biology, USA) and Dr Hongmin Wang (Sanford School of Medicine, The University of South Dakota, USA) for checking and revising the manuscript for English presentation.

Funding
This research was supported by the National Natural Science Foundation of China (No. 30770345, 30970454).

References
Awad JA, Morrow JD, Hill KE, et al. (1994) Detection and localization of lipid per oxidation in selenium and

160 of visceral injury in burned rats. Acta Nutrimenta Sinica 144: 339344. Gray LE, Ostery JS, and Kelece WR (1997) A doseresponse analysis of a single gestational dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male Long Evans Hooded rat offspring. Toxicology and Applied Pharmacology 146: 1112. Hassoun E, Bagchi D, and Stohs SJ (1995a) Evidence of TCDDinduced tissue damage in fetal and placental tissues and changes in amniotic fluid metabolises of pregnant CF1 mice. Toxicology Letters 76: 245250. Hassoun E, Bagchi D, Bagchi M, et al. (1995b) Effect of vitamin E succinate on smokeless tobacco-induced production of nitric oxide by rat peritoneal macrophages and J774A.1 macrophage cells in culture. Free Radical Biology and Medicine 18: 577583. Hassoun EA, Vodhanel J, and Abushaban A (2004) The modulatory effects of ellagic acid and vitamin E succinate on TCDD-induced oxidative stress in different brain regions of rats after subchronic exposure. Journal of Biochemical and Molecular Toxicology 18(4): 196203. Hassoun EA, Vodhanel J, Holden B, et al. (2006) The effects of ellagic acid and vitamin E succinate on antioxidant enzymes activities and glutathione levels in different brain regions of rats after subchronic exposure to TCDD. Journal of Toxicology and Environmental Health A 69(5): 381393. Hamm JT, Sparrow BR, Wolf D, et al. (2000) In uteri and lactation exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) after postnatal development of seminal vesical epithelium. Toxicology Science 54: 424430. Karanth S, Yu WH, Mastronardi CA, et al. (2003) Vitamin E stimulates luteinizing hormone-releasing hormone and ascorbic acid release from medial basal hypothalami of adult male rats. Experimental Biology and Medicine (Maywood) 228(7): 779785. Kovacs M and Schally AV (2001) Comparison of mechanisms of action of luteinizing hormone-releasing hormone (LHRH) antagonist cetrorelix and LHRH agonist triptorelin on the gene expression of pituitary LHRH receptors in rats. Proceedings of National Academy of Sciences USA 98(21): 1219712202. Kim SY and Yang JH (2005) Neurotoxic effects of 2,3,7,8tetrachlorodibenzo-p-dioxin in cerebellar granule cells. Experimental and Molecular Medicine 37(1): 5864. Latchoumycandane C, Chitra KC, and Mathur PP (2002a) Induction of oxidative stress in rat epididymal sperm after exposed to 2,3,7,8 tetrachlorodibenzo -p-dioxin. Archives of Toxicology 76(2): 113118. Latchoumycandane C, Chitra KC, and Mathur PP (2002c) The effect of 2, 3. 7, 8-tetrachlorodibenzo-p-dioxin on

Toxicology and Industrial Health 28(2) the antioxidant system in mitochondrial and microsomal fractions of rat testis. Toxicology 171(23): 127135. Latchoumycandane C and Mathur PP (2002b) Effects of vitaminE on reactive oxygen species-mediated 2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin toxicity in rat testis. Applied Toxicology 22(5): 345351. Li DJ, Xu ZS, Zhang ZH, et al. (2006) Antagonistic effects of vitamin E on the testicular injury by cyclophosphamide in mice. Zhonghua Nan Ke Xue 12(4): 318322. Mably TA, Bjerke DL, Moore RW, et al. (1992) In utero and lactational exposure of male rats to 2,3,7,8,tetrach-lorodibenzo-p-dioxin: Effect on spermatogenesis and reproductive capability. Toxicology and Applied Pharmacology 114: 118126. Moore JJ and Wray S. (2000) Luteinizing hormonereleasing hormone(LH-RH) biosynthesis and secretion in embryonic LH-RH neurrons. Endocrinology 141(12): 44864495. Moriguchi S and Muraga M (2000) Vitamin E and immunity. Vitamins and Hormones 59: 305306. Nau H. 2006. Impacts and impact mechanisms of dioxins in humans and animals. Dtsch Tierarztl Wochenschr 113(8): 292-297. Palaniappan M, Thirupathi M, and Karundevi B (2007) Effects of vitamins C and E on steroidogenic enzymes mRNA expression in polychlorinated biphenyl (Aroclor 1254) exposed adult rat Leydig cells. Toxicology 232(3): 170182. Poland A and Kimbrough RD (1984) Biological mechanisms of dioxin action. Bambury Report 18. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, p. 500. Rao MV and Sharma PS. (2001) Protective effect of vitamin Eagainst mercuric chloride reproductive toxicity in male mice. Reproductive Toxicology 15(6): 705712. Ronald D, Thomasa MR, Greena C W, et al. (2006) Cytochrome P450 expression and metabolic activation of cooked food mutagen 2-amino-1-methyl6-phenylimidazo[4,5-b]pyridine (PhIP) in MCF10A breast epithelial cells. Chemico-Biological Interactactions 160(3): 204216.Roman BL, Sommer RJ, Shinomiya K, et al. (1995) In utero and lactational exposure of the male rat to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin: impaired prostate growth and development without inhibited androgen production. Toxicology and Applied Pharmacology 134(2): 241250. Saitoh Y, Gibson MJ, and Silverman AJ (1992) Targeting of gonadotropin-releasing hormone axons from preoptic area grafts to the median eminence. Journal of Neuroscience Research 33(3): 379391.

Yin et al. Scholl TO, Chen X, Sims M, et al. (2006) Vitamin E: maternal concentrations are associated with fetal growth. American Journal of Clinical Nutrition 84(6): 14421448. Taketoh J, Mutoh J, Takeda T, et al. (2007) Suppression of fetal testicular cytochrome P450 17 by maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin: a mechanism involving an initial effect on gonadotropin synthesis in the pituitary. Life Science 80: 12591267. Tengerdy AE, Heinzerling RH, and Brown GL (1984) Enhancement of humoral immune response by vitaminE. International Archives of Allergy and Applied Immunology 44: 221226. Verma RJ and Nair A. (2001) Vitamin E ameliorates aflatoxin-induced biochemical changes in the testis of mice. Asian Journal of Andrology 3(4): 305309. Verma RJ and Nair A (2002) Effect of aflatoxins on testicular steroidogenesis and amelioration by vitamin. E Food and Chemical. Toxicology 40: 669672. Wejheden C, Brunnberg S, Hanberg A, et al. (2006) A rapid and sensitive response to dioxin exposure in the osteoblastic cell line UMR-106. Biochemical and Biophysical Research Communications 134(1): 116120.

161 Wu LL, Yan C, Su JF, et al. (2006) Effect of dan zhi xiaoyao san and its extracts on hypothalamus - pituitary gland- adrenal gland secretion in rats with chronic psychological stress. Journal of Guangzhou University of Traditional Chinese Medicine 23(5): 413415. Yang DF, Li YX, Yuan X, et al. 2001. Regulation of rat carboxylesterase expression by 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD): a dose-dependent decrease in mRNA levels but a biphasic change in protein levels and activity. Toxicology Science 64(1): 2027. Yao T (2005) Physiology. Beijing: People hygiene press, pp. 602603. Yin HP, Xu JP, Zhou XQ, et al. (2008) Antagonistic effects of vitamin E on transcription activity of CYP450 and antioxidant enzyme genes in the liver of mice treated acutely with TCDD. Acta Zoologica Sinica 54(6): 10381043. Yousef MI, Abdallah G.A, and Kamel KI (2003) Effect of ascorbic acid and vitaminE supplementation on semen quality and biochemical parameters of male rabbits. Animal Reproductive Science 76: 99111. Zhou XQ, Niu CJ, and Sun RY (2004) The effects of vitamin E on anti-acid stress ability in juvenile soft-shelled turtles, Pelodiscus sinensis. Comparative Biochemistry and Physiology Part C 137(4): 299305.

Copyright of Toxicology & Industrial Health is the property of Sage Publications, Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.