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INTRODUCTION
Adrenal Insufficiency (AI) is a serious condition where the adrenal glands do not make enough cortisol, a hormone needed for the body to work properly. If not treated, it can lead to a dangerous condition called adrenal crisis, which can be life-threatening (Kilberg and Vogiatzi, 2024). Currently, adrenal insufficiency affects more than 300 out of every 100,000 people and leads to more illness and a higher risk of death (Peel et al., 2024). Despite its clinical significance, there is limited preclinical research on drug-induced AI, particularly involving inhibitors of significant adrenal steroidogenic enzymes such as 11β-hydroxylase, 21-hydroxylase and 17-hydroxylase (Caroste et al., 2023; Guengerich et al., 2023; Pivonello et al., 2023).
Metyrapone is an adrenal steroidogenesis inhibitor that selectively inhibits 11b-hydroxylase (CYP11B1), the last enzyme in cortisol biosynthesis. This effectively prevents the conversion of 11-deoxycortisol into cortisol, as shown in Figure 1. It is also used to study the suppression of the Hypothalamo Pituitary and Adrenal (HPA) axis, although it has not been thoroughly studied (Detomas et al., 2022). The lack of adequate preclinical models hinders the development of novel diagnostic approaches and management strategies for drug-induced Adrenal Insufficiency.
This study establishes a reliable and reproducible model of Adrenal Insufficiency using metyrapone, which can be valuable tool for future pharmacological studies. It also helps to understand the pathophysiology of 11 β-hydroxylase inhibition and its effect on adrenal hormone production. The findings, which characterize biochemical, physiological, and histological abnormalities, can help to inform techniques for diagnosing and managing metyrapone induced Adrenal Insufficiency.
AI models are crucial for testing safety and efficacy of potential therapies, including hydrocortisone, prednisolone, and dexamethasone replacements (Nowotony et al., 2021). AI models help in investigating hormonal dynamics, including interactions between HPA axis and other systems. This model allows to induce the adrenal crisis and adrenal insufficiency, which are very difficult to study directly in humans.
MATERIALS AND METHODS
Animals
Wistar rats ranging 190-210 g were procured from Vaarunya biolabs Pvt. Ltd., Bangalore. Rats were housed in 12:12 hr light and dark, temperature (22±1ºC) and humidity (50±5%) controlled environment for one week before experimentation. Food (Standard rodent pellets) and water ad libitum.
Drugs and chemicals
Metyrapone was acquired from Merck, Bangalore, Sodium chloride, sodium phosphate, potassium phosphate were obtained from the store, Sri Adichunchanagiri College of Pharmacy.
Experimental design and procedures
40 rats were utilized in this study with ten in each group for four weeks. Group 1 serves as normal, Group 2, 3 and 4 are selected to induce adrenal insufficiency in Wistar rats at 100, 150 and 200 mg/kg of metyrapone, an 11 β-hydroxylase Inhibitor. The selected doses are well below the toxic level, ensuring safety during the experiment.
The LD50 for metyrapone tartrate was 760 mg/kg i.p (Nelson et al., 1980). A dose range from 100 mg/kg to 200 mg/kg provides a gradient to study potential dose dependent pharmacological effects. This approach allows to identify both sub-therapeutic and possibly maximal side effects.
Doses between 10 to 30% of the LD50 are commonly explored to access therapeutic effects while avoiding the acute toxicity. The highest dose, metyrapone 200 mg/kg is approximately 26% of the LD50 making it a rational choice for exploratory studies.
Current study aims to estimate cortisol, sodium, and potassium levels in the blood, as well as serotonin and dopamine levels in the brain.
Biochemical assays
For blood collection, a 23 G needle connected to a syringe was used to draw 1.5 mL via lateral tail vein. Rats were placed in restrainer individually to collect the blood. The blood samples were collected sent to the laboratory, Adichunchanagiri hospital and research centre for the analysis of Cortisol in the blood (Jameel et al., 2014).
Enzyme Linked Immunosorbent Assay (ELISA) was performed for the hormonal analysis, which is a sensitive, reliable, replicable, specialized, and speedy method. Sodium, and potassium were measured by spectrophotometer (Farahadi et al., 2019; Nassiri et al., 2017; Zhou et al., 2021).
Neurochemical analysis
Rats brain were dissected, homogenized with 0.1 M hydrochloric acid and butanol, the mixture was centrifuged for 10 min at 2000 rpm. 1 mL of the supernatant was taken, and it was agitated for 10 min before adding 2.5 mL of heptane and 0.31 mL of HCl (0.1 M). After that, the mixture is centrifuged.
Centrifugation was used to separate the two layers. Throughout the process, 0ºC is kept as the temperature. After removing the organic layer, 0.2 mL of the aqueous layer was extracted for the measurement of DA and 5-HT (Hira et al., 2020; Meissam et al., 2021; Saleem et al., 2021).
Determination of Serotonin.
0.2 mL of the aqueous phase was mixed with 0.25 mL of O-Phthaldialdehyde (OPT). After that, this combination was cooked for about 10 min at 100ºC. The substance was allowed to reach room temperature. In a spectrophotometer, absorbance was measured at 440 nm. 0.25 mL of HCl without OPT served as the blank solution.
Estimation of DA
For the process of oxidation, 0.05 mL of hydrochloric acid (0.4 M) and 0.1 mL of ethylenediaminetetraacetic acid were added to 0.2 mL of aqueous phase. And the mixture was then combined with 0.1 mL of iodine (0.1 M in ethanol) solution.
To stop the oxidation, 0.1 mL of sodium sulfite (Na2SO3) was added prior to the addition of 0.1 mL acetic acid. The reaction mixture was allowed to cool after heating for 6 min at 100ºC and measured at an absorbance at a wavelength of 350 nm. A blank solution was produced by adding the oxidation process's ingredients in the reverse order.
Histopathology
The Paraventricular Nucleus (PVN) of the hypothalamus, one of the brain's most important autonomic control regions were removed and immediately fixed in buffered neutral formalin, dehydrated and fixed with paraffin, sliced into 5 mm thickness with microtome and stained with eosin and hematoxylin. The sections prepared were visualized under light microscope.
Statistical analysis
All results were expresses as mean±SD. Two-way ANOVA was used to examine the biochemical changes. Differences between groups were tested by Tukey’s multiple comparison test. The statistical significance level (p value) for all analyses was set at 0.05.
RESULTS
Estimation of Cortisol
A crucial steroid hormone produced by the suprarenal glands, has diverse impact on metabolism, immune functions and various physiological processes. Cortisol levels were assessed for every seven days. It was observed that cortisol levels were declined in dose-dependent manner with a p value <0.0001 for mety 100, 150 and 200 mg/kg compared to normal in all the weeks. The results were depicted in Figure 2.
Sodium estimation
Sodium levels were assessed weekly up to 28th day. The values are significant at high doses represented in Table 1.
| Groups | Day 1 | Day 7 | Day 14 | Day 21 | Day 28 |
|---|---|---|---|---|---|
| Normal | 141.4±7.07 | 141.7±6.21 | 140.8±6.71 | 140.6±4.57 | 139.6±5.79 |
| mety 100 mg/kg | 136.2±3.8ns | 133.3±5.22* | 138.8±4.18ns | 136.1±4.14 ns | 132.9±6.10 ns |
| mety 150 mg/kg | 133±6.41* | 138.8±4.36* | 135.6±3.23ns | 120.5±8.89**** | 117±10.45**** |
| mety 200 mg/kg | 133.9±4.43* | 129.2±6.32*** | 130±4.66** | 117±9.49**** | 107.7±11.95**** |
Potassium estimation
Potassium levels are observed for every seven days up to 28th day. The values were represented in the Table 2.
| Groups | Day 1 | Day 7 | Day 14 | Day 21 | Day 28 |
|---|---|---|---|---|---|
| Normal Control | 4.882±0.27 | 4.856±0.32 | 4.753±0.36 | 4.768±0.41 | 4.811±0.30 |
| Mety 100 mg/kg | 4.773±0.33ns | 4.474±0.39* | 4.714±0.29 ns | 4.605±0.29 ns | 4.229±0.31** |
| Mety 150 mg/kg | 4.739±0.34 ns | 4.327±0.34** | 4.641±0.26 ns | 4.312±0.34* | 4.047±0.32**** |
| Mety 200 mg/kg | 4.366±0.43** | 4.183±0.30*** | 4.044±0.45**** | 3.741±0.44**** | 424±0.47**** |
Estimation of DA and 5-HT
Both DA and 5-HT levels were estimated at the end of the study and there were no significant changes in the serotonin at mety 100 mg/kg. At 150 and 200 mg/kg doses, there is a slight decline with the values of 5-HT represented in Figure 3.
Histopathology
The histopathology of rats’ brain focusing Paraventricular nucleus of hypothalamus represented in Figure 4.
DISCUSSION
Throughout the study, the normal rats cortisol levels remain constant at 80-84 ng/mL. Mety 100 mg/kg significantly reduces cortisol levels compared to the normal group while maintaining moderate levels between 59 and 64 ng/mL. Mety 150 mg/kg exhibited further cortisol suppression of 40-54 ng/mL, suggesting dose-dependent adrenal suppression.
With a statistical significance of p<0.0001 to normal, Mety 200 mg/kg exhibits the greatest suppression of cortisol during every stage of the research, suggesting acute adrenal insufficiency. The strongest suppression, which happens at 200 mg/kg, indicates severe AI.
Hyponatremia is a common electrolyte imbalance in adrenal insufficiency (Kumar et al., 2021). The sodium levels were constant at 139-141 mmol/L for normal group throughout the study. On days 7 and 21, there is a substantial drop in Mety 100 mg/kg from 133 to 138 mmol/L (p<0.05). Mety 150 mg/kg exhibits a steady decrease from 117 to 135 mmol/L, with a significant statistical shift starting on day 14 (p<0.01 to p<0.0001). By day 21, Mety 200 mg/kg demonstrated significant hyponatraemia (~107-134 mmol/L) with a p-value below 0.0001.
The potassium levels in the normal group remain consistent at 4.7-4.9 mmol/L throughout the research. On days 7 and 28, there is a slight decrease in Mety 100 mg/kg between 4.3 and 4.8 mmol/L that is statistically significant (p<0.05 to p<0.01). A progressive decrease (~4.4-4.6 mmol/L) was observed for mety 150 mg/kg, with a larger significance beginning on day 7 (p<0.01 to p<0.0001). Severe hypokalaemia (4.0-4.7 mmol/L) was observed by days 21 and 28, which was very significant (p<0.0001) for mety 200 mg/kg. Potassium levels decrease after prolonged use of mety (Kidwara et al., 2024).
The DA concentration is higher in the normal group. DA levels are reduced in a dose-dependent manner by Mety 100, 150, and 200 mg/kg. Significant drops (p<0.01, p<0.0001) are shown in the Mety 150 and Mety 200 groups, suggesting DA depletion.
5-HT is preserved by low dosages of mety, while it is depleted by high concentrations. Serotonin depletion occurs gradually as mety concentrations rise. Serotonin receptor sensitivity is decreased by chronic HPA suppression, which may be a factor in mood disorders.
CONCLUSION
Mety 200 mg/kg shows the highest reduction of cortisol, suggesting severe AI, glucocorticoid medication might be required to restore normalcy. Decreased levels of cortisol or corticosterone can lead to metabolic imbalance, depression-like behavior, and an impaired stress response. Hyponatremia and hypokalemia were observed at higher dosages. There was a dose-dependent decline in the DA and 5-HT. In conclusion, this study may result in acute AI compared to earlier studies, and more comprehensive research should be conducted for chronic conditions.