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Asian Pacific Journal of Reproduction
journal homepage: www.apjr.net
Original research http://dx.doi.org/10.1016/j.apjr.2016.07.001
Effects of potassium bromate on male rat growth and testicular histology
Adil Salim Elsheikh1*, Thoyba Faroug Fadul2'3, Eiman Mohamed-Elkheir Aboagla3, Ahmed Abdel Rahim Gameel4
1 Department of Applied Medical Sciences, Community College, Najran, Saudi Arabia 2Sudan Academy of Sciences, Khartoum, Sudan
3Animal Production Center, Ministry of Animal Resources, Kuku, Sudan
4Department of Pathology, Faculty of Veterinary Medicine, University of Khartoum, Sudan
ARTICLE INFO
ABSTRACT
Article history: Received 3 Apr 2016 Received in revised form 5 Jul 2016 Accepted 7 Jul 2016 Available online 6 Aug 2016
Keywords: Pubertal rat Potassium bromate Growth
Testis histology Infertility
Objective: To investigate the effects of supplementation of potassium bromate (KBrO3) to drinking water on the growth rate, pubertal weight, testis & epididymal weights and testicular histology of growing male rats.
Methods: Thirty male Wizard rat [age = 21 d, mean BW = (40.0 ± 5.3) g] were used. The rats were grouped into 5 treatment groups each group consist of 6 rats. T1 was offered drinking water supplemented with 100 mg, T2 200 mg, T3 300 mg, T4 400 mg/L KB1O3 for the duration of the experiment, while the control group was offered KBrO3 free water. The BW weights were taken weekly. Eight weeks after treatment the rats were sacrificed, testes & epididymae were excised and weighted. The testes were fixed and histopatho-logical sections of 5-6 mm were made, stained with H & E and examined under light microscope.
Results: The results showed that the growth rate, pubertal weight, testes & epididymal weights and testicular histology of growing male rats were significantly (P < 0.001) affected with KBrO3 supplementation to drinking water. The growth rate of the control group and T1 (100 mg/L KB1O3) fit well a sigmoid pattern growth curve and no differences in their growth rates were recorded, while the sigmoid pattern of the growth curves of treatment T2, T3 and T4 was disrupted. No difference (P > 0.05) in BW between the control and T1 was recorded. However, it is clear that T2, T3 and T4 significantly (P < 0.05) reduced the BW at puberty. Furthermore treatment groups recorded significantly (P < 0.001) low testicular and epididymal weights compared to the control. Supplementation of KBrO3 to drinking water caused serious changes in testicular tissue of the rats. The treatment rats' testes had distorted or even collapsed seminiferous tubules and narrow interstitial spaces. Upon magnification the seminiferous tubules appeared very narrow, mostly devoid of spermatogenesis and with no sperms.
Conclusion: Exposure of prepubertal rats to KBrO3 retards their growth, causes testic-ular hypoplasia and impairs spermatogenesis, which is a predictor of infertility or even sterility in the future.
1. Introduction
Infertility in men is steadily on the rise in many countries of the world [1,2]. Louis et al. has reported that the incidence of infertility among men in USA has reached 12% [3]. Many
Corresponding author: Adil Salim Elsheikh, Department of Applied Medical Sciences, Community College, Najran, Saudi Arabia. E-mail: adilelgarrai@yahoo.com
Peer review under responsibility of Hainan Medical College.
studies attributed the decline in male fertility to endocrine disrupting chemicals (EDCs) exposure especially the chlorinated compounds that are accused of inducing low sperm quality in men [2,4,5]. The EDCs are exogenous agents that interfere with synthesis, secretion, transport, metabolism, binding and elimination of natural blood hormones that are present in the body and are responsible for homeostasis, reproduction and developmental process [6]. The EDCs are also thought to act on nuclear hormone receptors such as androgen, estrogen, progesterone, thyroid, retinoid as well as
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on nuclear & non-nuclear and neurotransmitter receptors. Bromides are a member of the halide family a group of elements that includes fluorine, chlorine and iodine. The bromides are known of their binding to the same receptors that are used to capture iodine and interfere with thyroid hormones function resulting in a low thyroid state [6]. Thus bromides can be classified as a member of the EDCs. Potassium bromate (KBrO3), a member of the bromides group, is used in the maturation process of flour because of its oxidizing properties and has been used as a dough conditioner in the bread-making process for over 50 years. The WHO, Joint FAO/WHO Expert Committee on Food Additives [7] has temporarily recommended a maximum level of 75 ppm of KBrO3 for treating flour, provided that baking products prepared from such treated flour contain negligible residues of KBrO3 [7]. In Japan, the level has been set at 30 ppm under the same conditions as for WHO. In Canada KBrO3 is permitted as food additive, but it was delisted in 1994, however, it was reported as an impurity in packaging food papers [8]. Also KBrO3 has been introduced as an oxidizing agent, a primary standard, and a brominating agent in analytical chemistry. KBrO3 is also used in barley, cosmetics and water purification industries. In many animal experimental investigations KBrO3 has been classified as a possible carcinogenic substance [9-12]. Unfortunately in the under developing countries KBrO3 is used injudiciously as bread conditioner to maximize the bakeries profits. In Sudan, although supplementation of KBrO3 to bread is prohibited, the bakers illegally use unspecified quantities of KBrO3 as bread conditioner. This disaster is not limited to humans because in the periods of drought and scarcity of pasture the farm animals have to consume the dried surplus bread. Thus unknown amounts of KBrO3 reach the consumers through consumption of bread and/or animal products. Under this situation the consumers in Sudan are vulnerable to many of the health risks associated with consumption of KBrO3. Due to the sparse researches on the effects of KBrO3 on the growth and development of reproductive system, it is the objective of the current study to investigate the effects of KBrO3 on the growth rate, pubertal weight, testes & epididymal weights and testicular histology of growing male rats kept on KBrO3 during prepubertal period.
2. Materials and methods
2.1. Experimental animals housing and management
Twenty one day old male Wizar rats (n = 30) of mean BW of (40.0 ± 5.3) g were employed in this study. The rats were kept in rooms at 20-26 °C and a humidity of 40%-70% [13]. The lighting was set as described by Wong and Pace [14]. The rats were kept on a locally made pelleted ration (minced meat 1/ 2 kg, wheat flour 1/4 kg, salt 50 g and water). Fresh alfalfa was also provided daily. The body weight of the rats was monitored weekly using a digital balance and the weight was recorded in grams.
2.2. Supplementation of potassium bromate (KBrO3) to drinking water
The rats were identified and grouped into 5 treatments groups each group consist of 6 rats. T1 was offered drinking water
supplemented with 100 mg, T2 200 mg, T3 300 mg, T4 400 mg/ L KBrO3, while the control group was offered KBrO3 free water, for the duration of the experiment (5 weeks).
2.3. Histopathological samples
The rats were anesthetized with chloroform and sacrificed with cervical dislocation [15]. The testes and epididymidae were dissected and weighed. The testes were fixed and histopathological sections were made as described by Johannsen [16]. Briefly the tissues were dehydrated with alcohol and cleared with chloroform or xylene. After cleaning they were immersed in melted paraffin wax and quickly cooled to fill the intracellular spaces. Then section of 5-6 mm were made with rotary microtome, transferred to a warm water bath containing little amount of gelatin powder and left floating until mounted onto the slides. The slides were then incubation for 30 min at 50 °C to dry. The wax was removed and the slides were stained with H & E and examined under light microscope.
2.4. Experimental designs
This study is a one factorial design study to investigate the effects of supplementation of KBrO3 to drinking water on the growth rate, pubertal weight, testes & epididymal weights and testicular histology of growing male rats. Thirty male Wizard rat [age = 21 d, mean BW = (40.0 ± 5.3) g] were employed in this experiment to test the effects of KBrO3 on the above mentioned parameters. KBrO3 was supplemented to drinking water as described above. The BW of the rats was taken weekly and the BW at puberty was recorded 5 weeks after the treatment (the rats were 52 d the assumed age of puberty). The rats were sacrificed on week 8 of the treatment and their testes and epididymidae were weighted. Histopathological sections were prepared as above and examined under light microscope at 400x to investigate the changes that might happen in the STs and the process of spermatogenesis.
2.5. Statistical analysis
Data were subjected to ANOVA and differences at P < 0.05 were considered significant.
3. Results
3.1. The effect of KBrO3 on growth
As shown in Figure 1, KBrO3 treatment significantly (P < 0.001) influenced the growth rate of the growing rats. The growth of the control group and the rats in treatment 1 fit well a sigmoid pattern growth curve and no differences (P > 0.05) in their growth rate were recorded. The growth of rats in the remaining groups was significantly (P < 0.001) reduced by KBrO3 and the sigmoid pattern was disrupted.
3.2. The effects of KBrO3 on the weight at puberty
The puberty in male rat is around 52 d, since the rats were used after 3 week after birth, they will reach puberty on week 5 of the treatment. From Figure 2 it is clear that high KBrO3
Age (weeks)
Figure 1. The effect of КВгОз on the growth rate of male Wizard rats. C: Control group, Tr: 100 mg/L KBrO3, T2: 200 mg/L KBrO3, T3: 300 mg/L KBrO3, T4: 400 mg/L KBrO3.
Treatments
Figure 2. Effect of KBrO3 on weight at puberty of male wizard rats. C: Control group, T1: 100 mg/L KBrO3, T2: 200 mg/L KBrO3, T3: 300 mg/L KBrO3, T4: 400 mg/L KBrO3. a- b- c P < 0.001.
concentrations significantly reduced (P < 0.001) the BW at puberty. The mean BW of the rats in control group, T^ T2, T3 and T4 were (55.17 ± 3.72), (55.75 ± 1.77), (44.27 ± 0.67), (27.48 ± 1.13) and (27.75 ± 2.18) g, respectively. No difference (P > 0.05) in BW between the control and T1 was recorded. However, it is clear that the higher concentrations 200, 300, 400 mg/L KBrO3 significantly (P < 0.05) reduced the BW at puberty.
3.3. The effect of KBrOj on the testes weight
Figure 3 shows that the administration of potassium bromate reduced (P < 0.001) the testis weight of male rats. The mean testicular weight of control group, T1 T2, T3 and T4 were (0.73 ± 0.11), (0.49 ± 0.04), (0.24 ± 0.04), (0.24 ± 0.05) and (0.18 ± 0.03) g, respectively. No significant difference (P > 0.05) in the testicular weight among treatments was recorded.
3.4. The effect of KBrO3 on epididymal weight
The weight of the epididymae of the male rats were significantly (P < 0.001) influenced by potassium bromate (Figure 4). The mean epididymal weight of control group, T1, T2, T3 and T4
Treatments
Figure 3. Effect of KBrO3 on testicular weight of male rats.
C: Control group, T1: 100 mg/L KBrO3, T2: 200 mg/L KBrO3, T3: 300 mg/L
KBrO3, T4: 400 mg/L KBrO3. a- b- c P < 0.001.
T2 Treatments
Figure 4. Effect of KBrO3 on epididymal weight of male rats. C: Control group, T1: 100 mg/L KBrO3, T2: 200 mg/L KBrO3, T3: 300 mg/L KBrO3, T4: 400 mg/L KBrO3. a- b- c P < 0.001.
were (0.25 ± 0.04), (0.17 ± 0.03), (0.09 ± 0.02), (0.07 ± 0.02) and (0.05 ± 0.01) g, respectively. However, no differences (P > 0.05) between the weights of the epididymae of T2, T3 and T4 were observed.
3.5. Effect of KBrO3 on testicular histology
Figure 5 shows that KBrO3 altered the tissues of the testis of treated rats compared to that of the control. Plate 1 shows a testis of a control rat; the ST are round, the interstitial spaces are wide enough, the testes appear very active, with may cellular layers, spermatids, sperm formation and the tubules have many layers of germinal epithelium. However in plate 2 (T1) the ST of the testes appear elongate and there are few cellular layers and spermatids in ST. In plate 3 (T2) the ST had different shapes and the interstitial spaces were almost absent, so the tubules appear compact with very few spermatids formation. In plate 4 (T3) the STs are polyhedral and the interstitial spaces are narrow. Plate 5 (T4) shows that the STs are almost collapsed with peripheral dark stain and the tunica albuginea is shrunken. Plate 6 (T4) shows a magnified ST of plate 5, the tubule is devoid of sper-matogenesis with basal vacuolated small cells and large cells central cells and the lumen contains no sperms.
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Figure 5. Histopathological picture of the testes of rat treated with KBro3.
Plate 1. control with normal histological picture. Plate 2,3,4 and 5: testicular tubular structure of rats treated with potassium bromate. Notice the abnormalities (less spermatogonia layers, arrangement of tubules, narrow lumen and degeneration of interstitial tissue). Plate 6 shows the structure of a ST of rat in T4: notice the abnormalities (collapsed STs, no spermatogenesis, vacuolated cells, two layers of spermatogonia of different shape and size).
4. Discussion
The results of this study showed that KB1O3 supplementation to drinking water retarded the growth in growing rats and consequently reduced the pubertal, testicular and epididymal weights. Furthermore exposure to KBrO3 alters the histology of the rat testis and impaired spermatogenesis.
The growth of rats exposed to KBrO3 in this study was retarded and their testes weights were reduced. This was probably due to the binding of KBrO3 to the iodine receptors, minimizing iodine uptake by thyroid gland and causing iodine deficiency which lead to growth retardation [17]. Consequently the testis and the epididymal weights were reduced. The finding of this study is in agreement with toxicity and carcinogenicity studies on KBrO3 where short and long term oral administration of KBrO3 was proved to reduce the growth [10,18-20]. Contrarily KB1O3 was reported to have no effects on rat's body weight [21-23]. This discrepancy may be due to the differences in experimental rats age used and the dose of KBrO3 administered.
A remarkable decrease in pubertal body weight was observed in the current study. Similarly many investigators reported that EDCs reduce the pubertal body weight [24-26]. Factors that are known to modify the thyroid functions are also accused of limiting the biologically active iodine [27]. Sufficient iodine in tissues protects ATP (the biological units of energy) and helps cells to produce a healthy 36 ATPs through the Krebs cycle, which has profound implications on function of all cells. Also in the current study it is probable that the KBrO3 disrupted the antioxidant activity of iodine and its carbon-bonded products and iodolactones and consequently disturbed the thyroid gland functions [28] as a results the growth rate and epididymal & testicular weights were reduced. The low epididymal weights are due to growth retardation as well as azoospermia resulting from
testicular hypoplasia. The histopathological sections of the testes revealed severe hypoplasia of testicular structure and impairment of spermatogenesis process. These changes are due to a probable iodine deficiency induced by KBrO3 supplementation to water. Iodine helps to eliminate the oxidative stress, a known factor that induces germ cell apoptosis, because it neutralizes the hydroxyl ions [29]. Furthermore, it was reported that neonatal persistent and transient hypothyroidism cause prevalence of oxidative stress that is characterized by elevated lipid peroxide levels, protein carbonyl contents with decreased antioxidant enzyme levels. Since the testis is very rich in polyunsaturated fatty acids and has poor antioxidant defense system; the iodine deficiency induced by KBrO3 in the current study made the testes of the rats in this experiment vulnerable to oxidative stresses [29-31]. The iodine deficiency induced by KBrO3 perhaps hampered the biosynthesis of the thyroid hormones that are critical for coordination of physiology within and between cells and tissues and consequently affected the development and function of the gonads [32].
In conclusion, prepubertal exposure of male rats to KBrO3 retards growth, reduces testicular weight, alters testicular histology and impairs spermatogenesis. Thus it affects the male future fertility.
Conflict of interest statement
We declare that we have no conflict of interest. References
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