Effect of excess (within tolerable range) and excessive iodine for different durations on functional status of testis with possible mechanism

Transcription

1 Section III. Chapter 4 Effect of excess (within tolerable range) and excessive iodine for different durations on functional status of testis with possible mechanism Introduction Thyroid hormones regulate important physiologic processes like physical growth and reproductive function, brain development and metabolism. Deficiency of iodine leads to a spectrum of disorders collectively called IDDs. For correction of iodine deficiency, supplementation of iodine through salt in general and/or other iodine containing mediators resulted in excessive iodine intake of the population particularly in environmentally iodine sufficient regions and poses a serious public health concern (Alsayed et al, 2008; Camargo et al, 2008; Seal et al, 2006). Excess iodine intake causes thyroid dysfunctions namely, hypothyroidism, hyperthyroidism, autoimmune thyroid diseases, endemic goitre and even thyroid cancer (Zhao et al, 2010) including infertility (Paulíková et al, 2002). Still births, abortions and embryo toxicity are found as common incidences among the excess iodine-induced population (Han et al, 2012). Such facts point out a close relationship between thyroid and the gonadal functions (Wagner et al, 2008). Iodination mechanism is associated with production of certain reactive products such as, iodinium (I + ) and hypoiodite (IO - ) ions. As such, excessive ingestion of iodine alters pro- / antioxidant status in several tissues (Joanta et al, 2006) resulting in a state of cellular oxidative stress, generation of reactive oxygen species (ROS), finally apoptosis (Pereira et al, 1990). Most of the cellular products like, polyunsaturated fatty acids, proteins, and nucleic acids are potential targets of ROS (Griveau et al, 1995; Agarwal et al, 2006). The sperm plasma membrane contains high concentrations of polyunsaturated fatty acids that are highly susceptible to ROS, and this may impair sperm function (Aitken et al, 2006). Oxidative stress is also considered as one of the important factors related to defective sperm function. As ROS scavenging enzymes are low in testis (Kao et al, 2008), excessive ROS generation in spermatozoa or disruption of the antioxidant defense systems in the male reproductive tract makes this organ a favorable target for oxidative damage (de Lamirande and Gagnon, 1995; Turner and Lysiak, 2008). Excess iodine decreases sperm count and alters the mean weight of testis (Shoyinka et al, 2008). The effect of excess iodine induced impairment of testicular function is far from clear in the existing literary description. The present study has been designed to highlight whether oxidative mechanisms are involved in 116 P a g e

2 defective testicular activities after excess iodine ingestion. Therefore, a dose and time dependent investigation under the influence of excess iodine was carried out in adult male rats to study the functional status of testis by evaluating the activities of testicular steroidogenic enzymes viz., assay of testicular 5 3β hydroxysteroid dehydrogenase (HSD) and testicular 17β - HSD enzyme activities, serum testosterone as well as serum gonadotropin(s) viz. FSH and LH levels. ROS generating potentiality of excess iodine, if any, has also been investigated in vivo following the assay of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX) and lipid peroxidation (LPO) levels of testis. Experimental design and animal grouping for the study As mentioned in the previous chapter The materials and methods used have already been discussed in earlier chapters. A total of 36 adult male albino rats broadly divided into two groups according to the duration of the treatment i.e. for 30 days and 60 days respectively. The animals of each group (18 rats) was further subdivided into three subgroups (6 in each subgroup) the first subgroup of six animals were kept as control, the second subgroup of animals of six rats was administered excess iodine and the third subgroup of animals also consisted of six rats were supplemented with excessive iodine. All the animals were maintained with a normal diet and adequate iodine as mentioned earlier (Chakraborty et al, 2015). Treatment schedule for the iodine administered animals were for 30 and 60 days respectively to provide time for the occurrence of multiple seminiferous cycles. All animals were sacrificed 24 hours after the last treatment by cervical dislocation following protocol and ethical procedures. Blood samples for testosterone, FSH, LH, T 3 and T 4 hormone assay were collected from the hepatic portal vein of rats. Plasma samples were separated by centrifugation at 2000 RPM, and stored at -50 C until assayed. The right testes of the experimental animals were used for the measurement of biochemical parameters. All the methods used for biochemical enzymes and hormone assay have been described in the methodology section. 117 P a g e

3 Results Body and organ weight The alterations of body weights and organ weights after administration of excess (100EI) and excessive iodine (500EI) for different durations are shown in Tables 4.1 and 4.2. It was observed that body weight of the control group of rats were increased in a progressive manner during the entire course of investigation and showed a net body weight gain of +13.6% and +27.0% after 30 days and 60 days respectively. On the other hand, the net body weight gain of the animals treated with 100EI and 500EI respectively for 30 days was +7.6% and +6.2%. However, after the administration of the same dose for 60 days showed body weight gain of +16.0% and 11.6% which were noticeably less than the respective control group. Serum testosterone level In excess iodine administered groups serum testosterone level was decreased significantly in a dose and time dependent manner as compared to control rats (Fig. 4.1). The decrease was more in the group treated with 500EI for 30 and 60 days respectively. Table 4.1.: Alterations in the body weight (g) of experimental animals subjected to excess (100EI) and excessive (500EI) iodine for different durations * Parameters 30 days treatment 60 days treatment Control 100EI 30D 500EI 30D Control 100EI 60D 500EI 60D Body weight ( Initial ) ± ± ± ± ± ± 6.11 Body weight ( Final ) ± ± ± ± ± ± 6.18 Gain in body weight (%) 13.6% 7.6% 6.2% a 27.0% 16.0% a 11.6% ab * Data is presented as mean ± SD, n = 6 (100EI 30D= KI at dose of 0.7 mg KI/100g body weight for 30 days, 500EI 30D= KI at dose of 3.5 mg KI/100g body weight for 30 days, 100EI 60D= KI at dose of 0.7 mg KI/100g body weight for 60 days, 500EI 60D= KI at dose of 3.5 mg KI/100g body weight for 60 days). a when compared with control; b when compared with KI. 118 P a g e

4 Table 4.2.: Alterations in the testicular and accessory sex organ weights of experimental animals subjected to excess (100EI) and excessive (500EI) iodine for different durations* 30 days treatment 60days treatment Parameters Control 100EI 30D 500EI 30D Control 100EI 60D 500EI 60D Testicular weight ± ± ± 0.09 a 1.49 ± ± ± 0.09 a,b Ventral prostrate ± ± ±10.79 a ± ± 9.21 a,b weight ±11.04 a,b Coagulating gland ± ± ± 3.15 a ± ± ± a,b Seminal vesicles ± ± ± 5.57 a ± ± ± 6.24 a,b (without fluid) 2 Cauda epididymis ± ± ± 7.41 a ± ± a ± 7.30 a,b *Data is presented as mean ± SD, n=6. Values bearing superscripts are significantly different by ANOVA P < (100EI 30D= KI at dose of 0.7 mg KI/100g body weight for 30 days, 500EI 30D= KI at dose of 3.5 mg KI/100g body weight for 30 days, 100EI 60D= KI at dose of 0.7 mg KI/100g body weight for 60 days, 500EI 60D= KI at dose of 3.5 mg KI/100g body weight for 60 days) 1 g/100g body weight; 2 mg/100g body weight; a when compared with control; b when compared with KI. Testicular steroidogenic enzyme activities A significant decrease in the activities of the testicular 5 3β HSD and 17β - HSD enzyme were found in excess iodine administered groups, compared to control (Fig. 4.2). The decrease was found more in both the groups treated with 100EI and 500EI for longer duration (i.e. for 60 days). However, no significant change was found in the activities of those enzymes in 100EI administered group for 30 days. ELISA of serum FSH and LH Excess iodine-administration caused a significant reduction in serum FSH and LH levels in a dose and time dependent manner compared to control animals (Fig. 4.3). Testicular lipid peroxidation (LPO) level Testicular lipid peroxidation level after the exposure at different doses and durations in the experimental animals are shown in Table 4.3. The level was significantly increased after the 119 P a g e

5 exposure of 100EI for 60 days and after 500EI for 30 and 60 days respectively. However, no such change in LPO level was noticed in the group treated with 100EI for 30 days. Fig Effect of excess (100EI KI at dose of 0.7 mg KI/100g Bw) and excessive iodine (500EI KI at dose of 3.5 mg KI/100g Bw) for 30 days and 60 days respectively on serum testosterone level. Each bar denotes mean ± SD of six animals per group. Mean values are significantly different by ANOVA at P < a= control 30D versus 100EI 30D versus 100EI 60D versus Control 60D (not significant), b= Control 60D versus 100EI 60D (significant), c= 100EI 60D versus 500EI 60D versus control (30D & 60D) (significant) Fig Effect of excess (100EI KI at dose of 0.7 mg KI/100g Bw) and excessive iodine (500EI KI at dose of 3.5 mg KI/100g Bw) for 30 days and 60 days respectively on A. Testicular Δ 5 3β - hydroxysteroid dehydrogenase activity and B. Testicular 17β - hydroxysteroid dehydrogenase activity of adult rats. Each bar denotes mean ± SD of six animals per group. Mean values are significantly different by ANOVA at P < A & B a= control 30D versus 100EI 30D versus 500EI 30D versus Control 60D (not significant), b= Control 60D versus 100EI 60D (significant), c= 100EI 60D versus 500EI 60D versus control (30D & 60D) (significant) 120 P a g e

6 Testicular antioxidant enzyme activities The activities of antioxidant enzymes viz. superoxide dismutase, catalase and glutathione peroxidase after the administration of excess iodine on dose and duration dependent manner are shown in Table 4.3. Both 100EI and 500EI had elevated the activities of all these enzymes significantly in the groups treated for 30 days however significant inhibition in the activities of all those enzymes were found in a dose dependent manner when the treatment continued for 60 days only. Serum thyroid hormone levels Serum T 3 after 100EI and 500EI for 30 days were 2.24±0.14 and 0.51±0.09 respectively and for 60 days were 2.21±0.15 ng/ml and 0.34±0.12 ng/ml while that of the control group was 2.5±0.30 and 2.24±0.14 ng/ml respectively showing a significant decrease (P<0.05) only in 500EI treated groups. Serum T 4 after 100EI and 500EI for 30 days was 5.67±0.19 and 6.52±0.78 µg/dl respectively and for 60 days was 7.31±0.31 and 7.89±0.22 µg/dl, while that of the control group was 5.41±0.90 and 5.68±0.21 µg/dl respectively indicating significant (P<0.05) increase in only 500EI treated groups (Table 4.4). Table 4.3.: Effect of excess (100EI) and excessive (500EI) iodine on oxidative stress markers in testis of experimental animals for different durations Parameters 30 days treatment 60 days treatment Control 100EI 30D 500EI 30D Control 100EI 60D 500EI 60D LPO level # 4.14 ± ± 0.10 a 5.19± 0.38 a 4.19 ± ± 0.58 a b 10.8± 0.61 ab SOD activity* ± ± 0.37 a ± 0.39 a 12.54± ± 0.61 a 7.64 ± 0.17 ab Catalase activity $ 5.35± ± 0.17 a 8.64± 0.60 ab 5.44± ± 0.81 a 3.62± 0.24 ab Glutathione 0.66± ± 0.03 a 0.7± ± 0.01 a 0.54± 0.04 ab 0.41± 0.05 peroxidase *Data is presented as mean ± SD, n=6. Values bearing superscripts are significantly different by ANOVA P < (100EI 30D= KI at dose of 0.7 mg KI/100g body weight for 30 days, 500EI 30D= KI at dose of 3.5 mg KI/100g body weight for 30 days, 100EI 60D= KI at dose of 0.7 mg KI/100g body weight for 60 days, 500EI 60D= KI at dose of 3.5 mg KI/100g body weight for 60 days); # nmol TBARS/g of tissue, * unit/mg protein/min, $ nmoles/mg of protein/sec, μmoles of GSH consumed/min/mg of protein. a when compared with control; b when compared with KI. 121 P a g e

7 Fig Effect of excess (100EI KI at dose of 0.7 mg KI/100g Bw) and excessive iodine (500EI KI at dose of 3.5 mg KI/100g Bw) for 30 days and 60 days respectively on A. Serum FSH level and B. Serum LH level of adult rats. Each bar denotes mean ± SD of six animals per group. Mean values are significantly different by ANOVA at P < A. a= control 30D versus Control 60D (not significant), b= control 30D versus 100EI 30D versus 500EI 30D versus 100EI 60D (significant), c= 100EI 60D versus 500EI 60D versus control (30D & 60D) (significant), B. a= control 30D versus 100EI 30D versus Control 60D (not significant), b= 100EI 30Dversus 500EI 30D versus 100EI 60D (significant), c= 100EI 60Dversus 500EI 60D versus control (30D & 60D) (significant) Table 4.4.: Alterations in serum T 3 and serum T 4 of experimental animals subjected to excess (100EI) and excessive (500EI) iodine for different durations* Parameters 30 days treatment 60days treatment Control 100EI 30D 500EI 30D Control 100EI 60D 500EI 60D Serum T 3 (ng / ml) 2.5± ± ±0.09 a,b 2.44± ± ±0.12 a,b Serum T 4 (µg / dl) 5.41± ± ±0.78 a 5.68± ±0.37 a 7.89±0.22 a,b *Data is presented as mean ± SD, n=6. Values bearing superscripts are significantly different by ANOVA P < (100EI 30D= KI at dose of 0.7 mg KI/100g body weight for 30 days, 500EI 30D= KI at dose of 3.5 mg KI/100g body weight for 30 days, 100EI 60D= KI at dose of 0.7 mg KI/100g body weight for 60 days, 500EI 60D= KI at dose of 3.5 mg KI/100g body weight for 60 days) a when compared with control, b when compared with KI. 122 P a g e

8 Discussion To evaluate the effect of excess (100EI) and excessive (500EI) iodine on adult male reproductive physiology, potassium iodide (KI) at relatively high however nontoxic doses was administered for different durations and certain important parameters of testicular morphology and functions were investigated. Exposure of iodine in excess markedly reduced the net body weight gain of the experimental animals depending on the doses and durations when compared to the control animals. These findings are consistent with the earlier work where excess iodine was found to be associated with altered metabolic condition (Yang et al, 2006). Excess iodine caused a decrease in weight gain of calves (Paulíková et al, 2002), young rats and survival rate of pups (Schone et al, 2006). Reduced body weight after excess iodine might be for the suppression of thyroid hormone activity and consequential reduction in cellular oxidation (Lewis, 2004; Chakraborty et al, 2014). In consistent with decreased body weight a decrease in testicular as well as in accessory male sex organs weight viz. ventral prostate, seminal vesicle, coagulating gland and epididymis were also observed in a dose and time dependent manner in excess iodine administered animals. Iodine intake at relatively higher concentrations was found to affect the growth and differentiation of germ cells as well as their degeneration at different levels of their maturation. Perhaps the possible cause for reduction in the weight of the testis is due to reduction in number of germ cells and elongated spermatids as the weight of testis has been found to be largely dependent on the mass of differentiated spermatogenic cells. Presence of testosterone at optimum level is essential for completion of meiosis, formation of spermatids and conversion of pachytene spermatocytes to round spermatids (Chandra et al, 2007). Suppression of testosterone level under the influence of excess iodine as observed in this study might be the possible reason for the interruption of growth and differentiation of the germ cell as well as their degeneration. There are evidences that excess iodine induces hypothyroidism (Yang et al, 2007; Roti et al, 1991; Chow et al, 1991; Chakraborty et al, 2015); while plasma testosterone level and the testosterone binding globulin in plasma are decreased in hypothyroid condition (Cavaliere et al, 1988). There was no virtual change in serum T 3 level in 100EI groups. Thyroid functions remain unaltered even after continuous increased iodine intake with the use of 200 to 300 times higher dose than the daily requirement (Paulikova et al, 2002). However, altered synthesis of thyroid hormone was recorded only after long term supplementation of iodine at the dose 500 times than their daily requirement (Paulikova et al, 2002). In consistent with these observations significant decrease in serum T 3 level was found in 500 time s excess 123 P a g e

9 iodine-administered groups. On the other hand, unlike serum T 3, serum T 4 level was significantly higher in all the excess iodine-administered groups except the group treated with 100EI for 30 days. This is due to the inhibition of 5- deiodinase activity resulting in a decrease in the generation of T 3 from T 4 after high doses of iodine administration (Han et al, 2012). Testicular 5 3β hydroxysteroid dehydrogenase (HSD) and 17β HSD are the key enzymes of testosterone biosynthesis and the activities of those enzymes were decreased in a dose and time dependent manner in excess KI administered animals. Such effects were at par with the decreased testosterone synthesis as has been reflected by the decreased level of serum testosterone. Therefore, excess iodine not only decreases the synthesis of testosterone by developing a hypothyroid condition but also down regulates the activities of these two P- 450 enzymes in the testicular cells. Low serum testosterone stimulates the levels of LH and FSH which in turn enhance the production of more testosterone by feedback mechanism (Maneesh et al, 2006). However, low serum testosterone level did not enhance serum LH and FSH levels in excess iodine induced experimental animals as observed in this study. Earlier evidence shows an inappropriate response of pituitary gland for secreting serum LH and FSH in spite of a decrease in serum testosterone level and considered as a central effect on the interaction between the nervous system and endocrine system (Maneesh et al, 2006). This might be the reason of low LH and FSH levels in parallel to a low testosterone level as found in this study. During steroidogenesis certain reactive oxygen species are generated due to leakage of electron outside the electron transfer chain that normally is counteracted by antioxidant enzyme defense system present in testis (Chandra et al, 2007). Any disturbance may thrust these radicals not only to stimulate lipid peroxidation but also to produce an alteration in the level of protein and DNA causing cellular damage (Joanta et al, 2006). Therefore it is predicted that excess iodine caused an imbalance in the pro- and antioxidant system in testis resulting cellular damage in testicular germ cells by increasing lipid peroxidation (LPO). The role of sodium iodide symporter (NIS) in concentrating iodide in thyroid cells is well established (Russo et al, 2011). Recently, presence of NIS mrna and protein has been isolated from germinal and Leydig cells of fetal and adult testis of human, mouse and rat (Russo et al, 2011). Therefore iodine in excess amount gets concentrated in testicular tissues through NIS. This may further develop oxygen-derived free radicals forming molecular iodine reacting with iodinium cation (Joanta et al, 2006). Thus excess iodine may cause 124 P a g e

10 enhanced generation of free radicals resulting in the overall deterioration of testis as well as mammalian spermatozoa which are rich in poly unsaturated fatty acids (PUFA). A strong correlation exists between excess iodine and LPO in extra-thyroidal tissues other than testis (Joanta et al, 2006; Vitale et al, 2000) and that might be considered as a key mechanism of the ROS induced damage in both in testicular cells and sperms leading to their deformity, as found in this study. When ROS induced testicular damage occurs, a number of antioxidant enzymes participate to ameliorate this chain reaction for scavenging the ROS generation (Mates et al, 1991). Superoxide dismutase (SOD) is considered as the first line of defense followed by catalase and glutathione peroxidase (GPX), the important oxidative stress markers. All these enzymes act in a cooperative and synergistic way to ensure cellular protection to eliminate active oxygen species, failing which a deviation in homeostasis of intracellular components occurs (Chandra et al, 2010; Michiels et al, 1998). The overall defense mechanism was significantly enhanced in 30 days excess iodine treated group reflecting the compensatory efforts of antioxidant defense in testis during altered pro- and antioxidant balance environment. However, when the treatment was extrapolated for 60 days, all concerned antioxidant enzyme activities were failed to cope up the situation possibly due to breakdown of antioxidant defense mechanism creating a state of cytotoxicity and testicular damage. The overall results revealed that regular consumption of excessive iodine even in its tolerable range causes an imbalance in between pro- and antioxidant status of testis generates ROS and down regulates testosterone synthesis through hypothalamo-pituitary-testicular axis all these in turn impairs overall reproductive functions in adult male rats. The present study thus elevates apprehension about the reproductive health in populations consuming excess iodine for considerably long periods. 125 P a g e