Abstract

The present investigation was designed to investigate the curative and protective effect of thyme as a natural product on thyroid and liver status in hypothyroid rats induced by propylthiouracil (PTU). The rats were classified into 4 groups; group I: control, group II, was intraperitoneally injected with 10 mg PTU/kg, for 21 days, group III was i.p. injected with 10 mg PTU/kg, for 21 days followed by 5% thyme for four weeks, and group IV was i.p. injected with 10 mg PTU/kg, for 21 days followed by 10% thyme for four weeks. Serum total cholesterol, HDL, LDL, triglycerides, AST, ALT, alkaline phosphatase, total protein, albumin, urea, creatinine, Na⁺, K⁺, GSH, GSH-px, SOD, MDA, TNF-∝, serum triiodothyronine (T₃) and thyroxine (T₄), were evaluated. Propylthiouracil injection resulted in a significant increase in total cholesterol, LDL, triglycerides, AST, ALT, ALP, urea and creatinine, MDA, and TNF-∝, whereas HDL, total protein, albumin, Na+, K+, GSH, GSH-px, SOD, T₃ and T₄ decreased significantly compared to control. The high dose of thyme extract (10%) ameliorated most of the harmful effect of PTU, this may be due to its contents of flavonoids, thymol, phenols and luteolin.

1. Introduction

The thyroid gland synthesizes and releases thyroid hormones; triiodothyronine (T3) and thyroxine (T4). These amine hormones are containing iodine in the vertebrate ¹ and are necessary for optimal growth, development, and function of tissues. They have a great effect on oxygen consumption and metabolic rate of all cells including hepatocytes ^{2, 3}, thus alter hepatic function. The liver metabolizes T3 and T4 through conjugation, excretion, peripheral deiodination, in addition to the synthesis of thyroid-binding globulin, and thus controls their endocrine actions ⁴. Hence, normal levels of circulating thyroid hormones are necessary for the liver to work properly. Youssef and Mullen ⁵ suggesting that abnormal thyroid function may lead to hepatic dysfunction and vice versa ^{4, 6, 7}.

Hypothyroidism is a clinical syndrome caused due to deficiency of thyroid hormones i.e, characterized by decrease in serum T3 and T4 and increase in serum TSH concentration ⁸. Recent studies have shown that hypothyroidism accompanied by an increased production of reactive oxygen species ⁹. Many investigations have suggested that hypothyroidism may have symptoms that similar to liver disease (pseudo-liver disease): such as myalgias, fatigue and muscle cramps in the presence of an increased aspartate aminotransferase from a myopathy as well as coma associated with hyperammonaemia in myxoedema coma and myxoedema ascites. The liver biopsy findings revealed central congestive fibrosis in a number of patients ¹⁰.

Few studies have confirmed the pathophysiological influence of hypothyroidism, including its effect on liver function.

Propylthiouracil (PTU) has been involved in drug causing liver injury in hyperthyroid patients. Reported injury has ranged from a mild asymptomatic elevation of liver enzymes to acute liver failure ¹. Propylthiouracil has been used for hyperthyroidism treatment since the 1940s, but over the years reports of marked hepatotoxicity have come forth, particularly in children ^{2, 3}. This led to a similar warning by the European Medicines Agency and the United Kingdom Medicines and Healthcare Regulatory Agency later that year ¹¹. This research gives a concise review of the data on hepatotoxicity related to the anti-thyroid PTU ^{4, 12}. Generally, PTU is considered a drug treatment of the second degree except in patients suffering from allergy to methimazole.

Thyroid hormones influence all organs of vital systems and, in excess, can elevate metabolic and heart rates, ventricle contractility, and gastrointestinal motility as well as excitability of central nervous system and muscle ¹³.

Triiodothyronine (T₃) regulates the basal metabolic rate of all cells, including hepatocytes through thyroid hormone receptor and thereby modulate hepatic function ⁶. Based on this background, liver is a main target organ for thyroid hormone with important metabolic roles ⁴.

Recently, there is an ongoing studies in identifying the effect of various plant products as antioxidants, antimutagens, and anticarcinogens. Lamiaceae family like rosemary, thyme, sage, and oregano are well known for their antioxidant activity. The antioxidant action of many natural plant extracts is because of the presence of phenolic compounds or polyphenols ¹⁴.

Thyme (Thymus vulgaris) is belonging to the Lamiacea family, an aromatic native herb in the Mediterranean region but now can be found in many areas of the world with temperate climates ¹⁵. It is a perennial shrub with greenish-gray aromatic leaves. Thyme was now widely cultivated as spice, tea and herbal medicine ¹⁶. It has been commonly used in food industry especially for the flavor and preservation. The leafy parts of thyme are often added to food products and also used as a herbal medicine plant ^{17, 18}. Thyme essential oil (EO) contains many different ingredients, most of which possess antioxidant and antimicrobial properties and inhibits lipid peroxidation ¹⁹. The most important compounds of thyme are the phenols thymol (44-60%) and major active constituent carvacrol (2.2-4.2%), in addition to the monoterpene hydrocarbons p-cymene (18.5-23.5%) and c-terpinene (16.1-18.9%) ²⁰. Thyme oil, may help in antimicrobial fight. Aqueous extracts of oregano or thyme oil with good antibacterial activity could also be used in selected cases ²¹.

2. Materials and Methods

Forty male Rattus rattus albino rats weighting 120-150 g were purchased from the National Research Center, Egypt. The rats were adapted on a stock standard diet and tap water ad libitum. The study was conducted in accordance with the guidelines set by the CIOHS & ICLAS International Guiding Principles for Biomedical Research involving animals (2012), which accordance with the Guide for the Care and Use of Laboratory Animals (8^th Edition, 2011, published by The National Academies Press, 2101 Constitution Ave. NW, Washington, DC 20055, USA). This guide was approved by the Ethical Committee at National Center for Radiation Research and Technology, Egyptian Atomic Energy Authority, Cairo, Egypt (NCRRT- EAEA).

Propylthiouracil (PTU) was obtained from El-Gomhoria Egypt Company, dissolved in ethanol. PTU intraperitoneally injected as 10 mg/kg for 21days.

The dried leaf powder of thyme was purchased from the National Research Center (NRC), Dokki, . The powder was extracted with boiled distilled water for 10 minutes and then left to cool. After stirring, the mixture was kept for gavage. Thyme prepared in concentrations of 5% and 10% ²².

Animals were randomly classified into four equal groups. Group I, as a control. Group II was injected intraperitoneally by 10 mg PTU /kg, for 21 days. Group III was i.p. injected with 10 mg PTU/kg, for 21 days followed by oral administration of 5% thyme extract for 4 weeks. Group IV was i.p. injected with 10 mg PTU/kg, for 21 days followed by oral administration of 10% thyme extract also for 4 weeks.

Animals were sacrificed after termination of the experiment. Blood samples were withdrawn by cardiac puncture after light anesthesia. Blood was gathered in test tubes and centrifuged at 3000g for 10min. Sera were then separated and kept frozen for the following biochemical analysis. The dissected liver tissues were washed and homogenized in phosphate buffered saline (PBS; 10%). Liver homogenate was centrifuged at 5000g at 4°C for 15 min. and the supernatant was stored at -20C for various biochemical assays.

The collected sera were used for determination of serum total cholesterol, high and low density lipoproteins (HDL & LDL) according to Stein ²³. Triglycerides were evaluated by the method of Young ²⁴. Transaminases activities were measured by the method of Reitman and Frankel ²⁵. Serum alkaline phosphatase was determined by Moss ²⁶. Serum total protein and albumin levels were determined according to the method of Henery et al. ²⁷ and Dumas and Bigga ²⁸, respectively. Urea and creatinine were determined according to Patton ²⁹ and Henry, ³⁰ respectively. Na⁺ and K⁺ were evaluated according to Trinder ³¹ and Caley ³², respectively. Malondialdehyde and TNF-∝ were also determined in serum by Satoh ³³ and Maskos et al. ³⁴, respectively. Serum triiodothyronine (T₃) and thyroxine (T₄) levels were measured by radioimmunoassay (RIA) technique according to the method of Larsen ³⁵ and Tietz, ³⁶, respectively. Glutathione reduced (GSH) and glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activities and MDA were assayed in liver tissue according to the methods of Paglia and Valntine ³⁷, Marklund and Marklund ³⁸, and Yoshioka et al. ³⁹, respectively.

All data were statistically analyzed using SAS software ⁴⁰, as a one-way analysis of variance (ANOVA). Statistical significance was set at 0.05% probability.

3. Results

The following tables will illustrate the effect of PTU on the selected parameters and the ameliorative effect of thyme administration with the two doses compared to control group values.

Table 1 demonstrated a significant elevation (P≤ 0.05) in cholesterol, LDL and triglycerides accompanied with a significant decrease in HDL level in PTU treated group compared to control. Thyme decreased cholesterol and triglycerides levels compared to PTU group, while HDL significantly increased compared to PTU group after thyme administration. 10% thyme resulted in a marked improvement in cholesterol, HDL and LDL values compared to control values.

As seen in Table 2 AST, ALT and alkaline phosphatase were significantly increased (P≤ 0.05) in PTU treated group compared to control, whereas, significantly decreased (P≤ 0.05) in comparison with PTU group after thyme administration. 10% of thyme was more effective than 5% in lowering ALT and ALP. Total protein and albumin were significantly lowered (P≤ 0.05) in the PTU-treated group and returned back to normal values upon thyme administration.

Results shown in Table 3 revealed that urea and creatinine were significantly increased in PTU-group (P≤ 0.05) compared to control, whereas, they were significantly decreased after thyme treatment particularly 10% dose, compared to PTU group. Thyme administration improved the values near to control in both low and high doses 5% and 10%, particularly urea level. The levels of sodium and potassium levels were significantly decreased in PTU-group compared to control and turned back to control values after thyme treatment.

In Table 4 GSH, GSH_Px, SOD were highly significantly decreased in liver tissues, whereas MDA was highly significantly increased in serum and tissue in PTU group. Thyme administration mostly ameliorated those effects in the higher dose more than in the lower one. TNF was highly significantly increased (P < 0.05) in PTU group and its value decreased gradually as the thyme dose increases.

Table 5 showed that PTU significantly decreased the values of T₃ and T₄ (P≤ 0.05) compared to control. Thyme extract ameliorates thyroid hormones level, particularly T₃, which became near to the control value.

4. Discussion

Thyroid hormones modify the functions of body cells, tissues, and organs. They play vital roles in growth, differentiation, maturation, as well as metabolism ²⁷. Interaction between thyroid hormones and liver function was reported earlier ^{3, 20}. The effect of hypothyroidism on liver dysfunction has been evaluated ³. However, more studies about the effect of hypothyroid and hyperthyroid states on hepatic function are required.

In the present investigation, the injection with PTU significantly raised total cholesterol, triglycerides and LDL, when compared to control. A reduction in plasma TG led to a decrease in the TG content in the liver. Thyme supplementation led to a significant reduction in serum cholesterol, TG and LDL levels associated with a significant increase in HDL. It has been suggested that carvacrol and thymol are important essential oils of thyme ⁴¹.

Traditionally Thymus linearis Benth. have been used for treatment of various diseases. The extract also exhibited a reduction in serum cholesterol, triglycerides and LDL levels, while a significant increase in HDL level was observed. Moreover, these findings further authenticate the traditional use of this plant in folklore medicine ⁴².

Injection with PTU resulted in a significant increase in ALT and AST activities as compared with control, while 5% and 10% of thyme showed a significant reduction in liver enzymes as compared with PTU group. These results were in agreement with Al-Saimary et al. ⁴³ and Dias et al. ^{44, 45}. Frequent monitoring for hepatic dysfunction induced by PTU is of great importance because timely discontinuation of this drug and implementation of non-invasive therapeutic interventions may prevent liver failure or even death ^{2, 46, 47}. Co-administration of T. vulgaris extract with alcohol has a protective effect on hepatocytes manifested as remarkable decrease in ALP, AST and ALT activities and observable increase in total protein, albumin and globulin synthesis compared to control ⁴⁸. The results of the current study revealed that PTU lead to a significant increase of AST, ALT, urea and creatinine. The significant elevation in liver enzymes indicate alteration in the hepatic tissues and biliary system ⁴⁹, structural damaging of liver integrity, because these enzymes are released into plasma as a result of cellular damage ^{50, 51}. The improved biochemical parameters, histological picture of the liver and kidney in animals treated with PTU and administered thyme extract revealed the protective role of the extract against PTU-mediated liver injury in a dose dependent manner through its anti-oxidant and free radicals scavenging properties, which mainly due to the presence of phenolic compounds thymol and carvacrol ^{52, 53}. Although PTU-induced liver failure is not common in clinical examination, liver function should be appropriately evaluated during PTU treatment ^{54, 55}.

In the present study, serum urea was significantly increased in PTU group while decreased in 5% and10 % thyme-fed groups as compared with control. These results were in agreement with that reported by Al-Qattan et al. ⁵⁶ and Shirpoor et al. ⁵⁷. The increase in urea and creatinine levels in PTU treated rats is due to harmful and stressful effect on renal tissue. Taken together, the elevated level of urea and decreased level of total protein and albumin is an indicator of inhibition of protein anabolism and increase of protein catabolism and/or renal dysfunction. These results strongly indicated that PTU had harmful and stressful effects on the hepatic and renal tissues.

The significant decrease in serum total proteins may be due to the disassociation of polyribosomes from the endoplasmic reticulum and also due to defects in protein biosynthesis ⁵⁸. Consequently, albumin level was reduced as it represents the larger portion of serum proteins also due to increased renal loss of albumin due to the nephrotoxicity ⁵⁹. Sharpe et al. ⁶⁰, who reported that liver disorders are related to a decrease in the serum levels of total proteins. The anti-inflammatory effects of thyme extract should be interpreted with caution, due to its ambiguous dose-related effects.

Thyme intake enhance reduction in liver enzymes, urea and creatinine levels in hypercholesterolemic rats, accumulates cholesterol and develops inflammation of the liver and kidney. Accumulation of these components may result in hepatic satellite cells activation, and trigger the factors causing steatohepatitis such as steatosis, inflammation, fibrosis and cellular ballooning ⁶¹.

The essential oil of thyme plant had harmless effect on liver and kidneys because of inducing little changes in aminotransferase activity in rat plasma and desirable changes in total cholesterol ⁶². Creatinine level showed a highly significant increase in PTU group, while 10 % thyme-fed groups reduced creatinine as compared with control ⁵⁸.

Biologically important elements (Na⁺, K⁺) have also a marked effect on maintenance of homeostasis by participating in various physiological pathways ⁶³. Altered levels of these elements may induce a number of events including slow movement, postural abnormality, impaired balance, and extensive membrane damage.

Hypothyroidism is known to affect electrolyte levels in circulation. Though the effect on electrolytes and certain minerals is not clear and the underlying mechanisms responsible for these changes not well understood ⁶⁴. Serum potassium levels were found to be decreased in hypothyroid animals when compared to controls, though it was statistically significant. Thyroid hormones regulate the activity of sodium potassium pumps in most of the tissues ⁶⁵. Deficiency of thyroid hormones leading to low potassium levels in hypothyroidism, affect the Na-K ATPase activity leading to accumulation of water inside the cells and causing oedema. This could be one of the mechanisms responsible for weight gain seen in hypothyroid patients ⁶⁴. Our result is in accordance with study conducted by Montenegro et al. ⁶⁶, Kaur et al. ⁶⁷, Kavitha et al. ⁶⁸, and Bharti et al. ⁶⁹. Thyroid abnormalities may lead to renal function alterations and also may disturb electrolyte balance ⁷⁰.

Lipid peroxidation initiates complex reactions such as NO inhibition, electrolyte imbalance enhancement and leakage of lysosomal hydrolases via breakdown of lysosomal membrane. Similarly, Kim and Akera ⁷¹ documented that lipid peroxidation produced by free radical causes electrolyte imbalance not only by injuring Na+-K+ ATPase but also by interfering with normal interaction of membrane pumps (including Na–Ka–2Cl co-transporter and K+ channel) and production of protein radical in lipid membranes that affects normal ion transport. Na⁺ and K⁺ levels returned to normal values because of the antioxidant and free radical scavenging properties of thyme extract.

The current study showed that animals treated with PTU suffered from oxidative stress approved by the significant elevation of malondialdeyhide and the significant reduction of antioxidant enzymes such as SOD and GPx. These results are in line with those reported by Abdel-Wahhab et al. ⁴⁴, who suggested that oxidative stress may be attributed to direct action of PTU or by metabolites formed by free radicals generation. The oxidative damage to normal tissue was induced following exposure to hydroxyl (OH) free radical. Excess of free radicals may in turn lead to peroxidation impairment of lipid membrane and consequently, disrupt neural function and result in cell death ⁷².

Administration of thyme can ameliorate the disrupted liver function enzymes and bile system. These may relate to thyme enhancing GSH-px, GST and SOD activities and replenishing GSH storage ^{73, 74, 75}. As well as, Plant volatile compounds appear to accumulate in the cell membrane inhibiting the chain reaction of lipid peroxidation, stabilizing membrane activity ^{76, 77}.

According to the study of Sameh et al. ⁴⁸, rats co-administrated T. vulgaris with alcohol showed significant elevations in antioxidant enzymes activities; CAT, SOD, GR, GST and GPx were significantly elevated, while oxidative stress parameters; MDA and H₂O₂ concentration were significantly lowered corresponding to alcohol-control. Also, lipid profile was markedly improved and risk ratio was decreased by T. vulgaris extract co-administrated compared to alcohol-control.

Tumor necrosis factor alpha (TNF-α) is produced by macrophages and it plays an important role in tumor conditions ⁷⁸. It has been reported that TNF-α is an essential factor in tumor promotion ⁷⁹. In the current study, the injection of PTU significantly increased TNF-α suggesting that PTU preferentially affects macrophage functions. Indeed, TNF-α plays a causal role in the development of liver injury and hepatotoxicity ⁸⁰. Thyme extracts significantly reduced production and gene expression of the proinflammatory mediators TNF-α, this result may suggest that thyme extracts could have anti-inflammatory effects ⁸¹.

In the present research, the treatment with PTU resulted in a significant decrease in thyroid hormones. The hypothyroidism-associated oxidative stress in the consequences of both increased free radicals production and reduced capacity of the antioxidative defense ⁸².

PTU effect may inhibit the thyroid hormone synthesis by blocking the transformation of T₄ to T₃, through inhibiting the thyroid peroxidase enzyme, which adds iodide to tyrosine residues on the thyroxin hormone precursor thyroglobulin ⁸³. No case reports were found about hepatotoxicity from PTU for thyroid storm treatment. Thyroid storm itself can cause derangement of liver function tests and in rare cases result in fulminate hepatic failure ⁸⁴. Because of this, it can be challenging for the managing physician to distinguish between thionamide-mediated and hyperthyroidism mediated liver dysfunction. With rare complications like PTU-related hepatotoxicity, it might not be possible to address every minor side effect and study it. To do so would require a huge study population that might not be feasible or ethical to recruit ⁸⁵. It is important to highlight that thyme supplementation to hypothyroid rats can prevent the hypothyroidism occurrence. This agree with the results of Lima et al., ⁸⁶, who reported that, Flavonoids in thyme are able to increase iodide uptake and sodium-iodide symporter expression and thyroperoxidase, the key enzyme in thyroid hormones biosynthesis.

The treatment with a watery extract of thyme in rats that was treated with PTU resulted in an increase in T₃ and T₄ which is an indication for the amelioration of the oxidative stress which may be due to the free radical scavenging activity of the thyme extract.

Abeir and Hoda ⁸⁷, stated that thyme supplementation only after hypothyroidism induction can alleviate thyroid function and hepatic oxidative damage completely. In contrary, thyme administration before and after hypothyroidism induction regulated thyroid function and restored hepatic antioxidant capacity toward the normal. The present study suggests that supplementation of thyme extract displays a prophylactic role against hypothyroidism and hepatobiliary protective effects.

5. Conclusion

Thymus vulgaris possess various beneficial effects, like antioxidant properties. Also, it has lately recommended as a natural replacement for synthetic antioxidant. The data obtained showed that the watery thyme extract containing polyphenol, flavonoids have the protective effect on the toxicity produced by PTU which may be due to its antioxidant properties and free radical scavenger activity. The anti-inflammatory effects on liver, kidney, electrolyte imbalance and thyroid functions should be interpreted with caution, due to its ambiguous dose related. This underscores the importance of evaluating multiple doses of Thymus vulgaris in a variety of tissues not only for potential benefits but also for any toxic effects.

References