Ameliorative effects of piroxicam on perchloric acid-induced thyroid gland hormones disruption in male rats

1 Introduction

Male rodents are more sensitive to piroxicam and hydrocortisone as compared to female rodents (Saganuwan and Orinya, 2016). This may be due to higher ability for activation of metabolic process (NIEHS, 2000). The interest on toxicological effect of potent toxicant on living organism has increased (Forni, 2014). Hence laboratory dose is the point-of-departure for assessment of toxic chemicals. The increased attrition rate in development of a new anticancer is exasperating. Induction of thyroid disruption by fipronil was via increased elimination of thyroxine (T4) mediated by activation of hepatic enzyme. Fipronil sulfone, the metabolite of fipronil might have contributed to the disruption (Julien et al., 2009). High fat diet caused hypothyroidism mitigated by metformin(El-Sayed and Ibrahim, 2020). More so glucagon-like peptide may increase the risk of thyroid cancer. Perchloric acid could cause benign and malignant thyroid turmors and immunotoxicity. The oral median lethal dose (LD₅₀) of perchloric acid was 1100 and 250 mg/kg in rats and dogs respectively. The subcutaneous LD₅₀ in mice was 250 mg/kg. Chronic injection of perchloric acid of 600–1200 mg/kg was used to treat grave’s disease, in which immunoglobulin binds to thyroid stimulating hormones receptor, resulting in hypothyroidism (Baran and Gad, 2014). Hence the present study aims to assess mitigating effects of piroxicam on perchloric acid-induced thyroid gland hormones disruption in male rats.

2 Materials and methods

3 Results

Acute toxicity study of perchloric acid revealed median lethal dose (LD50) of 4207.5 ± 457.6 mg/kg body weight. The male and female rat administered 5000 mg/kg died within 14 days, after the administration of the chemical. Meanwhile, male and female rat administered 3415 mg/kg body weight survived the dose after 14 days. The effects of piroxicam on perchloric acid-induced thyroid hormones disruption in male rats is presented in the Table 1 . Groups treated with piroxicam showed significant decrease (p < 0.05) in thyroid stimulating hormones (TSH) and free triiodothyronine (fT3) whereas thyroxine (T4) increased significantly (p < 0.05), respectively (Table 1). Swelling of thoracic outlet and muscle of hindlimb were observed in some rats of group 2 and 3. Alanine amino transferase (AAT), aspartate amino transferase (AST) and alkaline phosphatase were significantly increased (p < 0.05) in the group administered perchloric acid only. The levels of urea, serum creatinine and plasma creatirine were significantly (p < 0.05) decreased (Table 2).

Haematocrit, erythrocytes and leucocytes counts were significantly (p < 0.05) decreased in perchloric acid and piroxicam treated groups, whereas monocytes counts were significantly increased (p < 0.05) in the groups treated with piroxicam respectively. Lymophocytes and neutrophils were significantly (p < 0.05) increased and decreased in the perchloric acid treated group respectively. Platelets and eosinophils were significantly decreased in all the groups (p < 0.05) except basophils, which decreased in 2.5 mg/kg piroxicam treated group (Table 3). The weight, width and length of heart were significantly (p < 0.05) increased. The significant increase in the weight and decrease (p < 0.05) in the length of right and left lobes of the liver were observed in the group administered piroxicam. However, the width of the right and left lobes of the liver was significantly (p < 0.05) decreased in the group administered piroxicam. Nevertheless the length and width of right and left kidneys, weight, length and width of spleen as well as length and width of right lung were significantly increased (p < 0.05) in the group administered piroxicam, whereas length of the lung was significantly decreased (p < 0.05). Meanwhile allometric ratio of the brain was significantly increased (p < 0.05) in the group administered piroxicam. Allometric ratios of the liver, heart, kidney, lung and spleen were increased significantly p < 0.05) in the group administered 5 mg/kg of piroxicam (Table 4).

3.1

3.1 Histopathology

Fig. 1 shows perivascular cuffs and normal neurological cells of the male rats administered perchloric acid and perchloric acid/piroxicam respectively. Fig. 2 shows zenker’s necrosis of myocardial cells of the rats administered, perchloric acid only.Congestion of the central vein and mononuclear cells infiltrations were observed in the group administered, perchloric acid and piroxicam (Fig. 3). Normal medullary rays, normal glomeruli surrounded by Bowman’s capsule and distal convoluted tubules with mononuclear cells infiltration were observed in the group administered perchloric acid and piroxicam, respectively (Fig. 4). White pulp with aggregation of lymphoid tissue and red pulp with aggregation of both white and red pulps, were observed in all the three groups (Fig. 5). Wide alveolar space was observed in the group administered piroxicam. Congestion and thickness of alveolar sac were observed in the group administered perchloric acid, whereas thick narrow air sacs with congested artery and large alveolar sac with thick wall comprising pneumocytes were observed in the group administered piroxicam, respectively (Fig. 6).

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Fig. 1

Shows perivascular cuffs (A&B), under perivascular space (C), glial cells (D), peripheral nerve (E) and mononuclear cells (F).

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Fig. 2

Shows Zenker’s necrosis (A), normal myocardium (B&D), and Zenker’s necrosis with mild mononuclear cellular infiltration (C).

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Fig. 3

Shows congestion of central vein (A), normal portal tract (B), mononuclear cell infiltration (C), whereas (D) is normal hepatocytes.

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Fig. 4

Shows normal medullary rays (A), glomeruli (B), parenchymal tubule (C), and distal convulated tubule with mononuclear cells infiltration (D).

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Fig. 5

Shows white pulp with aggregation of lymphoid tissue (A), red pulp with red blood cells and white blood cells (B), degeneration of red pulp (C) and wide spread combined degeneration of white pulp and red pulp (D).

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Fig. 6

Shows congested alveolar sac (A), wide alveolar space (B), congested smooth muscle (C), thick pneumocytes (D), thick air sac (E) and congested artery (F).

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Haemorrhages in the lung, spleen, kidney and heart, paleness of small and large intestine, shrinking of the stomach and diarrhoea where observed in the group administered 5 mg/kg body weight of piroxicam plus perchloric acid 100 mg/kg. Haemorhages were observed in the brain of the group administered 2.5 mg/kg of piroxicam plus perchloric acid. The animals were emaciated and the average weight reduced from 163.3 ± 10.5 g to 135.5 ± 11.8 g translating to 17 % of total body weight loss.

4 Discussion

The calculated LD50 of 4207.5 ± 457.6 mg/kg body weight of perchloric acid shows that the chemical is not safe. The finding agrees with the report of Saganuwan and Orinya (2016) indicating that toxic agent with LD₅₀<5000 mg/kg is not safe. Hypnosis, calmness and dullness observed in the present study show that perchloric acid may depress central nervous system.Meanwhile, the increased T₄ observed in all the groups on day 14 and 28 showed that both perchloric acid and piroxicam could cause increased thyroxine secrection. Perchlorate blocks the transport of iodide from entry into thyroid gland leading to iodine deficiency, which stimulates production of thyroid stimulating hormones (TSH) by the pituitary gland, invariably causing goitre. Perchlorate (HCLO4) absorbed through gastrointestinal tract was excreted in the urine within 48 h, with elimination half-life of 6.99 h. Goitrogens cause physiologic perturbations of TSH, which can lead to tumor associated with hormonal imbalance (Capen, 1997).

Decrease in triiodothyronine (T₃) and thyroxine (T₄) associated with increased TSH are the biomarkers of exposure to perchlorate. Perchlorate is highly persisted in surface water and ground water. Perchloric acid can react with aldehydes, alcohols and ketones, suggesting possible reaction with piroxicam (Baran and Gad, 2005). Iodine deficiency is a risk factor for thyroid cancer and anaplastic thyroid cancer characterized by chronic TSH stimulation and microcarcinoma with lower iodine intake (Zimmermann and Galetti,2015).

History of thyroid cancer in a family, diet and chemotherapy are predictors of thyroid cancer. Hence theoretic model of antineoplastic could be challenging in cancer chemotherapy (Saganuwan, 2019). Chemically induced cancer in lab animals portends potential hazards in human. TSH-producing pituitary cells undergo hyperplasia and neoplasia (Hill et al., 1998). Iodine ¹³¹I administered to patients suffering from thyroid toxicosis may cause failure of thyroid gland. Hepatocellular enzymes induction could cause hypertrophy of thyrotrophs in rats. Polychromated biphenyl (PCB) and prenenoline-160α carbonitrile (PCN) reduce thyroid hormone levels in rats. The mechanism of action of PCB is via induction of microsomal enzymes. Hyperplasia of parathyroid gland could be caused by X-irradiation on rats (Lindsay et al., 1961). However¹³¹I caused transitional changes to thyroid gland with associated iron T₄ (Rudquist et al., 2017).Thyrotoxicosis is associated with follicular cell hypertrophy, follicular cell hyperplasia, follicular cell adenoma, follicular cell carcinoma, C-cell hyperplasia, C-cell adenoma, C-cell carcinoma, hyperplasia, adenoma and carcinoma (Botts et al., 1991). Thyroid carcinogenesis is via inhibition of thyroid peroxide and 5-minodeodinate (Hurley et al., 1998). Pyrethrins and sodium phentobarbital caused thyroid hypofunction via induction of cytochrome ,CYP2B enzyme in rat liver (Frinch et al., 2006).Perfluorohexane sulfurates decreased thyroid hormones level and brain development is sensitive to T4 (Ramhoj et al., 2020). Progression of thyroid tumor to anaplasic thyroid carcinoma is via p53 in the mouse model of papillary thyroid cancer (Mc Fadden et al., 2014). Thyroid hormones are essential for growth, development of muscular, skeletal and neurological systems, erythropeisis and thermogenesis. About 99% of thyroid hormones are stored in protein bond-active form. Free T₄ and T₃ are basically active and stimulate feedback mechanism on the hypothalamus and pituitary gland (Thorson, 2014). Disruption of circadian rhythms leads to obesity, diabetes and cardiovascular disease (Bae and Androulakis, 2018).The decrease in renal parameters observed in the present study agrees with the report of Saganuwan (2018), indicating that creatinine could be used for identification of renotoxic agent.

The higher therapeutic effects produced by 2.5 mg/kg of piroxicam as compared to 5 mg/kg shows that piroxicam has hormetic dose response. Saganuwan and Orinya (2016) had earlier reported hormetic dose–response of piroxicam, which is toxico-neurological in rats. The stimulating effect was attributed to the parent drug, piroxicam (Saganuwan, 2017a), whereas depressant effect was attributed to piroxicam metabolites (Saganuwan, 2017a; Saganuwan, 2017b). The hormetic dose response can be due to tautomeric behaviour of piroxicam, that makes the drug isomers to be in equilibrium, and changed from acidic to basic or inert form (Saganuwan, 2016). Hormesis is a reversal between high and low doses of physical stressors, phytochemicals, chemicals or biological molecules, and the overall effects could be beneficial or hazardous (Jodynis-Liebert and Kujawska, 2020). Hormetic dose–response is exhibited by endocrine disruptors. The response could be low-moderate, low–high, low or high, represented by Arndt-Schulz curve and generalized adaptive curve. Therefore, the statistical methods used for hormesis are Cedergreen-Ritz-Strenbig model, Brain-Consens model, biologistic model and An-Johnson-Lovett model (Nweke et al., 2021) . Hormesis is constrained by plasticity, though evolutionary, frequent independent, remote-conditioning, risky, synergistic, resilient and requires assessment. The 2.5 mg/kg which produced better effect against disruption of thyroid hormones by perchloric acid is dose–response-time dependent. This type of response requires dose selection, repeated dosing, different inputs, rates and route of drug administration (Gabrielsson et al., 2019). Unfortunately, 25% of medical errors are due to drugs and 14% are attributed to poor drug calculations. The increased basophills, platelets, and eosinopils showed that piroxicam could be used in the treatment of histamine, serotonin and heparin induced reaction, whereas neutropenia observed is suggestive of inflammatory response and lymphocytosis observed may be due to stimulation of thymus. Lymphocytes are produced from B-lymphocytes (Watkins and Levy, 1988). IgA in the body fluid activates the complement system, 1gE acts on mast cells and basophills which play roles in inflammation and allergy, 1gD is a membrane receptor and 1gC is found on almost every cell (Percus, 2010).

Perivascular cuffs observed in the group of rats administered 100 mg/kg of perchloric acid and 2.5 mg/kg of piroxicam, show that both agents can cause acute and chronic perivascular cuffs, ameliorated by 5.0 mg/kg of piroxicam. The acute cuffs consist of lymphoid cells, and a few macrophages usually observed in nondemyelinated white matter and in the margin of plaque. The chronic cuffs consist of macrophages, plasma cells and plasmatoid cells seen in the plaques. Macrophages phagocytize myelins and axon (Tanaka et al., 1975), suggesting that piroxicam is a strong immunocytogenic agent. The observed Zenker’s necrosis in group administered 100 mg/kg of perchloric acid suggests that the chemical could cause waxy degeneration of heart striated muscle. The degeneration was ameliorated by 2.5 and 5.0 mg/kg of piroxicam. Zenker’s necrosis is caused by infiltration of albuminous material from outside heart cells (Wells, 1908). Since lactic acid may cause Zenker’s necrosis, perchloric acid may be the incriminating agent in the present study. Congestion of central veins caused by perchloric acid (100 mg/kg), and in addition to 2.5 mg/kg of piroxicam ameliorated by 5.0 mg/kg of piroxicam, shows that both perchloric acid and piroxicam have negative and positive effects on liver. Normal kidney architecture suggests beneficial effect of the two agents on the kidney. The observed dense white pulp and red pulp in the group administered perchloric acid (100 mg/kg) and in addition to 2.5 mg/kg of piroxicam show that the two agents could stimulate cellular immune system as evidenced by perivascular cuffing of the leucocytes observed in the brain. The mild degeneration of white and red pulps by piroxicam suggests suppressive potential of lymphoid tissues and erythrocytes.

5 Conclusion

Piroxicam mitigated disruption of thyroid gland hormones at 2.5 and 5 mg/kg body weight. Thyroxine, fT₃ and thyroid stimulating hormone_, creatinine, urea, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, mononuclear and polymorphonuclear cells and organ allometry are reliable parameters for identification of thyroid hormones disruptors. Hence piroxicam has neuro-endocrine-immunomodulatory potential and could be used in the management of goitre and treatment of autoimmune diseases.