Impact of damiana (Turnera diffusa) against amitriptyline induced heart injury, dysfunctions and DNA damage in male rats

1. Amitriptyline induced cardiac toxicity.
2. Amitriptyline toxicity is associated with elevation of cholesterol, TG and LDL-C.
3. Damiana supplementation enhancement of cardiac damage induced by Amitriptyline.

Introduction

Research has suggested that antidepressant use may significantly influence the dysregulation of the autonomic nervous system (ANS) seen in depressed or anxious people (1,2). Psychiatrists administer antidepressant drugs that are used to treat major depressive disorder, anxiety disorders, chronic pain, and addiction (1). Cardiac toxicity is typically included during medication development and risk assessment; tricyclic antidepressants adversely affect all body systems (2,3). Amitriptyline hydrochloride is a tricyclic antidepressant (TCA) and analgesic derived from dibenzocycloheptene. It is also used for migraine prevention, neuropathic pain syndromes, fibromyalgia, bipolar disorder, and nocturnal enuresis (4). Amitriptyline is a serotonin reuptake inhibitor that blocks histamine-H1 receptors, alpha 1 α1-adrenergic receptors, and muscarinic receptors, causing sedative, hypotensive, and anticholinergic effects (5,6). Amitriptyline, unfortunately, causes cardiovascular damage in psychiatric patients (7,8). Traditional medicine has been introduced with pharmaceuticals in recent years, based on their usage in combination with other medicines to improve medicinal efficacy, and they have established a solid reputation for effectiveness, causing a boom in the treatment area (9-13). Our study group has recently started investigating Turnera diffusa, also known as damiana, a therapeutic herb. Damiana, or Turnera diffusa Willd, has a long history of use as herbal aphrodisiac (14,15). Turnera diffusa is a member of the Turneraceae family of plants. It is a tiny shrub that grows in the tropical and subtropical regions of the United States. The Maya used it to treat various ailments (16,17). There are at least 20 compounds in Damiana leaves (1, 8-cineole, P-cymene, - and -pinene, thymol, -copaene, and calamine), as well as tannins, flavonoids, -sitosterol, Damiani, and the glycosides gonzalitosin, arbutin, and tetracyclin B (18). Damiana is safe in both men and animals.

This study aimed to determine the effect of damiana as an adjuvant herbal therapy for amitriptyline-induced cardiac damage in rats.

Materials and methods

Ethical approve

The research’s approach was approved by the Institutional Ethical Committee for Animal Care and Use, with a code of Faculty of Science, Tanta University, Egypt (IACUC-SCI-TU-0241).

Experimental animals

36 male albino rats were brought from the National Research Center in Giza, Egypt, at 10-12 weeks and weighing roughly 150 grams. Before the experiment began, the rats were divided six groups, each with six rats. Rats remained in the animal house of our faculty for one week before starting the experiment. They were kept on a standard rodent diet and water ad libitum, normal temperature conditions (23±2°C) in a 12h light/dark cycle, and a minimum relative humidity of 40%.

Damiana’s aqueous extract production

Damiana dried leaves from a neighbourhood market of clinical plants in Egypt were shacked to powder and soaked in boiled water for 24 hours at 37ºC, then extracted and stored at -30ºC in the dark until used (15,19).

Experimental groups

Group 1 control group (animals not given any drug). Group 2 damiana group (80 mg damiana/kg body weight/daily/ orally via a stomach tube) for four weeks (17). Group 3 amitriptyline (Tryptizol; El Kahira Pharm and Chem Ind Co) group (70 mg amitriptyline/kg body weight/daily/intraperitoneally injected) for four weeks (17). Group 4 co-treated group (70 mg amitriptyline/kg body weight/daily/intraperitoneally injected) + (80 mg damiana/kg body weight/daily/orally) for four weeks. Group 5 post-treated Co-treated group (70 mg amitriptyline/kg body weight/daily /intraperitoneally injected) for four weeks followed by (80 mg damiana/kg body weight/daily/orally) for another four weeks. Group 6 self-treated group (70 mg amitriptyline/kg body weight/daily/intraperitoneally injected) for four weeks and left for self-recovery without any drugs for successive 4 weeks.

At the end of the experimental period, rats were fasted overnight. Rats from each group were euthanized with intraperitoneal sodium pentobarbital (30 mg/kg; Danish company Lundbeck) for complete dissection.

Blood and serum samples

Blood was collected from the inferior vena cava and separated by centrifugation at 3000 rpm for 15 minutes. The collected serum was stored at -18°C until analysis to estimate blood parameters.

Cardiac function biomarkers

Serum lactate dehydrogenase (LDH) activity was estimated by a kinetic method described by Zilva and Pannall (20). In contrast, creatine kinase (CPK) levels in sera were determined via an akinetic method as described by Alotaibi et al. (21). Creatine kinase myoglobin (CK-MB) and myoglobin levels in sera were estimated as described by Elgharabawy et al. (22). Serum aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activities were detected using a colorimetric method according to Moustafa et al. (23) and Saggu et al. (24), respectively. Serum of Total protein levels according to Tousson et al. (25).

Electrolyte estimation

The electrolyte levels as potassium ions and sodium ions were detected according to Abd Eldaim et al. (26) by using profitable kits from India known as Sensa core for electrolytes.

Lipid profile estimation

Complete lipid profiles [triglyceride, cholesterol, high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C)] in sera were detected according to Aldubayan et al. (27) and Salama et al. (28) by using kits from ELLTECH.

Comet assay

The comet assay (single-cell gel electrophoresis) technique was used to analyze and quantify DNA damage in the cardiac tissues according to the approach derived from Abd Eldaim et al. (29).

Histopathological examination

Cardiac tissue samples were fixed with 10% formalin (natural buffer) solution for 48 hours and then processed for paraffin sectioning, stained with hematoxylin and eosin (H and E) for histopathological investigation, according to Tousson (30).

Immunohistochemical detection of proliferating cell nuclear antigen (PCNA)

The detection of PCNA expressions was assayed according to Tousson et al. (31) using the avidin-biotin complex method. The stained cells were quantitated by color thresholding using the Image -J- program.

Statistical analysis

The data was presented as means and SEM. Using one-way ANOVA, data were compared between the Amitriptyline and the other five groups. When P≤0.05, the P value became significant. Analysis was conducted utilizing (Graphpad Prism, Graphpad Software, Inc., La Jolla, CA, USA).

Results

Changes in cardiac enzymes

Table 1 revealed that amitriptyline (AMT) induced significant elevation in CK-MB, myoglobin, CPK, LDH, AST, and ALP levels compared to control. In contrast, there was a substantial decrease in CK-MB, myoglobin, CPK, LDH, AST, and ALP in co-treated (DE+AMT) and post-treated (AMT+DE) groups. Low significance improvement appeared in the treated group (ST AMT) as compared to co-treated (DE+AMT) and post-treated (AMT+DE) groups.

Changes in lipid profile

Table 2 revealed a significant elevation in cholesterol, triglycerides, LDL, and HDL depletion in intoxicated rats by amitriptyline (AMT) compared with control. On the other hand, there was a significant deficit in cholesterol, triglycerides, LDL, and a significant elevation in HDL in co-treated (DE+AMT) and post-treated (AMT+DE) groups when compared with AMT and ST AMT.

Changes in serum electrolytes

Table 3 revealed that serum sodium ions are significantly elevated with a significant decrease in the levels of potassium ions in intoxicated rats by amitriptyline (AMT and ST AMT) when compared with Control. On the other hand, there was a significant decrease in the levels of sodium ions with a substantial increase in potassium ions in co-treated (DE+AMT) and post-treated (AMT+DE) groups when compared with AMT and ST AMT.

Table 1: Variations in AST (U/I), ALP (U/I)), CPK (U/I), CK-MB (ng/ml), Myoglobin (ng/ml), and LDH (U/I)

Data were expressed as mean±SE of 6 observations. *: Significant difference from the control group at P<0.05, ^#: Significant difference from the AMT group at P<0.05.

Table 2: Variations in the levels of cholesterol (mg/dl), Triglycerides (mg/dl), HDL (mg/dl), and LDL (mg/dl)

Data were expressed as mean±SE of 6 observations. *: Significant difference from the control group at P<0.05, ^#: Significant difference from the AMT group at P<0.05.

Table 3: Variations in sodium ions (mEq/l) and potassium ions (mEq/l)

Data were expressed as mean±SE of 6 observations. *: Significant difference from the control group at P<0.05, ^#: Significant difference from the AMT group at P<0.05.

Comet assay results

Intoxicated (AMT+ST AMT) groups exhibited a significant increase in DNA damage (P<0.05) that was indicated by an increase in tail length, tail DNA%, and tail moment as compared to control and Damiana (DE) groups. Co-treated (DE+AMT) and post-treated (AMT+DE) groups revealed a significant decrease in this elevated DNA damage, with the best results in the co-treated (DE+AMT) group (Table 4 and Figure 1). On the other hand, no significant difference in DNA damage (tail length) was observed between control and Damiana.

Table 4: Comet assay parameters obtained by image analysis in cells of all groups after the treatment experiment

Significant difference from the control group (G1) at *P<0.05. Significant difference from amitriptyline group (G3) at #P<0.05.

Figure 1: Photomicrograph representation of DNA damage in the heart, using the comet assay. G1, control group; G2, Damiana group; G3, Amitriptyline group; G4, co- treated group; G5, post-treated group; G6, self-treated groups.

Histopathology results

Heart sections in control and damiana groups showed single, oval, and centrally located nuclei of cardiac myocytes with regularly arranged cardiac myofibres (Figures 2A and B). Heart sections in the Amitriptyline group revealed severe tissue injury with marked myocardial hypertrophy, cytoplasmic vacuoles, and collections of nuclei that showed deformation in sizes and shapes (Figure 2C). Heart sections in co-treated amitriptyline combined with the damiana group (G4) revealed moderate damage and clear improvement of all changes in the form of nucleus and cardiac muscle tissue (Figure 2D). Also, the heart sections post-treated with amitriptyline with the damiana group (G5) revealed mild to moderate damage and mild myocardial hypertrophy, a few myofibrillar structures with striations, branched appearance and continuity with adjacent myofibrils, nuclear pyknosis and mild cytoplasmic vacuoles (Figure 2E). Heart sections in the self-treated Amitriptyline (G6) group revealed strong injury with revealed hydrophobic changes of myofibrillar structure with striations, marked myocardial hypertrophy, nuclear pyknosis, moderate cytoplasmic vacuoles (Figure 2F).

Figure 2: Photomicrographs of rat heart sections in the different groups stained with Haematoxylin and eosin. A and B: Heart sections in control (G1) group and damiana (G2) group showed normal, oval, and centrally located nuclei of cardiac myocytes with regularly arranged cardiac myofibres. D: Heart sections of the Amitriptyline group (G3) revealed marked hypertrophy, vacuolation, deformation in sizes and shapes of the nucleus, and disturbance of cardiac myofibers (black arrows). F: Heart sections in co-treated Amitriptyline with Damiana group (G4) showed moderate regular distribution in nuclei of cardiac myocytes (black arrows). G: Heart sections in post-treated Amitriptyline with Damiana group (G5) showed moderate myofibers and nucleus. H: heart sections in the self-treated Amitriptyline group (G6) revealed marked hypertrophy.

PCNA expression changes in the heart

Heart sections in control (G1) damiana (G2) groups showed moderate positive reactions for PCNA expressions in cardiac nuclei (Figures 3A and B). Heart sections in the Amitriptyline-treated rat group (G3) showed vital positive responses for PCNA expressions in the cardiac seats (Figure 3C). On the other hand, heart sections in co-treated and post-treated Amitriptyline with the damiana group (G4 and G5) revealed mild to moderate reaction for PCNA expressions (Figures 3D and 3E). Heart sections in the self-treated Amitriptyline-treated rat group (G6) showed a moderate positive reaction for PCNA expressions in the cardiac nuclei (Figure 3F).

Figure 3: Photomicrograph of rat heart sections is stained with PCNA expression in different groups. A and B: Moderate positive reaction for PCNA expressions in control (G1) and Damiana (G2) groups. C: Strong positive reaction for PCNA expressions in Amitriptyline-treated rat group (G3). D and E: Mild to moderate reaction for PCNA expressions heart sections in co-treated and post-treated Amitriptyline with Damiana group (G4, G6). F: Mild positive reaction for PCNA expressions in self-treated Amitriptyline rat group (G6) group.

Discussion

Amitriptyline is widely prescribed for depressed patients and other mental illnesses. The mechanism of the myocardial cell damage associated with amitriptyline treatment is uncertain. Damiana is a heart muscle tonic in traditional medicine and has proven to have potential therapeutic properties on cardiac toxicity (32). However, little is known about herbal plants as protective agents against the cardiac toxicity caused by Amitriptyline. This study was designed to study the impact of Damiana (Turnera diffusa) on Amitriptyline-induced cardiac dysfunctions in rats. Current results revealed that treated rats with Amitriptyline induced a significant elevation in the cardiac markers (CK, CK-Mb, LDH, and myoglobin) and cardiac enzymes (AST and ALP) levels by comparison to the control group, in contrast, a significant depletion in cardiac markers and enzymes after Co- or post-treatment of Amitriptyline with Damiana as compared to Amitriptyline group. Our results agree with Acosta and Ramos (33), who revealed that amitriptyline induced severe abnormalities of the sarcolemmal integrity using LDH release of cultured myocardial cells. In addition, these results were by Boles et al. (34) and Sorodoc et al. (35), who reported that Amitriptyline induced elevation in cardiac enzymes, Mattila and Saarnivaara (36), who reported that rabbits injected with amitriptyline showed myocardial dysfunction and death. Likewise, Kaur et al. (37) showed that Amitriptyline may be responsible for cardiac dysfunction. Also, Tsujikawa et al. (38) reported that antidepressant drugs (TCAs) are a major cause of fatal drug poisoning due to their cardiotoxicity.

The existing drug appears to affect the heart muscle due to changes in the level of cholesterol and triglycerides (13,39). The current study showed a significant decrease in cholesterol, triglycerides, and LDL and increased HDL levels in the amitriptyline group. In contrast, a significant increase in cholesterol, triglycerides, LDL and a significant decrease in HDL was shown after Co- or post-treatment of amitriptyline with Damiana. These results were consistent with Kopf et al. (40) and Eker et al. (41), who reported that antidepressant treatment significantly increased lipid profiles.

Electrolytes such as sodium, potassium, magnesium, and calcium are involved in heart contraction and relaxation. Significant changes are observed in Na and K ions in the amitriptyline group compared to the control. On the other hand, rats treated with damiana showed substantial improvement in the level of Na and K ions compared to the amitriptyline group.

Current histopathological results revealed that the cardiac tissue in treated rats with amitriptyline revealed severe tissue injury with marked myocardial hypertrophy and DNA damage with strong positive PCNA expression, indicating the myocardial injury and deformation of nuclei of cardiomyocytes and disarrangement or disordered cardiac myofibres. Treatment of amitriptyline with damiana showed significant improvement in cardiac tissues compared to the amitriptyline group. The current result was by Tousson et al. (17), who reported that amitriptyline induced testicular injury and DNA damage, and the treatment with damiana modulates the alterations in testicular functions and structure.

The current result was by Sorodoc et al. (35), who concluded in their study that the use of a high dose of amitriptyline led to diffuse myocardial alterations such as dilated capillaries, hemorrhagic areas with variable sizes and, in over 30% of the treated animals, few small foci of acute myocardial necrosis characterized by pyknosis or karyolysis. Marti et al. (42) demonstrated that long-term amitriptyline treatment induced myocardial cell damage in patients.

Current results agree with Cohen et al. (43), who have an association between tricyclic antidepressants and an increased risk of myocardial infarction. Furthermore, the increase of cardiac enzymes, AST, ALP, and AST values with increased lipid profile levels, heart damage indicators, and histopathological and PCNA immunoreactivity alterations supported this conclusion, and the treatment with damiana improved cardiac functions and structure.

Conclusion

Heart diseases in psychiatric patients that use amitriptyline are life-threatening. So, it is essential to assess the cardiac health status of the patient, and control the complication of amitriptyline on the heart using an herbal substance that enhances its action without any side effects. This study suggests amitriptyline-induced cardiac toxicity and uses damiana, promising in protecting heart functions and structure against antidepressants such as amitriptyline.

Acknowledgment

Thanks to Professor Dr. Tousson Ehab Lab's help in biochemical analysis.

Conflicting interests

The author(s) declared no potential conflicts of interest concerning this article's research, authorship, and publication.