EFFECT OF METHANOLIC EXTRACTS OF Artemisia vulgaris AND Bergenia ciliata AGAINST DIETHYL-NITROSAMINE (DEN) INDUCED TESTICULAR CHANGES IN MICE

EFFECT OF METHANOLIC EXTRACTS OF Artemisia vulgaris AND Bergenia ciliata AGAINST DIETHYL-NITROSAMINE (DEN) INDUCED TESTICULAR CHANGES IN MICE | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article EFFECT OF METHANOLIC EXTRACTS OF Artemisia vulgaris AND Bergenia ciliata AGAINST DIETHYL-NITROSAMINE (DEN) INDUCED TESTICULAR CHANGES IN MICE Sara Shamim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9686581/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Diethylnitrosamine (DEN) or N-Nitrosodiethylamine (NDEA) is used as a carcinogen in experimental animal model systems. The DEN treatment at a necrogenic dose can cause acute toxicity to various cell types in the adenohypophysis of the pituitary gland and alter serum levels of several hormones that can alter reproduction. Artemisia vulgaris and Bergenia ciliata are known for their antioxidant and protective effects against various chemicals. Objective The present study aims to elucidate the anti-carcinogenic activity of Artemisia vulgaris and Bergenia ciliata against diethylnitrosamine (DEN) induced changes in the reproductive system of male albino mice. Methods Mice (N = 120), maintained at room temperature (25°C) were divided into six groups (each group containing 20 mice) and treated with saline, DEN, Artemisia vulgaris extract, Bergenia ciliata extract, DEN+ Artemisia vulgaris extract and DEN+ Bergenia ciliata extract respectively for eight weeks. At the end of the experiment, the final body weight was measured and mice were dissected. The weight of both testes was measured, the testicular tissue was processed for histology and blood samples were collected for analysis of testosterone. Histological images were analyzed by ImageJ software for measurement of the area covered by seminiferous tubule (%), area of the interstitium (%), seminiferous tubule diameter (µm), and epithelial cell height (µm). Results A significant (P 0.05) in Bergenia co-treated group. Similarly, a low (P < 0.05) level of plasma testosterone (0.2–0.3 ng/ml) was observed in DEN and DEN+ Artemisia extract-treated groups compared to Artemisia and Bergenia alone, DEN + Bergenia groups. Histological evaluation revealed a significant (P < 0.05) reduction in the diameter of seminiferous tubules, epithelial cell height, and cell numbers in DEN treated group compared to the control group. These effects were restored (P 0.05) in the Artemisia vulgaris extract co-treated group. Conclusion The results of the present study revealed that Bergenia ciliata extract is efficient in reversing DEN-induced DEN-induced reproductive toxicity in male mice. Further studies are needed to evaluate the components of Bergenia Ciliata extracts useful in treating reproductive organ toxicity. Animal Science Diethyle nitrosamine (DEN) A. vulgaris B. ciliata Testicular toxicity Testosterone Mice Figures Figure 1 Figure 2 INTRODUCTION Diethylnitrosamine (DEN) is a strong hepatocarcinogen that has been used in rodent models to research liver cancer progression. It increases the clonal growth of started hepatocytes and produces oncogenic mutations [ 1 ] . One of the key proteins involved in cancer progression is cdk4, and its overexpression is caused by DEN treatment [ 2 ] . DEN exposure modifies liver bioenergetics by blocking mitochondrial complexes I and IV and causes histological alterations, including pre-neoplastic lesions [ 3 ] . It also impacts oxidative stress, while the liver initially has some ability to offset these effects through enhanced antioxidant enzyme activity [ 3 ] . DEN-induced hepatocarcinogenesis in rats mirrors human hepatocellular carcinoma, making it a useful model for liver cancer processes and treatments [ 1 ] . Diethyl nitrosamine (DEN) or N-Nitrosodiethylamine (NDEA) is found in a variety of products like beauty care products, agricultural chemicals, medicinal agents [ 4 ], processed meat, cheddar, soybeans, smoke, salted and dried fish, whisky and groundwater. There is sufficient evidence to consider DEN among the most critical environmental carcinogens known to induce unrest in the nuclear proteins required in DNA repair/replication [ 5 ] . Since DEN does not itself exert carcinogenicity, it needs to be bioactivated by cytochrome P450 (CYP) enzymes in the liver, resulting in DNA adducts (Segments of DNA bound to a cancer-causing chemical) that form reactive electrophiles through an alkylation mechanism. Alkylation of DNA at the O 6 position of guanine is an important step in the formation of mutations in cancer, primarily due to the tendency of O 6-methylguanine to pair with thymine during replication, resulting in the conversion of guanine: cytosine to adenine: thymine pairs in DNA. The reactive electrophiles cause oxidative stress, stimulating cytotoxicity, apoptosis, liver proliferation, and necrosis [6;7] . The carcinogenic effect of DEN can be attributed to its oxidative damage to bio-molecules like proteins, lipids, and DNA [ 8 ] leading to cellular injury [9; 10] . Research on diethylnitrosamine (DEN) and its impact on testicular toxicity and possible medications that mitigate it has recently been conducted. It has been demonstrated that DEN causes oxidative stress, interferes with spermatogenesis, and modifies the hormonal balance in male rodents [ 11;12 ] . In human and animal bodies a natural defense system exists that neutralizes reactive oxygen species in cells and protects them from damage [13;14] . However, if ROS production is very high, the natural defense system is rendered ineffective against oxidative stress. Artemisia vulgaris (Mugwort) and Bergenia ciliata (Winter begonia) are therapeutic plants that exhibit anti-cancerous, anti-inflammatory, and anti-bacterial properties [15; 16] . A. vulgaris has antiparasitic, antispasmodic, and anti-inflammatory properties and bronchodilator activity. Aqueous and methanolic extracts of A. vulgaris exhibited inhibitory effects on cell growth and are attributed to the induction of apoptosis, suppression of cell migration, and autophagy [ 17 ] . Various species of Artemisia have shown antiproliferative effects on cancer cells. Extract from A. maritima reduced cell migration triggered apoptosis, halted the G2/M cell cycle, and hindered lung cancer cell survival [ 18 ] . According to Lian et al. (2018), an extract from A. vulgaris also showed cytotoxicity against colon cancer cells by causing autophagy, lowering mitochondrial membrane potential, and preventing cell migration. Extraction from the leaves of A. vulgaris inhibited the growth and metastasis of lung cancer cells, brought about cell death, and reduced the activity of the Wnt (Complex network of proteins that aids in cellular regulation) signaling pathway [ 19 ] . These results indicate that Artemisia extracts may influence apoptosis, cell cycle progression, migration, and other cellular processes, which could make them useful anticancer drugs. B. ciliata possesses anti-tussive and cytoprotective properties and its rhizome works against urolithiasis. B. ciliata is a defensive agent against neoplastic activity [ 20 ] . Despite some reports of toxic effects, B. ciliata has shown hypoglycaemic effects in rats treated with streptozotocin and shows potential in treating a wide variety of illnesses, including kidney problems [21; 22] . A lot of work has been done on the antioxidant activities of plants against carcinomas in the liver, lungs, and breast. Still, not enough studies are available on testicular toxicity and its amelioration using natural antioxidants. To facilitate gaining a comprehensive understanding of its therapeutic potential and safety profile, additional research is required, as it has the potential to serve as a foundation for the development of anti-cancer treatments. Androgen and androgen receptor (AR) signaling have a crucial role in the progression of prostate, colon, and breast cancer [ 16 ] . Androgens act through the AR, a transcription factor that belongs to the nuclear receptor superfamily. The androgen receptor plays physiological and pathological functions by translocating to the nucleus upon binding to androgens, where it binds to specific DNA sequences [ 23 ] . The AR complex regulates the pathological expression of genes [ 24 ] . As androgen and AR signaling are intimately involved in cancerogenesis, it stands to reason that androgen in combination with DEN might play an important role in testicular toxicity or carcinogenicity [ 16 ] . A lot of work has been done on the antioxidant activities of plants against carcinomas in the liver, lungs, and breast, however, not enough studies are available on the anti-cancerous effect of Bergenia ciliata on testicular toxicity. It was hypothesized that A. vulgaris and B. ciliata can rectify the carcinogenic effect of DEN on the testicular toxicity of albino mice. The objective was to evaluate the impact of B. clliata and A. vulgaris against diethyl-nitrosamine induced changes in testicular weight, testicular tissue, and testosterone level in male albino mice. MATERIALS AND METHODS ETHICAL STATEMENT The study was approved by the ethical committee of PMAS Arid Agriculture University Rawalpindi for the use of animal experimentally. DRUGS AND CHEMICALS Diethylnitrosoamine and NaCl have been purchased from Sigma Aldrich St Louis USA) One percent DEN was prepared by adding 99 ml of normal saline (sodium chloride 0.9 percent) and 1 ml of concentrated diethyl-nitrosoamine solution (0.01μg/μl). EXTRACT PREPARATION The plant extracts ( Artemesia vulgaris and Bergenia ciliata ) were prepared through conventional extraction methods [25] . The crude extracts were filtered through Whatman filter I and concentrated in a vacuum. EXPERIMENTAL DESIGN Balb C mice with an average body weight of about 35-40 g were procured from the National Institutes of Health (NIH), Islamabad, and were kept in an animal house having a temperature of 20-23 °C. The mice were divided into 6 groups containing ten mice in each group. The first group served as control and received saline solution (3.5 µl/g) once a week for eight weeks; the second group was given diethyl-nitrosamine (3.5 µl/g) intraperitoneally once a week for eight consecutive weeks, the third and fourth group received plant extracts (extract 1 and extract 2 respectively, 150 mg/kg) once in a week. The fifth and sixth groups were treated with DEN (3.5 µl/g) along with extract 1 (fifth group) and extract 2 (sixth group) at the dose rate of 150 mg/kg once a week for eight consecutive weeks. At the end of the experimental period, the mice were weighed and dissected. The blood samples and testicular tissue were collected for biochemical and histopathological analysis. BODY WEIGHT AND TESTICULAR WEIGHT Relative body and organ weight were measured on an electronic balance. BIOCHEMICAL ANALYSIS At the time of dissection, blood samples were collected in tubes containing ethylene-diamine tetra acetic acid (EDTA). Blood serum was separated after centrifugation at 3000 rpm for 10 minutes and stored at -20 °C till further analysis. The serum level of testosterone was measured through an ELISA kit (Amgenix Inc, USA). HISTOPATHOLOGICAL ANALYSIS Both the testes were collected, weighed separately, and stored in the refrigerator until histological evaluation. Testicular Parameters: The testicular tissue was fixed in buffered formalin for one week, impregnated in equal amounts of xylene, and wax, and incubated for 2 h at 65°C. The sections were double stained in hematoxylin and eosin and mounted with DPX. Tissue sections were observed under a light microscope at 20X and 40X using a Leica microscope (Germany) and photomicrographs were taken at 20X and 40X with an automatic photoshoot system (Cannon, Japan). Slides were examined under a microscope furnished with a computerized camera. Morphometry was done utilizing J image software. The area of the seminiferous tubule and epididymis tubules was measured by planimetry. The morphometric analyses were performed on a cellular level, which allowed a better understanding of the structural alterations brought about by the treatments given to the mice. Statistical Analysis: The data on body weight, testicular weight, morphometry, and planimetry were analyzed by ANOVA using CRD and are presented as mean±SEM. When the F ratio was found significant, Tucky’s test was used to compare treatment means. A probability value of <0.05 was deliberated as significant. RESULTS 4.1. BODY WEIGHT AND TESTICULAR WEIGHT The data on initial and final body weight (Mean±SEM) and weight (Mean±SEM) of the right and left testis of experimental mice are presented in Table 4.1. Significant reduction (P < 0.001) in final body weight was observed in DEN-treated mice compared to the control. Similarly, the Artemisia- alone-treated mice gained a final body weight lower (P < 0.05) compared to the control. The final body weights in DEN+ Artemisia and DEN+ Berginia treated rats were significantly lower (P < 0.001) compared to the control group. The reduction in final body weight was more pronounced in the Artemisia-treated group compared to that treated with Bergenia . However, no significant difference was noticed in co-treated groups when compared to the DEN alone treated group (Table 4.1). The DEN treatment resulted in a significant reduction (P<0.001) in the weight (g) of the right as well as left testis of treated mice compared to the control. The testicular weight was similar in all other groups ( Artemisia and Bergenia alone, DEN + Bergenia ) and the control animals, except the DEN + Artemisia treated group. The results showed a significantly higher (P<0.001) weight of the testis in all treated groups ( Artemisia and Bergenia alone,DEN + Artemisia, DEN + Bergenia groups) compared to DEN-treated groups. No significant difference was noted in the DEN + Artemisia treated group at P<0.001 compared to the control group (Table 4.1.). 4.2. MORPHOMETRY OF TESTICULAR TISSUE The data on testicular morphometry are presented in Table 4.2. The area (%) covered by seminiferous tubule was significantly reduced (P<0.001) in the testis of DEN alone treated group, while, the area (%) covered by the interstitium was significantly higher (P<0.001) in DEN treated group compared to Artemisia alone , Bergenia alone DEN + Bergenia and DEN + Artemisia groups. No significant difference in the area covered by seminiferous tubule and interstitium was observed in plant extracts alone treated groups. However, in the DEN + Artemisia treated group area covered by seminiferous tubule and interstitium was significantly low compared to the control group. Compared to DEN alone treated group, the area of the interstitium and seminiferous tubules was not different in the Artemisia + DEN but was significantly high (P<0.001) in Artemisia alone , Bergenia alone, and DEN + Bergenia groups Table 4.2. Significant reduction in the diameter and epithelial height was noted in the DEN alone treated group compared to the control group. The diameter in the DEN + Artemisia treated group was also statistically reduced (P<0.01) compared to the control group however, the other groups ( Artemisia alone , Bergenia alone, DEN + Bergenia treatment) groups exhibited no significant change compared to the control. In comparison with DEN alone treated groups, all the groups ( Artemisia alone , Bergenia alone, DEN + Bergenia ) exhibited significantly high diameter (P<0.001 and P<0.05) except in the DEN + Artemisi a treated groups. The study found no significant variations in epithelial height across any of the treatment groups, except the diethylnitrosamine (DEN)-only group. This shows that the epithelial height was considerably lower in the DEN treatment group than in the control group, indicating a substantial effect. The statistical significance was determined at a probability threshold of P<0.001, underscoring the robustness of the findings. Other treatment groups, which included those that were given plant extracts or combinations with DEN, did not show significant changes in epithelial height compared to the control group. This indicates that, in contrast to the other treatments, DEN had a targeted impact on changing epithelial height (Table 4.2.). 4.3. CELL COUNT IN SEMINIFEROUS TUBULES The data on the number of cell types in the seminiferous tubules are presented in Table 4.2. The number of spermatogonia, spermatocytes, and spermatids was significantly reduced (P<0.001) in the DEN alone treated group compared to the control group. When compared to the control group, the total number of distinct cell types did not significantly differ in the groups treated with Artemisia and Bergenia alone. However, compared to the group treated with DEN alone, there was a statistically significant (P<0.001) increase in the quantity of spermatocytes and spermatids in these groups. Cell counts in the group that received both DEN and Artemisia treatment were comparable to those in the group that received DEN treatment alone, suggesting that there was no discernible difference. In contrast to the control group, the cell counts in the DEN + Artemisia group were significantly different (P<0.001) (Table 4.2.). In the group treated with Bergenia in combination with DEN, cell numbers were recovered and were not different compared to the control group however, the cell numbers were significantly higher compared with the DEN alone treated group (P<0.001, <0.05 respectively; Table 4.2.). 4.4. PLASMA TESTOSTERONE Testosterone concentration was significantly reduced (P<0.001) in DEN alone treated group and DEN + Artemisia treated groups. Statistically significant differences were found between the control group and the treatment groups upon examination of testosterone levels. These differences were seen in the groups that received Artemisia alone, Bergenia alone, and DEN plus Bergenia . Different thresholds of statistical significance were established: P<0.05 for the DEN + Bergenia group, P<0.01 for the Bergenia alone group, and P<0.001 for the Artemisia alone group. These results show that the treatment groups' testosterone levels were lower than the control group's whereas the difference was strong enough to be considered statistically significant, demonstrating that the treatments exerted an effect on the testosterone levels. The study found that the DEN and DEN + Artemisia groups carried much lower testosterone levels than the Bergenia group. The Bergenia group had higher testosterone levels, which shows that it possessed a protective effect. Figure 4.1. 4.5. HISTO-PATHOLOGICAL OBSERVATIONS Microscopic study of the testicular sections in different groups showed pathophysiological symptoms in the testis. Testis of the control group animals and animals treated with Artemisia and Bergenia alone showed normal morphology including compactly arranged seminiferous tubules and thick epithelium. The interstitial space of the testis was also thin and the lumen was sperm-filled. In the DEN alone treated group, seminiferous tubules were arranged loose and interstitium was large. The tubular lumen was empty and disorganized epithelial cells were visible in the seminiferous tubules. Sloughing and vacuolation in the epithelium were the prominent changes observed in the seminiferous tubules of the DEN-treated group (Figure 4.2). The observations indicated that the Artemisia co-treatment was less effective while co-treatment with Bergenia was more effective against the toxic effects of DEN treatment in the testis of mice (Figure 4.2). Table 4.1: Mean ± SEM initial and final body weight and testis weight in different treatment and control groups of mice. Sr.No Body Weight Testicular Weight Treatments Initial weight Final weight Right Testis Left Testis Control 35±0.91 43.8±1.71 0.238±0.01 0.238±0.007 DEN 34.7±1.00 29.4±0.68*** 0.17±0.003*** 0.238±0.004+++ Artemisia 35.6±0.81 37.4±1.63*++ 0.236±0.005+++ 0.238±0.004+++ Bergenia 34.3±1.03 38.4±1.63++ 0.234±0.004+++ 0.234±0.005+++ DEN + Artemisia 35.3±0.95 29.4±1.16*** 0.182.004*** 0.172±0.004*** DEN + Bergenia 35.2±0.85 34±1.00*** 0.22±0.006+++ 0.224±0.005+++ Values are expressed as Mean ± SEM *, **, *** represents significant difference at probability value P<0.05, 0.01 and 0.001 as compared to control +, ++, +++ represents significant difference at probability value P<0.05, 0.01 and 0.001 as compared to DEN treated groups Table 4.2: Mean ± SEM area and diameter of seminiferous tubules, interstitium, and numbers of different cells in the seminiferous tubules in different treatment and control groups of mice. Sr.No Testis Morphometry Cell Count in Seminiferous Tubules Treatments %Area covered by seminiferous tubule %Area of interstitium Seminiferous tubule Diameter (µm) Epithelial height (µm) Spermatogonia Spermatocytes Spermatids Control 81.13±1.99 18.87±0.199 139.9±0.83 59.53±1.94 58.94±1.11 71.08±1.61 245.92±2.02 DEN 63.88±0.56*** 36.11±0.56*** 118.75±0.59**- 46.42.58*** 49.4±1.18*** 59.2±2.30*** 225.84±2.14*** Artemisia 81.72±0.57+++ 18.28±0.57+++ 146.5±5.87+++ 58.3±1.83*+++ 57.8±1.06+++ 69.48±1.24+++ 243.76±3.03+++ Berginia 83.95±0.93+++ 16.04±0.93+++ 144.55±7.51+++ 56.53±1.59+++ 59.64±0.74+++ 70.32±1.41+++ 244.72±2.75+++ DEN + Artemisia 67.79±0.60*** 32.20±0.60*** 120.32±0.41** 52.46±1.71+ 50.68±1.29*** 60.58±1.92*** 227.8±2.26*** DEN + Berginia 76.90±0.46+++ 23.09±0.46+++ 136.65±0.94+ 56.1±0.40+++ 55.96±1.26+++ 67.18±1.24+ 236.84±2.23+ Values are expressed as Mean ± SEM *, **, *** represents significant difference at probability value P<0.05, 0.01 and 0.001 as compared to control. +, ++, +++ represent significant differences at probability value P<0.05, 0.01, and 0.001 as compared to DEN treated group. DISCUSSION Diethylenitrosamine (DEN), the potent environmental carcinogen has been known to induce damage in many enzymes involved in DNA repair/replication and is specially used to cause hepatocellular carcinoma in experimental animal models [4] . Rhodamine dyes, which can produce nitrite-rich water in concentrations as high as 75%, are one of the primary environmental sources of DEN [26] . Exposure to treated water could potentially endanger the health of populations. At least 18 animal species, including poultry and cats, have identified DEN as carcinogenic, indicating its potential as a human carcinogen [27] . The pervasive carcinogenic potential of DEN underscores the significance of understanding its sources and mechanisms of action for public health protection. The DEN bioactivation in rat liver involves α-hydroxylation of DEN by CYP2E1 and other P450 isozymes leading to the reactive ethyl diazonium ion [28] . It has also been reported that there is a close association between guanine O-alkylation and mutation frequency [29] . According to Tong (1982) [30] , O6-chloroethyl guanine may crosslink with the opposite cytosine residues, thereby blocking DNA replication. DEN exposure of cells disturbs the balance between antioxidants and reactive oxygen species and results in carcinogenesis [3] . In the present study, a significant reduction in body weight was observed in mice that got DEN treatment for eight weeks. Reduction in body weight can be attributed to collective tissue damage in the body by initial toxic lesions in liver cells, and reproductive cells, as well as due to reduction of food intake [31] . Plants of the genus Artemisia have revealed cytotoxic effects against cancer cells by inducing cell cycle arrest [32] . The anticancerous activity of Artemisia species has been observed against human cancer cell lines especially A549, HeLa, and MCF7 [33] . Artemisia has bioactive compounds like alkaloids, terpenoids, flavonoids, and coumarines and possesses potential antioxidant activity against aging, inflammation, and chronic infectious diseases. However, in the present study, Artemisia did not ameliorate the damaging effects of DEN on body weight in DEN-treated mice. It might be possible the “concentration of extract” of A. vulgaris that was used during the experiment acted as a pro-oxidant and lost its efficiency against the potent diethylnitrosamine. Bergenia ciliata is a perennial herb having antibacterial, antioxidant, anti-inflammatory, anti-diabetics, and antiviral activities [34] . Bergenia treatment was efficient in maintaining a normal body weight. However, the beneficial effect of Bergenia in terms of body weight might be attributed to its anti-oxidant activity [35] . There is substantial therapeutic potential in Bergenia species, as evidenced by numerous studies. Bergenia ciliata demonstrated prophylactic effects against nephrolithiasis in rats, which were presumably mediated by its antioxidant activity [36] . Likewise, bergenin, which was isolated from Bergenia ligulata , exhibited potent antioxidant and antilithiatic properties, effectively shielding against oxidative stress and renal dysfunction in hyperoxaluric rodents [37] . Our results are in line with those of the previous study that reported the projected effects of Bergenia against tissue/organ/animal body weight loss due to exposure to hazardous chemicals [38] . It was observed that DEN treatment induces degenerative changes in the testicular tissues of mice. These changes were successfully ameliorated by Bergenia while Artemisia exhibited less or no protective effect against DEN-induced reproductive toxicity the reason may be the same as above. The structural and functional integrity of testicular tissues depends on the circulating androgens and a small decrease in androgen content can result in a reduction in the weight of the testes [39] . In the present study, testicular weight was significantly reduced in the DEN-treated group as has previously been reported by [40] . The mice treated with Bergenia ciliata extract restored normal testicular weight compared to DEN treated group because it is an excellent antioxidant that has the potential to directly scavenge or prevent the generation of ROS [35] . It is relevant to mention that Bergenia protects tissue from tissue weight loss [41] . However, the effect of Artemisia was not pronounced which is contradictory to previous literature [42] . The structural and functional integrity of testicular tissues depends on the circulating androgens and a small decrease in androgen content can result in a reduction in the weight of the testes [39] . In the present study, testicular weight was significantly reduced in the DEN-treated group as has previously been reported by [40] . The mice treated with Bergenia ciliata extract restored normal testicular weight compared to DEN treated which can be attributed to the antioxidant activity of Bergenia to directly scavenge or prevent the generation of ROS [35] and to protect the tissue from tissue weight loss [41] . The seminiferous tubules function to produce, maintain, and store sperm. The reduction of the seminiferous tubule width in mice shows that the shriveling of these tubules leads to structural disruption in sperm production in the testis [43] . In the present study, the % area covered by seminiferous tubule was significantly reduced in the testis of the DEN alone treated group compared to the control group. The interstitial cells of leydig are responsible for testosterone production. Similarly, the % area covered by the interstitium was significantly higher in DEN treated group than control. The increased % area of interstitium might be due to inflammation. The protection provided by Bergenia ciliata against DEN-induced tubular lesions and reduction in epithelial height as well as diameter might be due to the potent antioxidant potential of the plant [35] . However, the results are not in line with Anibogwu (2021) [44] who reported the antioxidant effects of Artemisia in embryonic cells. As the DEN acts by inducing oxidative stress resulting in cell death [40] , the Artemisia however, did not protect the tissue either due to lack of its antioxidant potential or due to certain other mechanisms [44] . Bergenia co-treatment resulted in reversing the reduction in the number of cells in the seminiferous tubules but Artemisia failed to increase the number of cells. This protective effect of Bergenia might be due to its antioxidant potential [35] however, the lack of effect of Artemisia in protecting testicular tissue is contradictory to previous findings [44] . Further studies are required to confirm the effects of these plant extracts. In the target tissues, androgens enter the cell cytoplasm by simple diffusion across the cell membrane. Once inside the cell, the androgens bind and activate the androgen receptors. The androgen-receptor complex attaches to a specific DNA site and stimulates the production of messenger RNA, which, in turn, stimulates the production of the enzymes and proteins necessary to affect androgen action [45] . The level of testosterone was increased in all the low-dose DEN-treated groups except for chronic exposure to DEN. High-dose administration of DEN reduced the testosterone level in the blood [40] . A significant reduction in testosterone might have caused pathological changes in the testis tissue [46]. Plant extracts as well as other chemical agents cause hormonal imbalance as alkaloids and flavonoids reduce plasma concentrations of LH, estradiol, and FSH [47]. In this study Testosterone concentration was significantly reduced in the DEN alone treated group and DEN + Artemisia treated groups Diethyle nitrosamine is a potent genotoxin and being an endocrine disrupter might alter the nature of those enzymes that are responsible for androgen production. No significant difference was noted in other treated groups regards to control group. CONCLUSION In conclusion, the results of the present study indicate that DEN can induce reproductive toxicity by affecting the HPG axis and ultimately the seminiferous tissue. These effects are reversed by Bergenia co-treatment but could not be reversed by Artemisia co-treatments. Hence, Bergenia is a more potent protective agent than Artemisia against DEN-induced reproductive toxicity. Further studies are obligatory to endorse the effects of these extracts against other chemical toxicants. Declarations Conflict of Interest Authors have no conflict of interest to declare. AUTHORS CONTRIBUTION S.S., and T.A., were involved in practical work. S.S. and S.J. analyzed the data. S.S. and S.A. drafted the manuscript. Availability of Data and Material Additional information will be provided upon request. References Sotty J, Bablon P, Weiss P-H, Soussan P (2024) Diethylnitrosamine Induction of Hepatocarcinogenesis in Mice. Methods Mol Biol 2769:15–25. https://api.semanticscholar.org/CorpusID:267498753 Park D-H, Shin JW, Park S-K, Seo J, Li L, Jang J, Lee M (2009) Diethylnitrosamine (DEN) induces irreversible hepatocellular carcinogenesis through overexpression of G1/S-phase regulatory proteins in rats. 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Biomed \& Pharmacotherapy = Biomedecine \& Pharmacotherapie 97:708–721. https://api.semanticscholar.org/CorpusID:206096914 Claessens F, Verrijdt G, Schoenmakers E, Haelens A, Peeters B, Verhoeven G, Rombauts W (2001) Selective DNA binding by the androgen receptor as a mechanism for hormone-specific gene regulation. J Steroid Biochem Mol Biol 76(1–5):23–30. https://doi.org/10.1016/s0960-0760(00)00154-0 Bluemn EG, Nelson PS (2012) The androgen/androgen receptor axis in prostate cancer. Curr Opin Oncol 24(3):251–257. https://doi.org/10.1097/CCO.0b013e32835105b3 Devgun M, Nanda A, Ansari SH (2012) Comparison of conventional and non-conventional methods of extraction of heartwood of Pterocarpus marsupium Roxb. Acta Pol Pharm 69(3):475–485. http://europepmc.org/abstract/MED/22594262 Abidi SL, Fish US (1982) NITRITE-RICH WATER FOLLOWING . 16 Schmähl D, Habs M, Ivankovic S (1978) Carcinogenesis of N-nitrosodiethylamine (DENA) in chickens and domestic cats. 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J Hydrol 391(1):202–216. https://doi.org/https://doi.org/10.1016/j.jhydrol.2010.07.012 Taleghani A, Emami SA, Tayarani-Najaran Z (2019) Artemisia: a promising plant for the treatment of cancer. Bioorg \& Med Chem, 115180. https://api.semanticscholar.org/CorpusID:208497499 Bordoni V, Sanna L, Lyu W, Avitabile E, Zoroddu S, Medici S, Kelvin DJ, Bagella L (2021) Silver Nanoparticles Derived by Artemisia arborescens Reveal Anticancer and Apoptosis-Inducing Effects. International Journal of Molecular Sciences , 22 . https://api.semanticscholar.org/CorpusID:237331937 Kushwaha N, Singh A (2024) Bergenia ciliata—Phytochemistry and Pharmacology: A Review. Biomedical Mater \& Devices. https://api.semanticscholar.org/CorpusID:267684964 Venkatadri R, Guha G, Kumar R, Mathew L (2010) Evaluation of Antioxidant Activities of Bergenia ciliata Rhizome. Records Nat Prod 4:38–48 Saha S, Shrivastav PS, Verma RJ (2014) The antioxidative mechanism involved in the preventive efficacy of Bergenia ciliata rhizomes against experimental nephrolithiasis in rats. Pharm Biol 52:712–722. https://api.semanticscholar.org/CorpusID:23205837 Aggarwal D, Kaushal R, Kaur T, Kumar R (2014) The most potent antilithiatic agent ameliorating renal dysfunction and oxidative stress from Bergenia ligulata rhizome. J Ethnopharmacol 158:85–93. https://doi.org/10.1016/j.jep.2014.10.013 Attaguile G, Russo A, Campisi A, Savoca F, Acquaviva R, Ragusa N, Vanella A (2000) Antioxidant activity and protective effect on DNA cleavage of extracts from Cistus incanus L. and Cistus monspeliensis L. Cell Biol Toxicol 16(2):83–90. https://doi.org/10.1023/a:1007633824948 Kubota K, Ohsako S, Kurosawa S, Takeda K, Qing W, Sakaue M, Kawakami T, Ishimura R, Tohyama C (2003) Effects of vinclozolin administration on sperm production and testosterone biosynthetic pathway in adult male rat. J Reprod Dev 49(5):403–412. https://doi.org/10.1262/jrd.49.403 Palanisami K, Hassan SM, Mani S, Vaduganathan R, Murugan E, Kumaravel S (2014) Effect on Aqueous and Ethanolic Pomegranate Peel Extract on the Diethylnitrosoamine Induced Changes on Lipid Profile and Testicular Function of Albino rats . https://api.semanticscholar.org/CorpusID:182039400 Bashir S, Gilani AH (2009) Antiurolithic effect of Bergenia ligulata rhizome: an explanation of the underlying mechanisms. J Ethnopharmacol 122(1):106–116. https://doi.org/10.1016/j.jep.2008.12.004 Ben-Nasr H, Abderrahim MA, Ben, Salama M, Ksouda K, Zeghal KM (2013) Potential phytotherapy use of Artemisia plants: Insight for anti-hypertension. J Appl Pharm Sci 3(5):120–125. https://doi.org/10.7324/JAPS.2013.3523 Fabrícia S. Predes.1, Juliana, Monteiro C1, Tarcízio AR, Paula2 SLP (2007) da M. Evaluation of rat testes treated with. Braz. J. Morphol. Sci , 24 (2), 112–117 Anibogwu R, Jesus K, De, Pradhan S, Pashikanti S, Mateen S, Sharma K (2021) Extraction, Isolation and Characterization of Bioactive Compounds from Artemisia and Their Biological Significance: A Review. Molecules 26(22). https://doi.org/10.3390/molecules26226995 Giannitrapani L, Soresi M, La Spada E, Cervello M, D’Alessandro N, Montalto G (2006) Sex Hormones and Risk of Liver Tumor. Ann N Y Acad Sci 1089:228–236. https://doi.org/10.1196/annals.1386.044 Stefanović A, Kotur-Stevuljević J, Spasić S, Bogavac-Stanojević N, Bujisić N (2008) The influence of obesity on the oxidative stress status and the concentration of leptin in type 2 diabetes mellitus patients. Diabetes Res Clin Pract 79(1):156–163. https://doi.org/10.1016/j.diabres.2007.07.019 Bianco F, Basini G, Grasselli F (2006) The plant alkaloid Sanguinarine affects swine granulosa cell activity. Reprod Toxicol 21 3:335–340. https://api.semanticscholar.org/CorpusID:24794243 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9686581","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":638720150,"identity":"912a44af-9663-499f-a546-ffb0ad5cf702","order_by":0,"name":"Sara Shamim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIie2PMUvDUBDHL/zhshxmbYk2XyES6CKkH+ZBXLqIU+nQoaBj136SN3WIPLCLpWsgi1HIHkRQXMwrnZOMgu8H739vuB93R+Rw/FFwesDTa/uRi+GKzyq2Cg9ViAKZjmztVaLHQ/Vxt0v9YE3Txec8vWRC9VZ0KPHLbRJua4WRoay80qpdjJNk3qVQRqHkABl6LscarSIcdinRpsaP5CtExnu4H+tVv0JFxu0Ug9iAvUabfiUuar6RfI9rwwg9vRdGzy3RJkMp+VJNjsem+dbLWeCvq/fOxc4oG5BTDmi3pDa8r4HdDofD8b/4BdHpPpi/BWkiAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0002-1490-4070","institution":"Southeast University","correspondingAuthor":true,"prefix":"","firstName":"Sara","middleName":"","lastName":"Shamim","suffix":""}],"badges":[],"createdAt":"2026-05-12 05:07:33","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-9686581/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9686581/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109273909,"identity":"6bb9efc5-a562-4ab8-acc8-251bfa88ff7b","added_by":"auto","created_at":"2026-05-14 14:36:48","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":132276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 4.1:\u003c/strong\u003e Mean ± SEM plasma testosterone concentration (ng/ml) in control and different treatment groups of mice. *, **, *** represents significant difference at probability value P\u0026lt;0.05, 0.01 and 0.001 as compared to control. +, ++, +++ represent significant differences at probability values P\u0026lt;0.05, 0.01, and 0.001 as compared to the DEN-treated group. ANOVA followed by Tukey’s test.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9686581/v1/00f5f2fa3689df29d101d084.jpeg"},{"id":109296090,"identity":"c26100c4-8ede-4def-a194-1fd59f243571","added_by":"auto","created_at":"2026-05-15 08:45:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":262358,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 4.2:\u003c/strong\u003e Photomicrograph of seminiferous tubules of the control and different treatment groups. A: control testis having closely arranged tubules and sperm-filled lumen. B: DEN treated group with shrunk seminiferous tubules and empty lumen. C, D: seminiferous tubules similar to the control group. E: represents seminiferous tubules with degenerative changes like in the DEN alone treated group. F: represents seminiferous tubules with improved diameter and filled lumen.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9686581/v1/4ae96bd9948d08803420e0d2.jpg"},{"id":109405071,"identity":"8477160c-1a1a-461d-89a3-78afb256d019","added_by":"auto","created_at":"2026-05-17 12:54:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":689485,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9686581/v1/3e5e953c-a603-48ad-9806-50c8a12a7835.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEFFECT OF METHANOLIC EXTRACTS OF \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArtemisia vulgaris \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eAND \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBergenia ciliata\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e AGAINST DIETHYL-NITROSAMINE (DEN) INDUCED TESTICULAR CHANGES IN MICE\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDiethylnitrosamine (DEN) is a strong hepatocarcinogen that has been used in rodent models to research liver cancer progression. It increases the clonal growth of started hepatocytes and produces oncogenic mutations \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. One of the key proteins involved in cancer progression is cdk4, and its overexpression is caused by DEN treatment \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. DEN exposure modifies liver bioenergetics by blocking mitochondrial complexes I and IV and causes histological alterations, including pre-neoplastic lesions \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. It also impacts oxidative stress, while the liver initially has some ability to offset these effects through enhanced antioxidant enzyme activity \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. DEN-induced hepatocarcinogenesis in rats mirrors human hepatocellular carcinoma, making it a useful model for liver cancer processes and treatments \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDiethyl nitrosamine (DEN) or N-Nitrosodiethylamine (NDEA) is found in a variety of products like beauty care products, agricultural chemicals, medicinal agents \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e],\u003c/sup\u003e processed meat, cheddar, soybeans, smoke, salted and dried fish, whisky and groundwater. There is sufficient evidence to consider DEN among the most critical environmental carcinogens known to induce unrest in the nuclear proteins required in DNA repair/replication \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Since DEN does not itself exert carcinogenicity, it needs to be bioactivated by cytochrome P450 (CYP) enzymes in the liver, resulting in DNA adducts (Segments of DNA bound to a cancer-causing chemical) that form reactive electrophiles through an alkylation mechanism. Alkylation of DNA at the \u003cem\u003eO\u003c/em\u003e6 position of guanine is an important step in the formation of mutations in cancer, primarily due to the tendency of \u003cem\u003eO\u003c/em\u003e6-methylguanine to pair with thymine during replication, resulting in the conversion of guanine: cytosine to adenine: thymine pairs in DNA. The reactive electrophiles cause oxidative stress, stimulating cytotoxicity, apoptosis, liver proliferation, and necrosis \u003csup\u003e[6;7]\u003c/sup\u003e. The carcinogenic effect of DEN can be attributed to its oxidative damage to bio-molecules like proteins, lipids, and DNA \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e leading to cellular injury \u003csup\u003e[9; 10]\u003c/sup\u003e. Research on diethylnitrosamine (DEN) and its impact on testicular toxicity and possible medications that mitigate it has recently been conducted. It has been demonstrated that DEN causes oxidative stress, interferes with spermatogenesis, and modifies the hormonal balance in male rodents \u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e[\u003c/span\u003e11;12\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e]\u003c/span\u003e\u003c/sup\u003e. In human and animal bodies a natural defense system exists that neutralizes reactive oxygen species in cells and protects them from damage \u003csup\u003e[13;14]\u003c/sup\u003e. However, if ROS production is very high, the natural defense system is rendered ineffective against oxidative stress.\u003c/p\u003e \u003cp\u003e \u003cem\u003eArtemisia vulgaris\u003c/em\u003e (Mugwort) and \u003cem\u003eBergenia ciliata\u003c/em\u003e (Winter begonia) are therapeutic plants that exhibit anti-cancerous, anti-inflammatory, and anti-bacterial properties \u003csup\u003e[15; 16]\u003c/sup\u003e. \u003cem\u003eA. vulgaris\u003c/em\u003e has antiparasitic, antispasmodic, and anti-inflammatory properties and bronchodilator activity. Aqueous and methanolic extracts of \u003cem\u003eA. vulgaris\u003c/em\u003e exhibited inhibitory effects on cell growth and are attributed to the induction of apoptosis, suppression of cell migration, and autophagy \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Various species of Artemisia have shown antiproliferative effects on cancer cells. Extract from \u003cem\u003eA. maritima\u003c/em\u003e reduced cell migration triggered apoptosis, halted the G2/M cell cycle, and hindered lung cancer cell survival \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. According to Lian et al. (2018), an extract from \u003cem\u003eA. vulgaris\u003c/em\u003e also showed cytotoxicity against colon cancer cells by causing autophagy, lowering mitochondrial membrane potential, and preventing cell migration. Extraction from the leaves of \u003cem\u003eA. vulgaris\u003c/em\u003e inhibited the growth and metastasis of lung cancer cells, brought about cell death, and reduced the activity of the Wnt (Complex network of proteins that aids in cellular regulation) signaling pathway \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. These results indicate that \u003cem\u003eArtemisia\u003c/em\u003e extracts may influence apoptosis, cell cycle progression, migration, and other cellular processes, which could make them useful anticancer drugs.\u003c/p\u003e \u003cp\u003e \u003cem\u003eB. ciliata\u003c/em\u003e possesses anti-tussive and cytoprotective properties and its rhizome works against urolithiasis. \u003cem\u003eB. ciliata\u003c/em\u003e is a defensive agent against neoplastic activity \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Despite some reports of toxic effects, \u003cem\u003eB. ciliata\u003c/em\u003e has shown hypoglycaemic effects in rats treated with streptozotocin and shows potential in treating a wide variety of illnesses, including kidney problems \u003csup\u003e[21; 22]\u003c/sup\u003e. A lot of work has been done on the antioxidant activities of plants against carcinomas in the liver, lungs, and breast. Still, not enough studies are available on testicular toxicity and its amelioration using natural antioxidants. To facilitate gaining a comprehensive understanding of its therapeutic potential and safety profile, additional research is required, as it has the potential to serve as a foundation for the development of anti-cancer treatments.\u003c/p\u003e \u003cp\u003eAndrogen and androgen receptor (AR) signaling have a crucial role in the progression of prostate, colon, and breast cancer \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Androgens act through the AR, a transcription factor that belongs to the nuclear receptor superfamily. The androgen receptor plays physiological and pathological functions by translocating to the nucleus upon binding to androgens, where it binds to specific DNA sequences \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The AR complex regulates the pathological expression of genes \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. As androgen and AR signaling are intimately involved in cancerogenesis, it stands to reason that androgen in combination with DEN might play an important role in testicular toxicity or carcinogenicity \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. A lot of work has been done on the antioxidant activities of plants against carcinomas in the liver, lungs, and breast, however, not enough studies are available on the anti-cancerous effect of \u003cem\u003eBergenia ciliata\u003c/em\u003e on testicular toxicity. It was hypothesized that \u003cem\u003eA. vulgaris\u003c/em\u003e and \u003cem\u003eB. ciliata\u003c/em\u003e can rectify the carcinogenic effect of DEN on the testicular toxicity of albino mice. The objective was to evaluate the impact of \u003cem\u003eB. clliata\u003c/em\u003e and \u003cem\u003eA. vulgaris\u003c/em\u003e against diethyl-nitrosamine induced changes in testicular weight, testicular tissue, and testosterone level in male albino mice.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eETHICAL STATEMENT\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the ethical committee of PMAS Arid Agriculture University Rawalpindi for the use of animal experimentally.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDRUGS AND CHEMICALS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDiethylnitrosoamine and NaCl have been purchased from Sigma Aldrich St Louis USA) One percent DEN was prepared by adding 99 ml of normal saline (sodium chloride 0.9 percent) and 1 ml of concentrated diethyl-nitrosoamine solution (0.01μg/μl).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEXTRACT PREPARATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plant extracts (\u003cem\u003eArtemesia vulgaris\u003c/em\u003e and \u003cem\u003eBergenia ciliata\u003c/em\u003e) were prepared through conventional extraction methods \u003csup\u003e[25]\u003c/sup\u003e. The crude extracts were filtered through Whatman filter I and concentrated in a vacuum.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEXPERIMENTAL DESIGN\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBalb C mice with an average body weight of about 35-40 g were procured from the National Institutes of Health (NIH), Islamabad, and were kept in an animal house having a temperature of 20-23 °C. The mice were divided into 6 groups containing ten mice in each group. The first group served as control and received saline solution (3.5 µl/g) once a week for eight weeks; the second group was given diethyl-nitrosamine (3.5 µl/g) intraperitoneally once a week for eight consecutive weeks, the third and fourth group received plant extracts (extract 1 and extract 2 respectively, 150 mg/kg) once in a week. The fifth and sixth groups were treated with DEN (3.5 µl/g) along with extract 1 (fifth group) and extract 2 (sixth group) at the dose rate of 150 mg/kg once a week for eight consecutive weeks. At the end of the experimental period, the mice were weighed and dissected. The blood samples and testicular tissue were collected for biochemical and histopathological analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBODY WEIGHT\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAND\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTESTICULAR WEIGHT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRelative body and organ weight were measured on an electronic balance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBIOCHEMICAL ANALYSIS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the time of dissection, blood samples were collected in tubes containing ethylene-diamine tetra acetic acid (EDTA). Blood serum was separated after centrifugation at 3000 rpm for 10 minutes and stored at -20 °C till further analysis. The serum level of testosterone was measured through an ELISA kit (Amgenix Inc, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHISTOPATHOLOGICAL ANALYSIS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth the testes were collected, weighed separately, and stored in the refrigerator until histological evaluation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTesticular Parameters:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe testicular tissue was fixed in buffered formalin for one week, impregnated in equal amounts of xylene, and wax, and incubated for 2 h at 65°C. The sections were double stained in hematoxylin and eosin and mounted with DPX. Tissue sections were observed under a light microscope at 20X and 40X using a Leica microscope (Germany) and photomicrographs were taken at 20X and 40X with an automatic photoshoot system (Cannon, Japan). Slides were examined under a microscope furnished with a computerized camera. Morphometry was done utilizing J image software. The area of the seminiferous tubule and epididymis tubules was measured by planimetry. The morphometric analyses were performed on a cellular level, which allowed a better understanding of the structural alterations brought about by the treatments given to the mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data on body weight, testicular weight, morphometry, and planimetry were analyzed by ANOVA using CRD and are presented as mean±SEM. When the F ratio was found significant, Tucky’s test was used to compare treatment means. A probability value of \u0026lt;0.05 was deliberated as significant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003e4.1.\u0026nbsp; \u0026nbsp; \u0026nbsp;BODY WEIGHT\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAND\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTESTICULAR WEIGHT\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data on initial and final body weight (Mean\u0026plusmn;SEM) and weight (Mean\u0026plusmn;SEM) of the right and left testis of experimental mice are presented in Table 4.1. Significant reduction (P \u0026lt; 0.001) in final body weight was observed in DEN-treated mice compared to the control. Similarly, the \u003cem\u003eArtemisia-\u003c/em\u003ealone-treated mice gained a final body weight lower (P \u0026lt; 0.05) compared to the control. The final body weights in DEN+\u003cem\u003eArtemisia\u003c/em\u003e and DEN+\u003cem\u003eBerginia\u003c/em\u003e treated rats were significantly lower (P \u0026lt; 0.001) compared to the control group.\u0026nbsp;The reduction in final body weight was more pronounced in the \u003cem\u003eArtemisia-treated\u003c/em\u003e group compared to that treated with \u003cem\u003eBergenia\u003c/em\u003e. However, no significant difference was noticed in co-treated groups when compared to the DEN alone treated group (Table 4.1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe DEN treatment resulted in a significant reduction (P\u0026lt;0.001) in the weight (g) of the right as well as left testis of treated mice compared to the control. The testicular weight was similar in all other groups (\u003cem\u003eArtemisia\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Bergenia\u003c/em\u003e alone, DEN + \u003cem\u003eBergenia\u003c/em\u003e) and the control animals, except the DEN + \u003cem\u003eArtemisia\u0026nbsp;\u003c/em\u003etreated group. The results showed a significantly higher (P\u0026lt;0.001) weight of the testis in all treated groups (\u003cem\u003eArtemisia\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Bergenia\u003c/em\u003e alone,DEN + \u003cem\u003eArtemisia,\u003c/em\u003e DEN + \u003cem\u003eBergenia\u003c/em\u003e groups) compared to DEN-treated groups. No significant difference was noted in the DEN + \u003cem\u003eArtemisia\u003c/em\u003e treated group at P\u0026lt;0.001 compared to the control group (Table 4.1.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2. \u0026nbsp; MORPHOMETRY OF TESTICULAR TISSUE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data on testicular morphometry are presented in Table 4.2. The area (%) covered by seminiferous tubule was significantly reduced (P\u0026lt;0.001) in the testis of DEN alone treated group, while, the area (%) covered by the interstitium was significantly higher (P\u0026lt;0.001) in DEN treated group compared to \u003cem\u003eArtemisia\u0026nbsp;\u003c/em\u003ealone\u003cem\u003e, Bergenia\u0026nbsp;\u003c/em\u003ealone DEN + \u003cem\u003eBergenia\u003c/em\u003e and DEN + \u003cem\u003eArtemisia\u0026nbsp;\u003c/em\u003egroups. No significant difference in the area covered by seminiferous tubule and interstitium was observed in plant extracts alone treated groups. However, in the DEN + \u003cem\u003eArtemisia\u0026nbsp;\u003c/em\u003etreated group area covered by seminiferous tubule and interstitium was significantly low compared to the control group. Compared to DEN alone treated group, the area of the interstitium and seminiferous tubules was not different in the \u003cem\u003eArtemisia\u003c/em\u003e + DEN but was significantly high (P\u0026lt;0.001) in \u003cem\u003eArtemisia\u0026nbsp;\u003c/em\u003ealone\u003cem\u003e, Bergenia\u0026nbsp;\u003c/em\u003ealone, and DEN + \u003cem\u003eBergenia\u003c/em\u003e groups Table 4.2. Significant reduction in the diameter and epithelial height was noted in the DEN alone treated group compared to the control group. The diameter in the DEN + \u003cem\u003eArtemisia\u003c/em\u003e treated group was also statistically reduced (P\u0026lt;0.01) compared to the control group however, the other groups (\u003cem\u003eArtemisia\u0026nbsp;\u003c/em\u003ealone\u003cem\u003e, Bergenia\u003c/em\u003e alone, DEN + \u003cem\u003eBergenia\u003c/em\u003e treatment) groups exhibited no significant change compared to the control. In comparison with DEN alone treated groups, all the groups (\u003cem\u003eArtemisia\u0026nbsp;\u003c/em\u003ealone\u003cem\u003e, Bergenia\u003c/em\u003e alone, DEN + \u003cem\u003eBergenia\u003c/em\u003e) exhibited significantly high diameter (P\u0026lt;0.001 and P\u0026lt;0.05) except in the DEN + \u003cem\u003eArtemisi\u003c/em\u003ea treated groups. The study found no significant variations in epithelial height across any of the treatment groups, except the diethylnitrosamine (DEN)-only group. This shows that the epithelial height was considerably lower in the DEN treatment group than in the control group, indicating a substantial effect. The statistical significance was determined at a probability threshold of \u0026nbsp;\u0026nbsp;P\u0026lt;0.001, underscoring the robustness of the findings. Other treatment groups, which included those that were given plant extracts or combinations with DEN, did not show significant changes in epithelial height compared to the control group. This indicates that, in contrast to the other treatments, DEN had a targeted impact on changing epithelial height (Table 4.2.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3. CELL COUNT IN SEMINIFEROUS TUBULES\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data on the number of cell types in the seminiferous tubules are presented in Table 4.2. The number of spermatogonia, spermatocytes, and spermatids was significantly reduced (P\u0026lt;0.001) in the DEN alone treated group compared to the control group. When compared to the control group, the total number of distinct cell types did not significantly differ in the groups treated with \u003cem\u003eArtemisia\u003c/em\u003e and \u003cem\u003eBergenia\u003c/em\u003e alone. However, compared to the group treated with DEN alone, there was a statistically significant (P\u0026lt;0.001) increase in the quantity of spermatocytes and spermatids in these groups. Cell counts in the group that received both DEN and \u003cem\u003eArtemisia\u003c/em\u003e treatment were comparable to those in the group that received DEN treatment alone, suggesting that there was no discernible difference. In contrast to the control group, the cell counts in the DEN + \u003cem\u003eArtemisia\u003c/em\u003e group were significantly different (P\u0026lt;0.001) (Table 4.2.). In the group treated with \u003cem\u003eBergenia\u003c/em\u003e in combination with DEN, cell numbers were recovered and were not different compared to the control group however, the cell numbers were significantly higher compared with the DEN alone treated group (P\u0026lt;0.001, \u0026lt;0.05 respectively; Table 4.2.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4. PLASMA TESTOSTERONE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTestosterone concentration was significantly reduced (P\u0026lt;0.001) in DEN alone treated group and DEN + \u003cem\u003eArtemisia\u003c/em\u003e treated groups. Statistically significant differences were found between the control group and the treatment groups upon examination of testosterone levels. These differences were seen in the groups that received \u003cem\u003eArtemisia\u003c/em\u003e alone, \u003cem\u003eBergenia\u003c/em\u003e alone, and DEN plus \u003cem\u003eBergenia\u003c/em\u003e. Different thresholds of statistical significance were established: P\u0026lt;0.05 for the DEN + \u003cem\u003eBergenia\u003c/em\u003e group, P\u0026lt;0.01 for the \u003cem\u003eBergenia\u003c/em\u003e alone group, and P\u0026lt;0.001 for the Artemisia alone group. These results show that the treatment groups\u0026apos; testosterone levels were lower than the control group\u0026apos;s whereas the difference was strong enough to be considered statistically significant, demonstrating that the treatments exerted an effect on the testosterone levels. The study found that the DEN and DEN + \u003cem\u003eArtemisia\u003c/em\u003e groups carried much lower testosterone levels than the \u003cem\u003eBergenia\u003c/em\u003e group. The \u003cem\u003eBergenia\u003c/em\u003e group had higher testosterone levels, which shows that it possessed a protective effect. \u0026nbsp;Figure 4.1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5. HISTO-PATHOLOGICAL OBSERVATIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicroscopic study of the testicular sections in different groups showed pathophysiological symptoms in the testis. Testis of the control group animals and animals treated with \u003cem\u003eArtemisia\u003c/em\u003e and\u003cem\u003e\u0026nbsp;Bergenia\u0026nbsp;\u003c/em\u003ealone showed normal morphology including compactly arranged seminiferous tubules and thick epithelium. The interstitial space of the testis was also thin and the lumen was sperm-filled. In the DEN alone treated group, seminiferous tubules were arranged loose and interstitium was large. The tubular lumen was empty and disorganized epithelial cells were visible in the seminiferous tubules. Sloughing and vacuolation in the epithelium were the prominent changes observed in the seminiferous tubules of the DEN-treated group (Figure 4.2). The observations indicated that the \u003cem\u003eArtemisia\u003c/em\u003e co-treatment was less effective while co-treatment with \u003cem\u003eBergenia\u003c/em\u003e was more effective against the toxic effects of DEN treatment in the testis of mice (Figure 4.2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.1: Mean\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn; SEM initial and final body weight and testis weight in different treatment and control groups of mice.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"588\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSr.No\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBody Weight\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTesticular Weight\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInitial weight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFinal weight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRight Testis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLeft Testis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e35\u0026plusmn;0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e43.8\u0026plusmn;1.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.238\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.238\u0026plusmn;0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e34.7\u0026plusmn;1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e29.4\u0026plusmn;0.68***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.17\u0026plusmn;0.003***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.238\u0026plusmn;0.004+++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eArtemisia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e35.6\u0026plusmn;0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e37.4\u0026plusmn;1.63*++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.236\u0026plusmn;0.005+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.238\u0026plusmn;0.004+++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBergenia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e34.3\u0026plusmn;1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e38.4\u0026plusmn;1.63++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.234\u0026plusmn;0.004+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.234\u0026plusmn;0.005+++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDEN + \u003cem\u003eArtemisia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e35.3\u0026plusmn;0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e29.4\u0026plusmn;1.16***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.182.004***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.172\u0026plusmn;0.004***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDEN + \u003cem\u003eBergenia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e35.2\u0026plusmn;0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e34\u0026plusmn;1.00***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.22\u0026plusmn;0.006+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.224\u0026plusmn;0.005+++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues are expressed as Mean \u0026plusmn; SEM\u003c/p\u003e\n\u003cp\u003e*, **, *** represents significant difference at probability value P\u0026lt;0.05, 0.01 and 0.001 as compared to control\u003c/p\u003e\n\u003cp\u003e+, ++, +++ represents significant difference at probability value P\u0026lt;0.05, 0.01 and 0.001 as compared to DEN treated groups\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.2: Mean\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn; SEM area and diameter of seminiferous tubules, interstitium, and numbers of different cells in the seminiferous tubules in different treatment and control groups of mice.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"986\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSr.No\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 507px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTestis Morphometry\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 382px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell Count in Seminiferous Tubules\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e%Area covered by seminiferous tubule\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e%Area of interstitium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003eSeminiferous tubule Diameter (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eEpithelial height (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eSpermatogonia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eSpermatocytes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003eSpermatids\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e81.13\u0026plusmn;1.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e18.87\u0026plusmn;0.199\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e139.9\u0026plusmn;0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e59.53\u0026plusmn;1.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e58.94\u0026plusmn;1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 125px;\"\u003e\n \u003cp\u003e71.08\u0026plusmn;1.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e245.92\u0026plusmn;2.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eDEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e63.88\u0026plusmn;0.56***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e36.11\u0026plusmn;0.56***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e118.75\u0026plusmn;0.59**-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e46.42.58***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e49.4\u0026plusmn;1.18***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e59.2\u0026plusmn;2.30***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e225.84\u0026plusmn;2.14***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eArtemisia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e81.72\u0026plusmn;0.57+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e18.28\u0026plusmn;0.57+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e146.5\u0026plusmn;5.87+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e58.3\u0026plusmn;1.83*+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e57.8\u0026plusmn;1.06+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e69.48\u0026plusmn;1.24+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e243.76\u0026plusmn;3.03+++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eBerginia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e83.95\u0026plusmn;0.93+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e16.04\u0026plusmn;0.93+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e144.55\u0026plusmn;7.51+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e56.53\u0026plusmn;1.59+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e59.64\u0026plusmn;0.74+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e70.32\u0026plusmn;1.41+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e244.72\u0026plusmn;2.75+++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eDEN + Artemisia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e67.79\u0026plusmn;0.60***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e32.20\u0026plusmn;0.60***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e120.32\u0026plusmn;0.41**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e52.46\u0026plusmn;1.71+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e50.68\u0026plusmn;1.29***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e60.58\u0026plusmn;1.92***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e227.8\u0026plusmn;2.26***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eDEN + Berginia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e76.90\u0026plusmn;0.46+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e23.09\u0026plusmn;0.46+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e136.65\u0026plusmn;0.94+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e56.1\u0026plusmn;0.40+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e55.96\u0026plusmn;1.26+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e67.18\u0026plusmn;1.24+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e236.84\u0026plusmn;2.23+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues are expressed as Mean \u0026plusmn; SEM\u003c/p\u003e\n\u003cp\u003e*, **, *** represents significant difference at probability value P\u0026lt;0.05, 0.01 and 0.001 as compared to control.\u003c/p\u003e\n\u003cp\u003e+, ++, +++ represent significant differences at probability value P\u0026lt;0.05, 0.01, and 0.001 as compared to DEN treated group.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eDiethylenitrosamine (DEN), the potent environmental carcinogen has been known to induce damage in many enzymes involved in DNA repair/replication and is specially used to cause hepatocellular carcinoma in experimental animal models\u0026nbsp;\u003csup\u003e[4]\u003c/sup\u003e. Rhodamine dyes, which can produce nitrite-rich water in concentrations as high as 75%, are one of the primary environmental sources of DEN \u003csup\u003e[26]\u003c/sup\u003e. Exposure to treated water could potentially endanger the health of populations. At least 18 animal species, including poultry and cats, have identified DEN as carcinogenic, indicating its potential as a human carcinogen \u003csup\u003e[27]\u003c/sup\u003e. The pervasive carcinogenic potential of DEN underscores the significance of understanding its sources and mechanisms of action for public health protection.\u003c/p\u003e\n\u003cp\u003eThe DEN bioactivation in rat liver involves α-hydroxylation of DEN by CYP2E1 and other P450 isozymes leading to the reactive ethyl diazonium ion \u003csup\u003e[28]\u003c/sup\u003e. It has also been reported that there is a close association between guanine O-alkylation and mutation frequency \u003csup\u003e[29]\u003c/sup\u003e. According to Tong (1982) \u003csup\u003e[30]\u003c/sup\u003e, O6-chloroethyl guanine may crosslink with the opposite cytosine residues, thereby blocking DNA replication. DEN exposure of cells disturbs the balance between antioxidants and reactive oxygen species and results in carcinogenesis \u003csup\u003e[3]\u003c/sup\u003e.\u0026nbsp;In the present study, a significant reduction in body weight was observed in mice that got DEN treatment for eight weeks. Reduction in body weight can be attributed to collective tissue damage in the body by initial toxic lesions in liver cells, and reproductive cells, as well as due to reduction of food intake \u003csup\u003e[31]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePlants of the genus \u003cem\u003eArtemisia\u0026nbsp;\u003c/em\u003ehave revealed cytotoxic effects against cancer cells by inducing cell cycle arrest \u003csup\u003e[32]\u003c/sup\u003e. The anticancerous activity of \u003cem\u003eArtemisia\u0026nbsp;\u003c/em\u003especies has been observed against human cancer cell lines especially A549, HeLa, and MCF7 \u003csup\u003e[33]\u003c/sup\u003e. \u003cem\u003eArtemisia\u0026nbsp;\u003c/em\u003ehas bioactive compounds like alkaloids, terpenoids, flavonoids, and coumarines and possesses potential antioxidant activity against aging, inflammation, and chronic infectious diseases. However, in the present study,\u003cem\u003e\u0026nbsp;Artemisia\u003c/em\u003e did not ameliorate the damaging effects of DEN on body weight in DEN-treated mice. It might be possible the “concentration of extract” of \u003cem\u003eA. vulgaris\u003c/em\u003e that was used during the experiment acted as a pro-oxidant and lost its efficiency against the potent diethylnitrosamine.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBergenia ciliata\u0026nbsp;\u003c/em\u003eis a perennial herb having antibacterial, antioxidant, anti-inflammatory, anti-diabetics, and antiviral activities \u003csup\u003e[34]\u003c/sup\u003e. \u003cem\u003eBergenia\u003c/em\u003e treatment was efficient in maintaining a normal body weight. However, the beneficial effect of \u003cem\u003eBergenia\u003c/em\u003e in terms of body weight might be attributed to its anti-oxidant activity \u003csup\u003e[35]\u003c/sup\u003e. There is substantial therapeutic potential in Bergenia species, as evidenced by numerous studies. \u003cem\u003eBergenia ciliata\u003c/em\u003e demonstrated prophylactic effects against nephrolithiasis in rats, which were presumably mediated by its antioxidant activity \u003csup\u003e[36]\u003c/sup\u003e. Likewise, bergenin, which was isolated from \u003cem\u003eBergenia ligulata\u003c/em\u003e, exhibited potent antioxidant and antilithiatic properties, effectively shielding against oxidative stress and renal dysfunction in hyperoxaluric rodents \u003csup\u003e[37]\u003c/sup\u003e. Our results are in line with those of the previous study that reported the projected effects of \u003cem\u003eBergenia\u003c/em\u003e against tissue/organ/animal body weight loss due to exposure to hazardous chemicals \u003csup\u003e[38]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIt was observed that DEN treatment induces degenerative changes in the testicular tissues of mice. These changes were successfully ameliorated by \u003cem\u003eBergenia\u0026nbsp;\u003c/em\u003ewhile \u003cem\u003eArtemisia\u0026nbsp;\u003c/em\u003eexhibited less or no protective effect against DEN-induced reproductive toxicity the reason may be the same as above. The structural and functional integrity of testicular tissues depends on the circulating androgens and a small decrease in androgen content can result in a reduction in the weight of the testes \u003csup\u003e[39]\u003c/sup\u003e. In the present study, testicular weight was significantly reduced in the DEN-treated group as has previously been reported by \u003csup\u003e[40]\u003c/sup\u003e. The mice treated with \u003cem\u003eBergenia ciliata\u003c/em\u003e extract restored normal testicular weight compared to DEN treated group because it is an excellent antioxidant that has the potential to directly scavenge or prevent the generation of ROS \u003csup\u003e[35]\u003c/sup\u003e. It is relevant to mention that \u003cem\u003eBergenia\u003c/em\u003e protects tissue from tissue weight loss \u003csup\u003e[41]\u003c/sup\u003e.\u0026nbsp;However, the effect of \u003cem\u003eArtemisia\u003c/em\u003e was not pronounced which is contradictory to previous literature \u003csup\u003e[42]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe structural and functional integrity of testicular tissues depends on the circulating androgens and a small decrease in androgen content can result in a reduction in the weight of the testes \u003csup\u003e[39]\u003c/sup\u003e. In the present study, testicular weight was significantly reduced in the DEN-treated group as has previously been reported by \u003csup\u003e[40]\u003c/sup\u003e. The mice treated with \u003cem\u003eBergenia ciliata\u003c/em\u003e extract restored normal testicular weight compared to DEN treated which can be attributed to the antioxidant activity of\u0026nbsp;\u003cem\u003eBergenia\u003c/em\u003e to directly scavenge or prevent the generation of ROS \u003csup\u003e[35]\u003c/sup\u003e and to protect the tissue from tissue weight loss \u003csup\u003e[41]\u003c/sup\u003e. The seminiferous tubules function to produce, maintain, and store sperm. The reduction of the seminiferous tubule width in mice shows that the shriveling of these tubules leads to structural disruption in sperm production in the testis \u003csup\u003e[43]\u003c/sup\u003e. In the present study, the % area covered by seminiferous tubule was significantly reduced in the testis of the DEN alone treated group compared to the control group. The interstitial cells of leydig are responsible for testosterone production. Similarly, the % area covered by the interstitium was significantly higher in DEN treated group than control. The increased % area of interstitium might be due to inflammation. The protection provided by \u003cem\u003eBergenia\u003c/em\u003e ciliata against DEN-induced tubular lesions and reduction in epithelial height as well as diameter might be due to the potent antioxidant potential of the plant \u003csup\u003e[35]\u003c/sup\u003e. However, the results are not in line with Anibogwu (2021)\u003csup\u003e[44]\u003c/sup\u003e who reported the antioxidant effects of \u003cem\u003eArtemisia\u003c/em\u003e in embryonic cells. As the DEN acts by inducing oxidative stress resulting in cell death \u003csup\u003e[40]\u003c/sup\u003e, the \u003cem\u003eArtemisia\u003c/em\u003e however, did not protect the tissue either due to lack of its antioxidant potential or due to certain other mechanisms \u003csup\u003e[44]\u003c/sup\u003e. \u003cem\u003eBergenia\u003c/em\u003e co-treatment resulted in reversing the reduction in the number of cells in the seminiferous tubules but \u003cem\u003eArtemisia\u003c/em\u003e failed to increase the number of cells. This protective effect of \u003cem\u003eBergenia\u003c/em\u003e might be due to its antioxidant potential \u003csup\u003e[35]\u003c/sup\u003e however, the lack of effect of \u003cem\u003eArtemisia\u003c/em\u003e in protecting testicular tissue is contradictory to previous findings \u003csup\u003e[44]\u003c/sup\u003e. Further studies are required to confirm the effects of these plant extracts.\u003c/p\u003e\n\u003cp\u003eIn the target tissues, androgens enter the cell cytoplasm by simple diffusion across the cell membrane. Once inside the cell, the androgens bind and activate the androgen receptors. The androgen-receptor complex attaches to a specific DNA site and stimulates the production of messenger RNA, which, in turn, stimulates the production of the enzymes and proteins necessary to affect androgen action \u003csup\u003e[45]\u003c/sup\u003e. The level of testosterone was increased in all the low-dose DEN-treated groups except for chronic exposure to DEN. \u0026nbsp;High-dose administration of DEN reduced the testosterone level in the blood \u003csup\u003e[40]\u003c/sup\u003e. A significant reduction in testosterone might have caused pathological changes in the testis tissue [46]. Plant extracts as well as other chemical agents cause hormonal imbalance as alkaloids and flavonoids reduce plasma concentrations of LH, estradiol, and FSH [47]. In this study\u0026nbsp;Testosterone concentration was significantly reduced in the DEN alone treated group and DEN + \u003cem\u003eArtemisia\u003c/em\u003e treated groups Diethyle nitrosamine is a potent genotoxin and being an endocrine disrupter might alter the nature of those enzymes that are responsible for androgen production. No significant difference was noted in other treated groups regards to control group.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn conclusion, the results of the present study indicate that DEN can induce reproductive toxicity by affecting the HPG axis and ultimately the seminiferous tissue. These effects are reversed by \u003cem\u003eBergenia\u003c/em\u003e co-treatment but could not be reversed by \u003cem\u003eArtemisia\u003c/em\u003e co-treatments. Hence,\u003cem\u003e\u0026nbsp;Bergenia\u003c/em\u003e is a more potent protective agent than \u003cem\u003eArtemisia\u003c/em\u003e against DEN-induced reproductive toxicity. Further studies are obligatory to endorse the effects of these extracts against other chemical toxicants.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eAuthors have no conflict of interest to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAUTHORS CONTRIBUTION\u003c/h2\u003e \u003cp\u003eS.S., and T.A., were involved in practical work. S.S. and S.J. analyzed the data. S.S. and S.A. drafted the manuscript.\u003c/p\u003e\u003ch2\u003eAvailability of Data and Material\u003c/h2\u003e \u003cp\u003eAdditional information will be provided upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSotty J, Bablon P, Weiss P-H, Soussan P (2024) Diethylnitrosamine Induction of Hepatocarcinogenesis in Mice. Methods Mol Biol 2769:15\u0026ndash;25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://api.semanticscholar.org/CorpusID:267498753\u003c/span\u003e\u003cspan address=\"https://api.semanticscholar.org/CorpusID:267498753\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark D-H, Shin JW, Park S-K, Seo J, Li L, Jang J, Lee M (2009) Diethylnitrosamine (DEN) induces irreversible hepatocellular carcinogenesis through overexpression of G1/S-phase regulatory proteins in rats. 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Reprod Toxicol 21 3:335\u0026ndash;340. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://api.semanticscholar.org/CorpusID:24794243\u003c/span\u003e\u003cspan address=\"https://api.semanticscholar.org/CorpusID:24794243\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"PMAS-Arid agriculture University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diethyle nitrosamine (DEN), A. vulgaris, B. ciliata, Testicular toxicity, Testosterone, Mice","lastPublishedDoi":"10.21203/rs.3.rs-9686581/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9686581/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eDiethylnitrosamine (DEN) or N-Nitrosodiethylamine (NDEA) is used as a carcinogen in experimental animal model systems. The DEN treatment at a necrogenic dose can cause acute toxicity to various cell types in the adenohypophysis of the pituitary gland and alter serum levels of several hormones that can alter reproduction. \u003cem\u003eArtemisia vulgaris\u003c/em\u003e and \u003cem\u003eBergenia ciliata\u003c/em\u003e are known for their antioxidant and protective effects against various chemicals.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThe present study aims to elucidate the anti-carcinogenic activity of \u003cem\u003eArtemisia vulgaris\u003c/em\u003e and \u003cem\u003eBergenia ciliata\u003c/em\u003e against diethylnitrosamine (DEN) induced changes in the reproductive system of male albino mice.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eMice (N\u0026thinsp;=\u0026thinsp;120), maintained at room temperature (25\u0026deg;C) were divided into six groups (each group containing 20 mice) and treated with saline, DEN, \u003cem\u003eArtemisia vulgaris\u003c/em\u003e extract, \u003cem\u003eBergenia ciliata\u003c/em\u003e extract, DEN+\u003cem\u003eArtemisia vulgaris\u003c/em\u003e extract and DEN+\u003cem\u003eBergenia ciliata\u003c/em\u003e extract respectively for eight weeks. At the end of the experiment, the final body weight was measured and mice were dissected. The weight of both testes was measured, the testicular tissue was processed for histology and blood samples were collected for analysis of testosterone. Histological images were analyzed by ImageJ software for measurement of the area covered by seminiferous tubule (%), area of the interstitium (%), seminiferous tubule diameter (\u0026micro;m), and epithelial cell height (\u0026micro;m).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) reduction in body weight was observed in animals treated with DEN and \u003cem\u003eArtemisia\u003c/em\u003e co-treated group while body weight was not affected (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in \u003cem\u003eBergenia\u003c/em\u003e co-treated group. Similarly, a low (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) level of plasma testosterone (0.2\u0026ndash;0.3 ng/ml) was observed in DEN and DEN+\u003cem\u003eArtemisia\u003c/em\u003e extract-treated groups compared to \u003cem\u003eArtemisia\u003c/em\u003e and \u003cem\u003eBergenia\u003c/em\u003e alone, DEN\u0026thinsp;+\u0026thinsp;\u003cem\u003eBergenia\u003c/em\u003e groups. Histological evaluation revealed a significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) reduction in the diameter of seminiferous tubules, epithelial cell height, and cell numbers in DEN treated group compared to the control group. These effects were restored (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the group treated with \u003cem\u003eBergenia ciliata\u003c/em\u003e extract but were not restored (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in the \u003cem\u003eArtemisia vulgaris\u003c/em\u003e extract co-treated group.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe results of the present study revealed that \u003cem\u003eBergenia ciliata\u003c/em\u003e extract is efficient in reversing DEN-induced DEN-induced reproductive toxicity in male mice. Further studies are needed to evaluate the components of \u003cem\u003eBergenia Ciliata\u003c/em\u003e extracts useful in treating reproductive organ toxicity.\u003c/p\u003e","manuscriptTitle":"EFFECT OF METHANOLIC EXTRACTS OF Artemisia vulgaris AND Bergenia ciliata AGAINST DIETHYL-NITROSAMINE (DEN) INDUCED TESTICULAR CHANGES IN MICE","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-14 14:36:42","doi":"10.21203/rs.3.rs-9686581/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4690e9e1-a443-420f-bfbd-81aa99389184","owner":[],"postedDate":"May 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":67978415,"name":"Animal Science"}],"tags":[],"updatedAt":"2026-05-14T14:36:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-14 14:36:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9686581","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9686581","identity":"rs-9686581","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}