Physiological and Hormonal Effects of Titanium Dioxide Nanoparticles on Thyroid and Kidney Functions

Titanium dioxide nanoparticles (TiO 2 NPs) are generally used in different types of applications such as the industry of plastics, paper industry, paints, toothpaste, cosmetics, sunscreens, and in various lifestyles, because of the vast range of applications and our daily exposure to these nanoparticles and a lack of information on animal and human health this study was designed to reveal dose and time-dependent effects of TiO 2 -NPs on the thyroid gland and kidney functions in male rats. For this study 54, Sprague-Dawley albino adult male rats were classified into three main groups each of 18 rats treated for a particular duration (1,2, and 4) weeks respectively. Each group was subdivided into three subgroups each of six rats treated as follows; group (1) serve as normal control, group (2, and 3) intra-peritoneal treated with TiO 2 NPs (50,200) mg/kg respectively, rats are dissected at the end of each experiment and the weights of thyroid and kidney is measured. The result showed a highly significant decrease (p<0.01) in the thyroid gland and a highly significant increase (p<0.01) in kidney weights and TSH, blood urea, creatinine, and total protein, while a highly significant decrease (p<0.01) inT3 and T4 in all different doses (50,200) mg/kg at durations 1, 2 and 4 weeks. The outcomes of the present study illustrate a significant decrease in serum levels of T4 and T3 with exposure to TiO 2 NPS which disrupts thyroid function, while TiO 2 NPS raises the level of urea, total protein, and creatinine. This could be related to the high dose of TiO2-NPs and duration of the study, which caused degeneration and necrosis of kidney cells and damage to peritubules that led to the prevention of secretion which raised urea levels in the blood, also led to high levels of creatinine and total protein in serum because of the imbalance that occurred in the kidney functions.


Introduction
Nanoparticles: are small substances, that have at least one dimension in the range of (1-100)nm, the small size and high surface area of Nanoparticles make them principal entrants in all lineaments of modern life enforcement 1 . TiO 2 NPs are an odorless and noncombustible white powder that can crystallize in three structures brookite, rutile, and anatase, it is a semiconducting chemically inert material that displays photocatalytic activity when exposed to light 2 TiO 2 NPs have several applications in medical and engineering sciences and are used in removing pollution like organic toxic and heavy metals from the sewerage, and filtration of water and air 3 . The kidney is one of the vital organs in the body, excreting body waste products and drugs through highly specialized cells located in renal nephrons, kidney functions can be altered by many environmental contaminants, chemicals, and drugs 4 . Some studies showed that TiO2 NPs have possible adverse health effects after entering the body, initiate inflammatory mechanisms in cells, apoptosis, and generate oxygen free radicals which destructs the nucleus and DNA, also variable changes in the functions of cells 5 . When Gui et al 6 . suggested that TiO2-NPs induced kidney inflammation leading to tissue necrosis, disorganizing the renal tubules, and producing reactive oxygen species (ROS). NPs may alter the mitochondrial function of the thyroid epithelial cells, which might decrease cellular ATP production required as an energy source for synthesis and release of thyroid hormone 7 . The present study was aimed to evaluate the effects of

Materials and methods
Preparation of titanium dioxide nanoparticles (TiO 2 NPs ) solution TiO 2 NPs used in this study were obtained from Skyspring Nanomaterial company ( USA), they were in white powder Rutile 99.9 % purity, Particle size (30nm) diameter. The stock suspension was prepared by dissolving 1gram of powder TiO 2 NPs in 10 ml of distilled water and then mixed by vortex for 10 min to prevent agglomeration, from this stock suspension, two additional diluted TiO 2 NPs suspensions (low and high) doses were prepared: Group of 200 mg/kg of TiO 2 NPs (high dose) Group of 50mg/kg of TiO 2 NPs (low dose) Animals The study was conducted on (54) adult male Sprague-Dawley albino rats (Rattus norvegicus) their ages about (2.5-3) months as a mammalian model and average body weight of (250-260)gm. The animals were purchased from the Iraqi Center For Cancer And Medical Genetic research and then transferred to the animal house of the college of science, Mustansyria University. The males were kept for 10 days period of adaptation before starting treatment in clean plastic cages with metal network cover under the climate-controlled condition of the animal house with 22-25 temperatures. Animals were allowed to feed standard rats' pellets with free access to tap water.

Collection of Blood Samples and The Dissection of the Animals.
The end of each experiment was followed by weighing of the animals, they were completely anesthetized by diethyl ether for several minutes and blood samples were obtained by heart puncture were collected into non-heparinized tubes used in the hormonal examination. 5 ml of blood for the hormonal test collected from each rat was used to obtain sera (1.0-1.5) ml separated by centrifugation at 3000 rpm for 10 min, and then they were kept at -20ºC until analysis. The thyroid gland and kidney were removed by forceps and blade, then washed with normal physiological saline 0.9% (NaCl) to remove blood, blotted with filter paper to dry it for several minutes then weighed.

kidney Functions 1 Measurement of Blood Urea
The serum concentration of urea is determined using the Urease-method (enzymatic colorimetric method) according to linear company kit / Spain/2018.

Measurement of Serum Creatinine
Determination of creatinine levels in serum is done by using the kinetic colorimetric method, according to linear company kit / Spain/ 2018.

Measurement of Total protein
This analysis is done by using the Direct Biuret method for the determination of protein levels in serum, according to AGAPPE company kit /Switzerland /2018.

Statistical Analysis
The obtained data of this study are expressed as mean ± SE. The Statistical Analysis System-SAS (2012) program is used to observe the effect of different factors (concentration of TiO2 NPs and period) in the studied parameters. Two-way analysis of variance (ANOVA) is used depending on the least significant difference (LSD).

Thyroid weight and Functions
Results showed that TiO 2 -NPs had effects on thyroid weights as it was demonstrated in the Table. 1. Rats exposed to TiO 2 -NPs for 1 week demonstrated a highly significant decrease(p<0.01) in thyroid weight of treated groups with doses of 50 and 200 mg/kg (0.200±0.015) and (0.180±0.005) gm respectively compared to control groups (0.270±0.009) gm, as well, there was a highly significant decrease (p<0.01) in thyroid weights of experimental groups treated with TiO 2 NPs for 2 weeks in doses 50 and 200mg/kg (0.178±0.009) and (0.141± 0.020) gm respectively when compared to  Statistical analysis of the present study for the effect of TiO 2 -NPs on thyroid hormones that include TSH, T3, and T4 in Tables.2,3, and 4 Sequential reveals that: The values of TSH(µlU/ml) showed high significant increase (p<0.01) at different treatment durations (1, 2, 4) weeks exposing to TiO 2 -NPs at (50, 200) mg/kg as follows: during week 1 (0.600 ± 0.015) and (0.841± 0.037) compared to control group (0.051 ± 0.004), also during week 2 in different doses( 0.990 ± 0.003) and ( 1.50 ± 0.013) compared to control group (0.143 ± 0.091), in addition to week 4 at low and high doses ( 2.11 ± 0.013) and (2.83± 0.16) (µlU/ml) compared to control group,( 0.060 ± 0.005) (µlU/ml), demonstrated in Table. 2. High significant increase in the level of (TSH) (µlU/ml) also was observed when comparing between treated groups themselves depending on the concentrations as fixed factors while days were the variable factors in the concentrations (50 and 200) mg/kg exposed to TiO 2 -NPS with the increase of experimental duration 1, 2 and 4 weeks respectively.  High significant decrease (p<0.01) of (T3) (ng/ml) serum level of in both treated groups (50, 200) mg/kg (1.260±0.012),(1.060±0.051) (ng/ml) respectively exposed to TiO 2 -NPs for 1 week compared to the control groups (1.490±0.017) (ng/ml), also there was high significant decrease(p<0.01) in the level of (T3) in both doses (50, 200) mg/kg at 2 weeks (0.980±0.004),(0.810±0.005) (ng/ml) in comparison to control groups (1.231 ± 0.023), and at 4 weeks ( 0.77± 0.014), (0.416±0.01) (ng/ml) in comparison to control groups (1.46 ± 0.006) (ng/ml) demonstrated in Table.3. High significant decrease due to TiO 2 -NPS in serum level of (T3) (ng/ml) was observed when comparing between treated groups themselves depending on the concentrations as the fixed factors while days were the variable factors in the concentrations (50 and 200) mg/kg exposing to TiO 2 -NPS with the increase of experimental duration time 1, 2 and 4 weeks respectively.  values of T4(µg/dl) displayed non-significant decrease of treated groups (50) mg/kg (6.20±0.01) (µg/dl) , but showed significant decrease (p<0.05) of treated groups (200) mg/kg (5.08±0.79) (µg/dl) respectively at 1 week compared to control groups(6.91±0.01) (µg/dl), at week 2 also showed non-significant decrease(p<0.05) at dose (50) mg/kg ,(4.32±0.01) (µg/dl), while significant decrease (p<0.05) of treated groups (200)    The weight, size, and histology of the thyroid gland are affected by the production of thyroxin and its functional status, also some disorders of the thyroid gland such as overactive or underactive thyroid gland are established by enlargement of the thyroid gland as a part of the compensatory mechanism to maintain of thyroid hormone homeostasis 8 . Hypothyroidism due to thyroid gland hypertrophy shows a significant increase in thyroid gland weight in animals 9

Table 3. Effect of Dose and Time of TiO 2 -NPS on The Level of T3
. 10 Suggests that thyroid gland weight in hypothyroidism disorder rats demonstrated a significant increase. 11 After oral administration of TiO 2 -NPS (5 g / kg BW) for (65 days) in male rats, results illustrate that TiO 2 NPS effects became non-significant in the function of the thyroid gland, the difference may be due to the short duration, high dose and the type of treatment 12 .
The increase in time and doses of TiO 2 -NPS reduces thyroid weight, results of the present study showed a significant decrease in serum levels of T4 and T3 with exposure to TiO 2 NPS.

Kidney Weight and Functions
The statistical analysis of the weight of the right kidney is shown in Table.5      Statistical analysis of the present study of the effect of TiO 2 NPS on kidney functions that include Urea, creatinine, and total protein: Results of this study show a high significant increase (p<0.01) in urea level at different doses of TiO 2 NPS (50, 200)mg/ml at 1 week (49.70±0. 19),(58.00 ±0.01)mg/dl respectively compared with control groups 01±0.01)mg/dl, the level of urea at 2 week showed high significant increase (p<0.01) at different doses (50,200) mg/kg (60.00 ± 0.01),( 66.00 ± 0.01) mg/dl respectively compared with control groups( 41.00 ± 0.01)mg/dl, and at 4 week ( 68.00 ± 0.01),( 89.00 ± 0.03)mg/dl respectively when compared to control groups ( 40.67 ± 0.20)mg/dl showed in table. 7. High Significant increase in Urea level(mg/dl) is observed when comparing between treated groups themselves depending on the concentrations as fixed factors while days are the variable factors in the concentrations (50 and 200) mg/kg exposed to TiO 2 -NPS with the increase of experimental duration 1, 2 and 4 weeks respectively.   Table. 8. A significant increase in creatinine level(mg/dl) is observed when comparing between treated groups themselves depending on the concentrations as fixed factors while days are the variable factors in the concentrations (50 and 200) mg/kg exposed to TiO 2 -NPS with the increase of experimental duration 1, 2 and 4 weeks respectively. The statistical analysis results of total protein level show high significant increase (p<0.01) in level at different doses (50, 200) mg/kg for (1 , 2 ,4) weeks, at 1 week results are (7.50 ± 0.01), (9.00 ± 0.02) mg/dl respectively compared with control groups(5.30 ± 0.04)mg/dl, at 2 weeks treatment results were ( 8.70 ± 0.02, 11.60 ± 0.01mg/dl) respectively compared with control groups( 5.33 ± 0.01mg/dl), and at 4 weeks exposure results are(10.40±0.01),(12.60± 0.01)mg/dl respectively when compared with control groups( 5.32 ± 0.01)mg/dl demonstrated in Table. 9. High Significant increase in total protein level(mg/dl) is observed when comparing between treated groups themselves depending on the concentrations as fixed factors while days are the variable factors in the concentrations (50 and 200) mg/kg exposed to TiO 2 NPS with the increase of experimental duration 1, 2 and 4 weeks respectively. The effects of TiO 2 -NPS in the kidney, shown by 13 revealed that nanoparticles of TiO 2 -NPS have been depot in the cells of the kidney and caused the pathological changes and nephron-like toxicity in the form of inflammation in the glomeruli of the kidney, also 25 nm TiO 2 -NPS can significantly raise the urea level of serum compared with the control group, Kidney dysfunction is constructed in rats exposed to TiO 2 NPS, because of an increase in the level of blood urea and creatinine in the serum of mice. Treated groups of male rats using 3 doses of 30, 50, and 70 mg/kg TiO 2 NPS, observed no significant alterations when compared to their controls, but Twenty days after the last injection in the second stage of the treatment groups in all three doses of ( 30, 50, 70) mg/kg showed the increase (p<0.001) in serum urea level in exposed groups 14 .

Table 9. Effect of Dose and Time of TiO 2 -NPS on Total Protein Level
Meena et.al 15 Suggested that treated rats with 50 mg/kg of TiO 2 -NPS, increased levels of blood urea in serum which is directly correlated with the sign of glomerulonephritis toxicity, swelling in renal glomerulus, renal tubules crammed with the proteinic fluids because of the distribution of TiO 2 -NPS particles in kidneys. Another study showed that nanoparticles of TiO 2 -NPS have reduced the level of urea and creatinine 16 .The difference in the results obtained from animals treated with nanoparticles may be due to the type of animal, the route of administration to the nanoparticles orally,  19 .Responsibility for expelling the unsafe substances such as NPS from blood, after absorbance into the circulatory system it can be filtered by the renal system 20 .Some studies supported this result and suggest that TiO 2 -NPS exposure could induce apoptosis in different types of cells or organs, like the liver, spleen, and kidney 14 . 21 Showed that gold nanoparticles increase the level of urea, but urea level restored to normal after a few time, this is due to the initial shock of the kidney that gradually overcomes and the renal function returned to normal and explained the role of urea as a carrier of waste nitrogen, it plays some interactions in the system of nephrons. Both increase or decrease of the organ coefficients may be caused by -TiO 2 -NPS excretion or accumulation in the organs can lead to histopathological changes 22 . A single oral gavage of 5 g/kg TiO 2 -NPS particles stimulate liver and kidney damage, with hepatomegaly, hepatocyte necrosis, swollen renal glomerulus, and proteinic liquid aggregation in the renal tubules 13 . Filtration of blood in the glomeruli of the kidney nephron, propose the most common process to remove the nanoparticles through the kidneys 23 .So removal of creatinine in the bloodstream is done by the kidneys, the measurement of creatinine level in the blood can indicate the function of the kidneys 24 . If there is a failure in renal function, the creatinine level of serum increases 25 . Another study showed that the level of creatinine may be reduced after exposure to TiO 2 -NPS administration and this result is confirmed by Amara et al, who proposed that TiO 2 -NPS administration intraperitoneally at doses of 25 mg/kg (20-30 nm), after 7 days, as results showed a certain pathological change in the kidney of treated rats resulting in a high plasmatic uric acid level and a decrease of creatinine content 26 . While 27 revealed that the amount of TiO 2 -NPS in the mouse liver, spleen, lung, and kidneys reached high levels after 14 days of intraperitoneal administration. Researchers found that injected mice intraperitoneally with nano-titanium dioxide (20 mg/kg) in different sizes of the mice, after one week, they did not have any clear distinctive toxicity, mortality, and significant changes in liver and kidney 28 . Recent studies proposed that nanoparticles such as TiO2 -NPS after entry into cells, can induce an inflammatory response, (apoptosis) and generate the oxygen free radicals ROS which mutate DNA, and also change the functions of the cells body 29 . A cross-sectional study that included 100 individuals 50 hypothyroid cases and 50 normal controls, was done to determine thyroid dysfunction effects on serum values of Urea, creatinine, and uric acid, the results showed high significant increase in serum values of Urea, creatinine and uric acid in hypothyroid patients compared to controls which illustrates that hypothyroidism is associated with deteriorating renal function, hypothyroid-induced renal dysfunction may cause adverse clinical consequences, especially among patients on medications cleared by the kidneys, therefore these parameters should be regularly monitored in hypothyroid patients 30 . 31 observed a linear association between FT4 and Uric acid(UA) level in the population of the study, also reported a correlation between the prevalence of hyperuricemia and elevated FT4 levels, that may be impute to the effects of FT4 on purine nucleotide turnover and UA excretion. In a cross-sectional study that included108 individuals 56 cases with hypothyroidism and 52 controls; aged between 20 and 60 years (52 men and 56 women), after applying inclusion and exclusion criteria, serum TSH, T4, T3, uric acid, and creatinine were estimated, the results showed significant elevation in uric acid and creatinine levels as compared to control group, that explains hypothyroidism causes significant increase in serum uric acid and creatinine levels 32 . Autoimmune thyroiditis (AIT) is correlated with hypothyroidism, and different hypotheses have been put forward concerning the correlation between AIT and glomerulopathies, and several potential mechanisms for this relationship have been considered 33 . Iglesias et al., 34 Reported that renal development, kidney hemodynamics, glomerular filtration rate and sodium and water homeostasis are influenced by thyroid hormones, so hyperthyroidism and hypothyroidism affect renal function by direct renal effects. In many ways thyroid and kidney are interdependent on each other for optimal functioning of either organs, urinary loss of thyroid hormones and thyroid binding globulins in proteinuria cases in substantial amount leading to subclinical/overt hypothyroidism 35 . Subclinical hypothyroidism in SRNS is temporary and may improve with remission. 36 Revealed that prolonged proteinuria in steroid resistant nephrotic syndrome patients (SRNS) may cause renal tubules progressive damage and impaired absorption of low molecular weight proteins that will exhaust the thyroid reserve and result into overt hypothyroidism. Hypothyroidism, is a well-known complication of nephrotic syndrome (NS), it is a common feature of primary and secondary glomerular diseases and includes loss of protein in the urine and elevated urinary excretion of thyroid hormones and thyroxine-binding globulin 37,38 .
The outcomes of the present study illustrate an increment in kidney weight, Urea, Total protein, and Creatinine. This could be related to the high dose of TiO 2 -NPs and duration of the study, which caused degeneration and necrosis of kidney cells and damage to peritubules that led to the prevention of secretion which raised Urea levels in the blood, also led to high levels of Creatinine and Total protein in serum because of the imbalance that occurred in the kidney functions.

Conclusion
The outcomes of the present study illustrate the significant decrease in serum levels of T4 and T3 with exposure to TiO 2 NPS which disrupts thyroid function, while TiO 2 NPS rises the level of urea, total protein, and creatinine. This could be related to the high dose of TiO2-NPs and duration of the study, which caused degeneration and necrosis of kidney cells and damage to peritubules that led to the prevention of secretion which raised urea levels in the blood, also led to high levels of creatinine and total protein in serum because of the imbalance that occurred in the kidney functions.