An International PeerAn International Peer--Reviewed Scientific JournalReviewed Scientific Journal http://jjbs.hu.edu.jo/ Jordan Journal of Biological Sciences Financed by the Scientific Research and Innovation Support FundFinanced by the Scientific Research and Innovation Support Fund Hashemite Kingdom of Jordan المجلة الأردنية للعلوم الحياتية Jordan Journal of Biological Sciences (JJBS) http://jjbs.hu.edu.jo Jordan Journal of Biological Sciences (JJBS) (ISSN: 1995–6673 (Print); 2307-7166 (Online)): An International Peer- Reviewed Open Access Research Journal financed by the Scientific Research and Innovation Support Fund, Ministry of Higher Education and Scientific Research, Jordan and published quarterly by the Deanship of Scientific Research , The Hashemite University, Jordan. Editor-in-Chief Professor Wedyan, Mohammed A. Environmental Biochemistry, The Hashemite University Assistant Editor Professor Muhannad, Massadeh I. Microbial Biotechnology, The Hashemite University Editorial Board (Arranged alphabetically) Professor Al-Eitan, Laith Biotechnology and Genetic Engineering Jordan University of Science and Technology Professor Al-Khateeb , Wesam M. Plant Genetics and Biotechnology Yarmouk University Professor Al-Ghzawi , Abdul Latief A. Plant biotechnology The Hashemite University Professor Al-Najjar , Tariq Hasan Ahmad. Marine Biology The University of Jordan/ Aqaba Professor Khleifat, Khaled M. Microbiology and Biotechnology Mutah University Professor Odat , Nidal Plant biodiversity Al Balqa Applied University Associate Editorial Board 0BProfessor Al-Hindi, Adnan I. Parasitology The Islamic University of Gaza, Faculty of Health Sciences, Palestine 1BProfessor Krystufek, Boris Conservation Biology Slovenian Museum of Natural History, Slovenia 2BDr Gammoh, Noor Tumor Virology Cancer Research UK Edinburgh Centre, University of Edinburgh, U.K. 3BDr Rabei, Sami H. Plant Ecology and Taxonomy Botany and Microbiology Department, Faculty of Science, Damietta University,Egypt 4BProfessor Kasparek, Max Natural Sciences Editor-in-Chief, Journal Zoology in the Middle East, Germany 5BProfessor Simerly, Calvin R. Reproductive Biology Department of Obstetrics/Gynecology and Reproductive Sciences, University of Pittsburgh, USA Editorial Board Support Team Language Editor Professor Shadi Neimneh Publishing Layout Eng.Mohannad Oqdeh Submission Address Professor Wedyan, Mohammed A. The Hashemite Universit y P.O. Box 330127, Zarqa, 13115, Jordan Phone: +962-5-3903333 ext.4147 E-Mail: jjbs@hu.edu.jo http://jjbs.hu.edu.jo/ mailto:jjbs@hu.edu.jo المجلة الاردنية للعلوم الحياتية Jordan Journal of Biological Sciences (JJBS) http://jjbs.hu.edu.jo International Advisory Board (Arranged alphabetically) Professor Abdelaziz M. Hussein Mansoura University, Egypt Professor Adnan Bashir Al- lahham German Jordanian University, Jordan Professor Ahmed Amri genetic resources ICARDA in Morocco, Morocco Professor Amir Menwer Al-Hroob Al-Hussein Bin Talal University, Jordan Professor Elif Demirkan Bursa Uludag University Turkey, Turkey Professor Erhan Nurettin ÜNLÜ Turkey Dicle University,Turkey Professor Hassan Mohammed M. Abd El-Rahman Awad National Research Centre, Egypt Professor Khalid M. Al-Batayneh Yarmouk University, Jordan Professor Laith Abd Jalil Jawad School of Environmental and Animal Sciences, Unitec Institute of Technology Auckland ,New Zealand Professor Maroof A. Khalaf Jordan University/ Aqaba , Jordan Professor Mohammed H. Abu-Dieyeh Biological and Environmental Sciences, Qatar University, Qatar Professor Nour Shafik Emam El-Gendy Egyptian Petroleum Research Institute, Egypt Professor Omar F. Khabour Jordan University of Science and Technology, Jordan Professor Saleem Hmood Aladaileh Al-Hussein Bin Talal University, Jordan Professor Walid Al Zyoud German Jordanian University, Jordan Professor Abhik Gupta School of Environmental Sciences, Assam University, India Professor Ahmed Deaf Allah Telfah Leibniz-Institut für Analytische Wissenschaften - ,Germany Dr. Amalia A Tsiami University of West London, London Professor David Modry Masaryk University, Science Department, Czech Professor Emad Hussein Malkawi Yarmouk University, Jordan Professor Gottfried Hartmut Richard Jetschke Friedrich-Schiller-University of Jena, Germany Professor Ihsan Ali Mahasneh Al al-Bayt University, Jordan Professor Khalid Majid Hameed Dept. of Biological Sciences, Duke University, USA Dr Maizirwan Bin Muhammad Mel International Islamic University Malaysia, Malaysia Professor Mohamed Emara Chartered Management Institute, UK Professor Nabil Joseph Awadalla Girgis King Khalid University, Saudi Arabia Professor Olga Anne Marine Technology and Natural Sciences of Klaipėda University, Lithuania Dr Roy Hendroko Setyobudi University of Muhammadiyah, Indonesia Dr. Salem M Akel St, Jude’s Children’s Research Hospital, USA Professor Yacob Hassan Yacob Al al-Bayt University, Jordan Instructions to Authors Scopes Study areas include cell biology, genomics, microbiology, immunology, molecular biology, biochemistry, embryology, immunogenetics, cell and tissue culture, molecular ecology, genetic engineering and biological engineering, bioremediation and biodegradation, bioinformatics, biotechnology regulations, gene therapy, organismal biology, microbial and environmental biotechnology, marine sciences. 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Reference to a chapter in an edited book: Mettam GR and Adams LB. 2010. How to prepare an electronic version of your article. In: Jones BS and Smith RZ (Eds.), Introduction to the Electronic Age. Kluwer Academic Publishers, Netherlands, pp. 281–304. Conferences and Meetings: Embabi NS. 1990. Environmental aspects of distribution of mangrove in the United Arab Emirates. Proceedings of the First ASWAS Conference. University of the United Arab Emirates. Al-Ain, United Arab Emirates. Theses and Dissertations: El-Labadi SN. 2002. Intestinal digenetic trematodes of some marine fishes from the Gulf of Aqaba. MSc dissertation, The Hashemite University, Zarqa, Jordan. Nomenclature and Units Internationally accepted rules and the international system of units (SI) should be used. If other units are mentioned, please give their equivalent in SI. 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Galley Proofs The Editorial Office will send proofs of the manuscript to the corresponding author as an e-mail attachment for final proof reading and it will be the responsibility of the corresponding author to return the galley proof materials appropriately corrected within the stipulated time. Authors will be asked to check any typographical or minor clerical errors in the manuscript at this stage. No other major alteration in the manuscript is allowed. After publication authors can freely access the full text of the article as well as can download and print the PDF file. Publication Charges There are no page charges for publication in Jordan Journal of Biological Sciences, except for color illustrations, Reprints Ten (10) reprints are provided to corresponding author free of charge within two weeks after the printed journal date. 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Indexing JJBS is indexed and abstracted by: CABI DOAJ ( Directory of Open Access Journals) EBSCO Google Scholar CAS ( Chemical Abstract Service) Journal Seek ETH- Citations HINARI Open J-Gat Index Copernicus SCImago NDL Japanese Periodicals Index Clarivate Analytics ( Zoological Abstract) SCIRUS Scopus OAJSE AGORA (United Nation's FAO database) ISC (Islamic World Science Citation Center) SHERPA/RoMEO (UK) Directory of Research Journal Indexing (DRJI) Ulrich's JJBS Volume 16, Number 3, September 2023 ISSN 1995-6673 Jordan Journal of Biological Sciences CONTENTS Original Articles 379 – 387 1 Extraction, Characterization, Amino Acid Profile of Halal Gelatin from Kampong and Broiler Chicken Feet Skin Noor Harini, Manar Fayiz Mousa Atoum, Swastika Tri Aji Wulandari, Vritta Amroini Wahyudi , Asad Jan, and Irum Iqrar 389 – 401 2 Molecular Simulations of Moringa oleifera Phytochemicals as Potential Antagonists of the Proinflammatory NF-κB p50 Transcription Factor Kathleen Cole C. Tung, Romeric F. Pobre, Glenn G. Oyong 403 – 412 3 Diversity and Seasonal Variation of Fish Assemblages of Dingapota Haor an Eutrophic Wetland of Northeastern Bangladesh Md. Nahiduzzaman, Ehsanul Karim, Md. Nazmul Hossen, Nazia Naheen Nisheeth and Yahia Mahmud 413 – 424 4 Moderately Thermophilic Bacteria from Jordanian Hot Springs as Possible Sources of Thermostable Enzymes and Leukemia Cytotoxic Agents Maher Obeidat , Belal Al-Shomali 425 – 430 5 First Record of the Scorpion Vachoniolus globimanus (Scorpiones: Buthidae) from Jordan Bassam Abu Afifeh, Mohammad Al-Saraireh and Zuhair S. Amr 431 – 437 6 GC-MS Analysis of Various Crude Extracts from the Leaves, Flowers, and Stems of Datura metel Linnaeus 1753 and the Potential Activity as Anesthetic Agents on Fish Rindhira Humairani , Nanda Rizki Purnama, Herpandi Herpandi, Mochamad Syaifudin, Ilham Zulfahmi, Yusrizal Akmal, Muliari Muliari, Agung Setia Batubara 439 – 443 7 The Effect of 17β-Estradiol and Genistein on the Prostate Gland and Testes of Aged Rats Falah Shidaifat , Mohammed Khalifeh and Yousef Yasin 445 – 454 8 Immunomodulatory Properties of Citrus limon Extracts on BALB/c Mouse Lymphoid and Myeloid Lineage Cells Wira Eka Putra, Ken Retno Puspaningrum, Arief Hidayatullah, Muhaimin Rifa’i 455 – 465 9 Heavy Metals Effect on the Rat Uterus and Effectiveness of Vitamin E Treatment Sikora K, Lyndin M, Sikora V, Hyriavenko N, Piddubnyi A, Lyndina Y, Awuah WA, Abdul-Rahman T, Korobchanska A, Alexiou A, Romaniuk A 467 – 475 10 Heavy metal contamination and potential health risk assessment associated with selected farmed fish in Rajshahi, Bangladesh Jibon Kumar Ghosh, Md. Shahidul Islam, Md. Tariqul Islam, Md. Mahedul Islam Murad and Md. Mahabubur Rahman 477 – 502 11 Comparative Analysis of Cichorieae Tribe (Asteraceae) Chloroplast Genomes: Insight to Structure, Repetitive DNA, and Phylogeny. Rubar Hussein M. Salih 503 – 511 12 Biochemical Profile of Five Species of Cultured Marine MicroalgaeP 1 Teja. Gurugubelli, Yedukondala Rao. Poturaju and Rukmini Sirisha. Imandi 513 – 518 13 A Histological Examination of the Sublethal Effects of Methyl Parathion on the Liver, Gills and Gonads of Alburnus tarichi (Güldenstädt, 1814) Ertuğrul KANKAYA and Güler ÜNAL 519 – 527 14 Formulated Hand Sanitizer Utilizing Passion Fruit (Passiflora edulis) Leaf Extract as an Active Ingredient Cabral, Daisy Anne M., Hernandez, Marianne D., Martinez, Leslie Ann B.and Sangalang, Reygan H. 529 – 536 15 Telmisartan Enhances the Accumulation of Doxorubicin as a Combination Therapy for the Management of Triple Negative Breast Cancer Wala’a Al.Safadi, Belal Omar Al-Najjar, MoathAlqaraleh, Anas Ibrahim Abed, Walhan Alshaer, Dana Alqudah, Fadwa Daoud,Razan Mohammad Obeidat, Obada Abdulmalek Sibai, Zainab Zaki Zakaraya 537 – 564 16 Streptomyces–Alginate Beads Formula Promote Maize Plant Growth and Modify the Rhizosphere Microbiome Okto Asriatno, Abdjad Asih Nawangsih, Rika Indri Astuti, Aris Tri Wahyudi JJBS Volume 16, Number 3,September 2023 ISSN 1995-6673 Pages 455 – 465 https://doi.org/10.54319/jjbs/160309 Jordan Journal of Biological Sciences Heavy Metals Effect on the Rat Uterus and Effectiveness of Vitamin E Treatment Sikora K1,*, Lyndin M1,2, Sikora V3,1, Hyriavenko N1, Piddubnyi A1,4,5, Lyndina Y1, Awuah WA1, Abdul-Rahman T1, Korobchanska A6, Alexiou A8,9, Romaniuk A1. 1 Sumy State University, Sumy, Ukraine; 2 University of Duisburg-Essen, Essen, Germany; 3 University of Foggia, Foggia, Italy; 4 Umeå University, Umeå, Sweden; 5 Ukrainian-Swedish research center SUMEYA, Sumy, Ukraine; 6 Kharkiv National Medical University, Kharkiv, Ukraine; 7 Novel Global Community Educational Foundation, Hebersham, Australia; 8 AFNP Med Austria, Wien, Austria Received: November 29, 2022; Revised: January 26, 2023; Accepted: February 7, 2023 Author Contributions: Conceptualization, K.S. and A.R.; methodology, K.S., M.L., A.W., T.A.R, A.K. and N.H.; software, K.S., V.S. and A.P.; formal analysis, K.S., M.L., A.A. and Y.L.; investigation, K.S., M.L., V.S., A.W., T.A.R, N.H., and Y.L.; resources, K.S., V.S., M.L. and A.A..; data curation, K.S., M.L., V.S., A.P., A.A. and A.R.; writing— original draft preparation, K.S., M.L., V.S., N.H., Y.L., A.W., T.A.R, A.K. and A.P.; writing—review and editing, K.S., M.L., V.S., A.A. and A.R.; visualization, K.S., M.L., V.S. and A.P.; supervision, A.R.; project administration, K.S., M.L., V.S., A.A. and A.R.. All authors have read and agreed to the published version of the manuscript. Acknowledgments: This research has been supported by the Ministry of Education and Science of Ukraine [Grant № 0121U100472 and Grant № 123U100111] and research theme of the Department of Pathological anatomy of Sumy State University [№ 0119U100887]. Institutional Review Board Statement: The study was approved by the Bioethics Committee of the Medical Institute of Sumy State University (protocol No. 2/10 from 10.10.2019). Compliance with Ethical Standards Disclosure of p otential conflicts of i nterest: The authors report no conflict of interest. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Research involving Human Participants and/or Animals: All applicable international, national, and/or institutional ethical guidelines for the care and use of animals were followed. Data availability: All data generated and analyzed during this study are included in this published article and its supplementary information files. Abstract Environmental pollution by heavy metals (HMs) is an increasingly critical problem that is posing a growing threat to reproductive health. Consequently, the aim of the current research was to study changes in rat uterus under 90 days of HMs exposure and estimate the efficacy and benefits of vitamin E treatment. Female rats were randomly divided into three groups: untreated animals (control group); animals orally treated with the HMs mixture (HM group); and animals treated simultaneously with HMs and vitamin E (HM+E group). The toxic effects of the HMs (comprising Zn, Cu, Mn, Fe, Pb, and Cr) on the uterus of rats were investigated by histological, morphometrical, spectrophotometrical, and statistical methods. Long-term HMs exposure triggered pathological (degenerative, inflammation, and atrophic) changes in the rat uterus together with a significant reduction of the uterine-wall thickness (37.99%, p<0.0001) compared to the control. In contrast, there was a lower intensity of morphological lesions and wall thickness decrease (26.03%, p<0.0001) in the uterus, in rats that underwent treatment with vitamin E. A substantial bioaccumulation of zinc, copper, manganese, iron, lead, and chromium general levels in the rat uterus was demonstrated in both the HM group (74.46%, p<0.0001) and the HM+E group (49.81%, p<0.0001), as compared to the control group. The lowest accumulative potential belonged to Zn and the highest to Pb. The results obtained showed a significant decline in the weight of animals treated by HMs in both HM (18,21%, p<0.01) and HM+E (13,09%; p<0.05) groups compared to the control. Our findings have demonstrated that treatment with vitamin E in HM-induced intoxication has a significant restrain of HMs accumulation (up to 16.46%, p<0.0001) together with morphometric variations (less on 16.17%, p<0.01). In summary, long-term exposure to the HMs mixture had a pernicious toxic effect on the morphology and chemical content of the uterus of rats (strong negative correlations). Treatment with vitamin E significantly reversed the HMs impact on the uterus but did not demonstrate absolute protection. * Corresponding author. e-mail: k.sikora@med.sumdu.edu.ua. https://doi.org/10.54319/jjbs/160309 © 2023 Jordan Journal of Biological Sciences. All rights reserved - Volume 16, Number 3 456 Keywords: uterus; heavy metals; reproductive health; vitamin E; antioxidant; detox treatment 1. Introduction In recent years, significant insights have been gained in understanding the roles of the uterus in menstruation, fertility, and pregnancy together with orchestrating the development, differentiation, and maturation of the reproductive system. However, in the context of the deterioration of reproductive health and infertility increase (up to 21.9%), the study of uterus pathology became relevant (Cedars et al., 2017; Ho et al., 2017; Nik Hazlina et al., 2022). Indeed, certain conditions and diseases of the uterus can cause reproductive-health disorders and dysfunction of the synergistic action of the procreative system, disrupting the reproduction and survival of species (Peters et al., 2016; Cedars et al., 2017; Hanson et al., 2017). Any disturbance of estrogenic/anti-androgenic endocrine activity and enzyme mechanisms that can act by mimicking or inhibiting the actions of endogenous hormones may be accompanied by uterus lesions (e.g., benign and malignant tumors, congenital uterine malformation, infertility, ectopic gestation, pregnancies abort and poor pregnancy outcomes). Moreover, such damage of the uterus can be manifested in subsequent generations (Newbold et al., 2006; Gore et al., 2015; Rosenfeld, 2015; Katz et al., 2016; Hanson et al., 2017; Ho et al., 2017). In addition, uterus macroscopic and microscopic lesions (atrophic and hyperplastic) can be caused by the development of pathological processes in this organ (e.g., endometritis, endometriosis, reproductive tract infection (about 33% in females), microflora imbalance, and benign and malignant tumors) or in other organs (e.g., diabetes, obesity, cardiovascular disease, oxidative stress, chronic stress, reproductive tract obstruction, and neurological disorders) (Gore et al., 2015; Peters et al., 2016; Katz et al., 2016; Cedars et al., 2017; Chen et al., 2017; Hanson et al., 2017; Lin et al., 2018; Vannuccini and Petraglia, 2019). It is important to note that uterine lesions can also be provoked by factors with exogenous origins, such as viruses, bacteria, parasites, ionizing/non-ionizing radiation, and pollutants (Newbold et al., 2006; Gore et al., 2015; Cedars et al., 2017; Chen et al., 2017; Hanson et al., 2017; Ho et al., 2017; Lytvynenko et al., 2017; Lin et al., 2018; Nwosu et al., 2018). It is known that long-term exposure of the organism to exogenous pollutants can contribute to epigenetic modifications that are reflected in subsequent generations (i.e., transgenerational epigenetic inheritance). The effects of various chemicals (e.g.., heavy metals (HMs), bisphenol A, genistein, phytoestrogens, diethylstilbestrol, phthalates, and polyaromatic hydrocarbons) can simulate the impact of sex hormones and morphogens (Romaniuk et al., 2015; Katz et al., 2016; Ho et al., 2017; Mohammad Hosseini et al., 2019). The increased morbidity risk due to xenobiotic contamination of the environment has encouraged the study of their effects. Among the most common pollutants that have a detrimental effect on organisms are HMs (Romaniuk et al., 2015; Hamid et al., 2016; Romaniuk et al., 2017; Mohammad Hosseini et al., 2019). However, HMs are not always toxic — most of them are essential trace elements. They are involved in numerous enzymatic, hormonal, redox, and other processes at all developmental stages. However, exceeding the threshold level in the body results in their accumulation in tissues, and they can acquire toxic properties (Singh et al., 2011; Hamid et al., 2016; Nwosu et al., 2018). In addition, some metals are always toxic (Pb, Cd, Cr, Ti, Si, Rb, Sr, Al, As, and Sn). The HMs effect depends on their properties, concentration, type, density, duration of exposure, molecular stability, partition coefficient, polarity, the interaction between metals, distribution, and transport into the ecosystem (Singh et al., 2011; Jaishankar et al., 2014; Nakade et al., 2015; Hamid et al., 2016; Nwosu et al., 2018; Mohammad Hosseini et al., 2019). It is important to note that the long-term effects of various HMs are reflected in the abnormal variability of biochemical, functional (inhibition of menstruation, decrease in the frequency of implanted ova and of pregnancies, intrauterine growth restriction, preterm delivery, and spontaneous abortions), morphological (histopathological changes in the endometrium, myometrium and perimetrium; inflammation; reduction in the uterine gland, and decrease in the height of columnar cells, etc.), and molecular genetic (degeneration of hormones receptors and decrease of their sensitivity, oxidative stress, altering enzymes, growth factors, proliferation activities, tumor suppressor genes, cytokines, lymphokines, transport proteins and proteases, etc.) parameters of the uterus (Jaishankar et al., 2014; Nakade et al., 2015; Hamid et al., 2016; Katz et al., 2016; Hanson et al., 2017; Ho et al., 2017; Mohammad Hosseini et al., 2019). However, some recent data revealed discrepancies regarding these changes due to the effect of the most common HMs and their accumulation (Nakade et al., 2015; Mohammad Hosseini et al., 2019; Lee et al., 2021). On the one hand, this might have been due to the one or several effects of HMs. On the other hand, these changes might have depended on the variability of the xenobiotic concentrations (Singh et al., 2011; Jaishankar et al., 2014; Hamid et al., 2016; Su et al., 2017; Mohammad Hosseini et al., 2019; Lee et al., 2021). In addition, most previous research has considered pollutants and their concentrations in specific geographic locations. HMs accumulation in the organism differs globally depending on the pollution source and the ways of environmental spread (Singh et al., 2011; Jaishankar et al., 2014; Romaniuk et al., 2015; Nakade et al., 2015; Hamid et al., 2016; Romaniuk et al., 2017; Su et al., 2017; Lee et al., 2021). Consequently, the HMs effects on the body are extremely unpredictable and may negatively affect reproductive health (such as breast, endometrial, fallopian tubes or ovarian cancers, endometriosis, endometritis, menstrual disorders, infertility and spontaneous abortions, as well as pre-term deliveries, stillbirths) (Jaishankar et al., 2014; Peters et al., 2016; Hamid et al., 2016; Cedars et al., 2017; Doncova et al., 2019; Dutta S et al., 2022). Nevertheless, there have been increasing numbers of reports of the successful use of various (natural or artificial compounds) supplementation, which can withstand the adverse impact of HMs. These agents have detoxifying and antioxidant properties and can reduce the intensity of xenobiotics' effects as prophylactics and for the treatment of HMs-related disorders. Most of these protective substances have a direct antagonistic relationship with © 2023 Jordan Journal of Biological Sciences. All rights reserved - Volume 16, Number 3 457 HMs and have high efficiency (Al-Attar, 2011; Jaishankar et al., 2014; Romaniuk et al., 2018; Sahiti et al., 2020). However, multiple mechanisms of action of each trace element (especially in combination) can complicate the search for universal natural compounds that will neutralize the accumulation of HMs in body tissues and/or completely block their effect. One of the most discovered naturally occurring supplementation is vitamin E (α-tocopherol) which consists of tocopherols and tocotrienols. This effective lipid-soluble non-enzymatic antioxidant can reduce radical-induced peroxidation in biological membranes and blood, stimulate the activation of antioxidant enzymes, suppress inflammation, accelerate structural recovery, protect cellular membranes and reduce the intensity of oxidative stress caused by HMs-induced toxicity. This enables free radicals to acquire a hydrogen atom from antioxidant molecules, effectively countering lipid peroxidation and safeguarding unsaturated membrane lipids due to its oxygen-scavenging capability. (Al-Attar, 2011; Mohd Mutalip et al., 2018; Sahiti et al., 2020). Moreover, various studies have shown that adequate intake of vitamin E solves reproductive health problems (an essential dietary factor required to maintain normal reproduction), such as enhancing term delivery, sustaining the endometrial membrane, preventing breast cancer growth, decreasing the level of fetal death and spontaneous abortion, etc. (Al-Attar, 2011; Mohd Mutalip et al., 2018; Sahiti et al., 2020). Based on these, vitamin E is often used in researches that describe its effectiveness and protective effects from oxidative stress specifically caused by the impact of various HMs (Al-Attar, 2011; Romaniuk et al., 2018; Sahiti et al., 2020). However, till today, there is no clear information regarding the beneficial effect of vitamin E on the uterus induced by HMs exposure. Summarizing all the above, the aim of our current research was to study the changes in rats' uterus under 90 days of HMs exposure and estimate the efficacy and benefits of vitamin E treatment. 2. Materials and Methods 2.1. Animals For this study, we used 12-week-old healthy Wistar female rats with an average weight of 221.7±17.1 g, which were purchased from the Animal Experimental Unit of the Medical Institute, Sumy, Ukraine. The rats were selected after physical and behavioral examinations (body weight and health state, posture, and response to handling). The animals were acclimated for 7 days before any experimental procedures. All animals were housed in same-sex sub-groups (4 animals in 1 cage) in polypropylene cages with individual ventilation and were maintained under environmentally controlled laboratory conditions of temperature 22°C±1°C, relative humidity 55±5%, and 12 hours light/dark cycle. During the experiment, the animals had ad libitum access to standard pellets and water. Cage cleaning was performed daily. Individual animal bodyweights were recorded at weekly intervals. All necessary procedures were adopted to keep the rodents free from stress. Nulliparous and non-pregnant female rats were used in the study. The estrous-cycle monitoring was performed by daily vaginal smears. These were collected every morning at 9.00 and were analyzed by light microscopy (Sikora et al., 2021). The results before and during experiments were presented according to four phases (proestrus, estrus, metestrus, and diestrus). However, we used data only from the estrus phase to avoid the cyclic hormonal changes in female rats that could be associated with the estrous cycle and confound the results. 2.2. Experimental design The female rodents were randomly assigned to three groups (eight rats per group). Group I (Control) comprised normal (untreated) rats that received ordinary food and drinking water. Group II (HM) comprised rodents that were orally treated with HMs substances for 90 days. Group III (HM+E) comprised animals that received water with HMs and vitamin E within 90 days. The experimental animals were euthanized by CO2 inhalation followed by cervical dislocation and their uteruses were immediately exposed by low abdominal midline incision. The uteruses were then collected and trimmed of fascia and fat. From each rat, the uterine wall of 1.0 cm in length was excised from each uterine horn (proximal part) in the direction from the partial caudal fusion to the ovaries. One random uterine horn was assigned to atomic absorption spectrometry and the other horn was fixed in formaldehyde for later use. A total of 48 uterine horns from 24 rats were assigned to the investigation. 2.3. Experimental substances The experimental model comprised six of the most common (dangerous and potentially dangerous) HMs (Jaishankar et al., 2014; Nakade et al., 2015; Romaniuk et al., 2017; Su et al., 2017; Romaniuk et al., 2018; Lee et al., 2021) at the following concentrations: zinc (ZnSO4×7H2O) – 5 mg/l, copper (CuSO4×5H2O) – 1 mg/l, iron (FeSO4) – 10 mg/l, manganese (MnSO4×5H2O) – 0.1 mg/l, lead (Pb(NO3)2) – 0.1 mg/l, and chromium (K2Cr2O7) – 0.1 mg/l. The concentrations of mentioned above HMs were comparable to those found in the environment according to the results of the epidemiological examination of the environment of the Northern regions of Ukraine and in accordance with preliminary reports (Romaniuk et al., 2015; Romaniuk et al., 2017). The list of chemical elements and their concentrations were confirmed and approved by the Bioethics Committee of the Medical Institute of Sumy State University (No. 2/10 from 10.10.2019). The HMs mixture was dissolved in ordinary water and prepared each three days. Contaminated water was supplied in a drinking bottle in ad libitum access for oral administration annually within 90 days. As antagonist supplementation, we used alpha- tocopherol (vitamin E) due to its antioxidant properties at an average daily prophylactic dose (9.1 mg/kg to rats' bodyweight considering species' characteristics). Conversion of human doses to rat doses was as following: Animal equivalent dose (mg/kg) = Human dose (mg/kg) × Km ratio (Nair and Jacob, 2016). Based on this, considered the coefficient of species characteristics of rats (6.0) and humans (37.0) with average human body weight (70 kg), the dose for rats was followed: 37,0/6,0 = 6,2; 1,47 mg/kg x 6,2= 9,1 mg/kg. The average weight of animals was 221.7 ± 17.1 g. Therefore, animals received vitamin E at an average dose of 2.02 mg per rat bodyweight. Animals © 2023 Jordan Journal of Biological Sciences. All rights reserved - Volume 16, Number 3 458 were administered vitamin E via the oral gavage technique (daily at 10.00 am) for 90 days. Selected antioxidant was estimated based on the literature and manufacturer's recommendations (Al-Attar, 2011; Nair and Jacob, 2016; Romaniuk et al., 2018; Sahiti et al., 2020). 2.4. Tissue processing, histology, and morphometric scoring of the uterus The fresh rat uterine horns were fixed in 10% neutral buffered formaldehyde for 24 hours, dehydrated in ethanol (70–96%), and embedded in paraffin wax blocks. Formalin-fixed paraffin-embedded tissue blocks were sectioned using a rotational microtome Shandon Finnesse 325 (Thermo Scientific, USA). Transverse sections of uterine horns with a thickness of 5 μm were placed on SuperFrost Plus™ Adhesion slides (Thermo Scientific, USA) and dried overnight. The next day, the samples were submerged in xylene (dewaxing – 2 times per 5 minutes each), descending grades of ethanol (rehydration – 100% (1 time per 5 minutes), 95% (1 time per 5 minutes), 70% (1 time per 5 minutes)) and washed in running tap water (2 times per 5 minutes each). Immediately after, samples were immersed in hematoxylin solution for 4 min and followed by immersion in eosin solution for 2 min. The sample were washed in running tap water (2 times per 10 minutes each) after incubation of both hematoxylin and eosin solutions. Finally, sections were dipped in 96% (2 times per 5 minutes) and 100% (1 time per 5 minutes) ethanol, cleared up with xylene (1 time per 5 minutes), and mounted on Histomount Mounting Solution (Thermo Scientific, USA). Two independent pathologists additionally evaluated the histopathological examination. In case, two pathologists did not reach a consensus regarding the results, we sought the help of a third pathologist. The microscopy and morphometric scoring of the rats' uteruses were performed with the Zeiss Axio Primo Star microscope, Zeiss AxioCam ERс 5s digital camera, and ZEN 2 (blue edition) software package (Germany). 2.5. Atomic absorption spectrometry of uterine tissues The atomic absorption spectrometry of uterine tissues was performed according to the following protocol. Tissue samples were scaled, weighed on an analytical balance, dried (at 105°C), and burnt in porcelain crucibles at 450°C (48 h). Ash was dissolved into hydrochloric and nitric acids at 50°C overnight. After dilution with distilled water, the samples were measured. The sample solution was evaporated with the flame atomizer. Thereafter, the electrothermal atomic absorption spectrophotometer C- 115M1 (Ukraine) with the analytic software package AAS SPEKTR (Ukraine) was used to determine the number of chemical elements according to their wavelength as follows: zinc (213.9 nm), copper (324.7 nm), iron (248.3 nm), manganese (279.4 nm), lead (283.3 nm), and chromium (357.9 nm). 2.6. Statistical analysis All results are expressed as the mean ± standard deviation (M ± SD). Distribution type was estimated with the Shapiro–Wilk test. The differences between groups for normally distributed datasets were determined by the independent student's t-test. The one-way analysis of variance (ANOVA) followed by Bonferroni's post-hoc comparisons test was performed to compare variables among groups. Analysis of the strength and direction of the relationships between two variables was performed using the Pearson's (r) correlation coefficient. Differences in values were considered significant at p<0.05. Data analysis and graphs were prepared with GraphPad Prism® 6.0. 2.7. Ethics approval All animals handling and experimental procedures fully adhere to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines 2.0 (Percie et al., 2020). The experiment has been conducted in the European Community Guide for the Care and Use of Laboratory Animals guidelines, ethical and responsible manner, and is in full compliance with all relevant codes of experimentation (institutional and national) and legislation. This study was approved by the Bioethics Committee of the Medical Institute of Sumy State University (No. 2/10 from 10.10.2019). 3. Results 3.1. Liveweight, histopathologic and morphometric changes in rat uterus caused by HMs HMs administration induced body weight loss in both experimental groups. Indeed, the liveweight proportion of female rats was less in HM group (HMs exposure only) (decrease of 18,21%; p < 0.01) and in HM+E group (HMs exposure with vitamin E treatment) (decrease of 13,09%; p < 0.05) than in the Control group. There was no significant difference between HM group and HM+E group. A difference in body weight was first noticed in the third week of the experiment and it increased in the following weeks. Other visual changes were not detected. Long-term HMs exposure (HM group) contributed to pathological changes in the initial part of the uterine horn and reduction of the organ's wall thickness (see Figure 1 and Figure 2). However, these changes were found in both the endometrium and the myometrium. Detailed morphological analysis indicated a nonspecific versatility of these changes: dystrophy of the prismatic (vacuolar degeneration) and exocrine cells (cystic transformation of goblet cells) of the mucous membrane and myometrial myocytes. Atrophic changes in the epithelium were also observed — that are, reduced and uneven height of the superficial (cylindrical epithelium changes to cubic) and glandular epithelium due to a reduction of cytoplasm volume; reduction of endometrial gland number, size, and lumen; and cystic enlargement of single glands. The uterine mucosa had a decreased number of folds and a significantly increased intrauterine lumen. Focal inflammatory infiltration was found mainly in the endometrium. However, the myometrium was also locally involved in the inflammatory process. This was accompanied by connective tissue disorganization, microcirculatory disorders, and slight edema along the entire wall of the uterine horn. © 2023 Jordan Journal of Biological Sciences. All rights reserved - Volume 16, Number 3 459 Figure 1. The HMs (Zn, Cu, Mn, Fe, Pb, and Cr) effect on the histopathologic changes in rats uterine horns: control group (A), HM group (B), and HM+E group (C). Staining with hematoxylin and eosin. Magnification: ×40 and ×200. Scale bar – 50 µm. Figure 2. Variability of rat uterus wall thickness under HMs exposure in HM and HM+E groups, compared to the control. Data are expressed as Mean ± SD (Bars – T style with above direction). Values were analyzed by One-way ANOVA followed by Bonferroni's post- hoc comparisons test (n=24): *p < 0.05; **p < 0.01; ***p < 0.001. Additionally, compared to the control group, the morphometric analysis revealed a significant reduction of the uterine-wall thickness (37.99%, p<0.0001) under HMs long-term exposure. Among all of the uterine layers, the endometrium had the highest reduction of the thickness (40.28%, p<0.0001), followed by the myometrium (35.28%, p<0.0001), and the perimetrium (2.88%, p>0.05). In contrast, we detected moderate degenerative and atrophic changes in the rat uteruses of the HM+E group. On the one hand, the intensity of pathological transformations and morphometric variations (less on 16.17%, p<0.01) of the uterus wall were lower than those in the HM group. On the other hand, the morphometric scoring showed a reduction of rat uterine thickness (26.03%, p<0.0001) in the HM+E group due to a decrease of the endometrium and myometrium sizes by 27.88% and 23.56% (p<0.0001), respectively, compared to the control group. The difference in perimetrium thickness was not statistically significant (1.95%, p>0.05). 3.2. Imbalance of chemical contents in rat uterus tissues caused by HMs The detection limits, distribution, and variability of the HMs concentration levels in rat uteruses are shown in Figure 3 and Table 1. The HM and HM+E groups had a wide range of variations of HMs accumulation in uterus tissues. According to atomic absorption spectrometry, the mean concentration of each element (Zn, Cu, Fe, Mn, Pb, and Cr) differed significantly (74.46%, p<0.0001) from the control values, even in the group with treatment by vitamin E (49.81%, p<0.0001). The highest concentration was estimated for Fe and the lowest for Pb. However, we detected a tendency for a general increase of HMs in rat uterus tissues as follows (listed in descending order): Pb (88.11%, p<0.0001) > Fe (86.26%, p<0.0001) > Cr (73.09%, p<0.0001) > Mn (63.6%, p<0.0001) > Cu (61.8%, p<0.0001) > Zn (49.34%, p<0.0001) for HM group vs Pb (62.24%, p<0.0001) > Fe (58.81%, p<0.0001) > Cr (55.58%, p<0.0001) > Cu (46.17%, p<0.0001) > Mn (44.77%, p<0.0001) > Zn (29.4%, p<0.0001) for the HM+E group relative to the control group. Therefore, the HMs bioaccumulation in the HM+E group was lower than that in the HM group (16.46%, p<0.0001). © 2023 Jordan Journal of Biological Sciences. All rights reserved - Volume 16, Number 3 460 Figure 3. The imbalance of HMs concentration in rat uterine tissues in HM and HM+E groups. The Zn, Cu, Mn, Fe, Pb, and Cr concentrations were significantly higher in the HM group than in the control and HM+E group. The HMs concentration in the HM+E group was significantly higher than in the control group. Data are expressed as Mean ± SD (Bars – T style with above direction). Values were analyzed by One-way ANOVA followed by Bonferroni's post-hoc comparisons test (n=24): *p < 0.05; **p < 0.01; ***p < 0.001. Table 1. Variability of HMs concentration (μg/g) in rats uterine tissues. HM group HM+E group Control group Pb 0.269 ± 0.009***1/***2 0.232 ± 0.009***1 0.143 ± 0.006 Fe 163.18 ± 12.650***1/***2 139.13 ± 10.37***1 87.61 ± 4.38 Cr 1.582 ± 0.037***1/***2 1.422 ± 0.034***1 0.914 ± 0.01 Mn 3.91 ± 0.23***1/***2 3.46 ± 0.15***1 2.39 ± 0.15 Cu 7.78 ± 0.35***1/***2 7.06 ± 0.21***1 4.83 ± 0.15 Zn 56.69 ± 3.8***1/***2 49.74 ± 4.01***1 37.96 ± 2.45 Total 233.41 ± 8.84***1/***2 200.42 ± 9.86***1 133.83 ± 5.5 Note: 1 – compared to control group. 2 – compared to HM+E group. *p < 0.05; **p < 0.01;***p < 0.001. 3.3. Correlation analysis There were strong negative correlations between rats' uterine thickness and HMs accumulation in both experimental groups — HM and HM+E (see Table 2). Thus, each individual metal had a different strength of influence as follows: Zn (r=−0.89), Cu (r=−0.93), Mn (r=−0.91,), Fe (r=−0.95), Pb (r=−0.93), Cr (r=−0.95) vs Zn (r=−0.77), Cu (r=−0.95), Mn (r=−0.87), Fe (r=−0.97), Pb (r=−0.91), Cr (r=−0.93) (p<0.0001), respectively. It is important to note that the strength of these relationships was different in the endometrium, myometrium, and perimetrium. The correlation between the uterus membranes thickness and the HMs concentration was slightly lower in the HM+E group (r=−0.91, p<0.0001), compared to the HM group (r=−0.96, p<0.0001). Table 2. The strength of Pearson's correlations (r) between HMs accumulation and uterus thickness. Endometrium Myometrium Perimetrium HM group HM+E group HM group HM+E group HM group HM+E group Pb -0.92*** -0.85*** -0.87*** -0.84*** 0.16 -0.21 Fe -0.94*** -0.85*** -0.9*** -0.83*** 0.38 -0.19 Cr -0.93*** -0.87*** -0.9*** -0.84*** 0.24 -0.14 Mn -0.9*** -0.8** -0.85*** -0.82*** 0.14 -0.18 Cu -0.91*** -0.9*** -0.87*** -0.84*** 0.25 -0.05 Zn -0.88*** -0.74** -0.81*** -0.65*** 0.13 -0.05 Total -0.95*** -0.86*** -0.9*** -0.82*** 0.33 -0.17 Note: *p < 0.05; **p < 0.01; ***p < 0.001 – compared to the control group. © 2023 Jordan Journal of Biological Sciences. All rights reserved - Volume 16, Number 3 461 4. Discussion Industrialization and urbanization have affected many organisms' natural lifestyles, violating the evolutionarily programmed organism existence and corresponding complex (genetic) diseases (Saeb and Al-Naqeb, 2016). The progressive accumulation of pollutants in the environment poses a great threat. Therefore, humanity should focus on mitigating (degassing and deactivation) existing and preventing future pollution (Jaishankar et al., 2014; Hamid et al., 2016; Ho et al., 2017; Zhang et al., 2019; Sahiti et al., 2020). Many previous studies have shown the effects of chemical toxins on organisms. Such 'coexistence' may depend on the origin, ways of pollutants influence, individual characteristics of each species, the effectiveness of individual protection or prevention, social behavior, health outcomes, social and demographic features (Saeb and Al-Naqeb, 2016). In general, four factors can contribute to the violation of physiological homeostasis in the body: genetic, hormonal, ontogenetic, and life/health factors (Jaishankar et al., 2014; Saeb and Al-Naqeb, 2016; Ho et al., 2017; Su et al., 2017; Zhang et al., 2019; Lee et al., 2021). HMs are among the top exogenous pollutants worldwide that can spread and bioaccumulate in terrestrial, aquatic, and airborne environments. In such natural conditions, essential and toxic trace elements have a long half-life, and they can accumulate and change their nature (Singh et al., 2011; Jaishankar et al., 2014; Romaniuk et al., 2015; Nakade et al., 2015; Hamid et al., 2016; Romaniuk et al., 2017; Nwosu et al., 2018; Mohammad Hosseini et al., 2019; Zhang et al., 2019; Sahiti et al., 2020). It should be noted that the geochemical cycling of HMs on the planet has both artificial and natural compounds (weathering of metal-bearing rocks and volcanic eruptions, etc.). This increases their spread even in regions with low urbanization and technological progress levels (Singh et al., 2011; Jaishankar et al., 2014; Nakade et al., 2015; Hamid et al., 2016; Ali et al., 2019; Zhang et al., 2019; Lee et al., 2021). Moreover, polyelemental additive metal contamination can contribute to the suppression and/or stimulation of each compound or even change its properties (Lodovici and Bigagli, 2011; Singh et al., 2011; Jaishankar et al., 2014; Nwosu et al., 2018; Ali et al., 2019). The main effects of HMs on the body are the development of oxidative stress (hyperproduction of free radicals, reactive oxygen and nitrogen forms and lipid peroxidation, and inhibition of antioxidant mechanisms), inhibition of enzymatic activity, hormonal disorders, disruption of cell integrity, imbalance of cell division and apoptosis, impaired gene expression (blocking of signal pathways), chromosomal aberrations, pathological methylation and accumulation of damaged DNA, etc. (Singh et al., 2011; Jaishankar et al., 2014; Romaniuk et al., 2015; Hamid et al., 2016; Romaniuk et al., 2017; Chen et al., 2019). However, the mechanisms of HMs effect on the reproductive system are not fully understood. On the one hand, the toxicity at low exposure concentrations of metals such as cadmium, lead, aluminum, metalloid arsenic is more or less clear (cytotoxic, carcinogenic, and genotoxic effects). On the other hand, the excessive concentrations of essential elements (such as copper, zinc, manganese, nickel, iron, etc.) can act through complex direct and indirect pathways (Fenton-type reaction, or depletion of antioxidant systems). It is related to their mandatory physiological participation in antioxidant protection (Bielen et al., 2013; Jaishankar et al., 2014; Hamid et al., 2016; Romaniuk et al., 2017; Chen et al., 2019). Antioxidant deficiency, chronic diseases, or toxicants exposure are accompanied by an increased free radical concentration. It contributes to the violation of redox regulation and the development of oxidative stress, damage to the integrity of lipids, proteins, and DNA. On the one hand, the imbalance of redox systems occurs by direct inhibition of enzymatic protective (antioxidant) mechanisms (superoxide dismutase, ascorbate peroxidase, catalase, and glutathione peroxidase), non-enzymatic metabolic antioxidants (lipoic acid, glutathione, L- arginine, coenzyme Q10, melatonin, uric acid, bilirubin, metal-chelating proteins, transferrin, etc.) and nutrient non-enzymatic antioxidants (vitamin E, vitamin C, carotenoids, trace metals (selenium, manganese, zinc), flavonoids, omega-3 and omega-6 fatty acids, etc.). On the other hand, it is achieved by stimulation of enzymes that produce free radicals (hydroxyl (OH•), superoxide (O2•–), nitric oxide (NO•), nitrogen dioxide (NO2•), peroxyl (ROO•) and lipid peroxyl (LOO•)) and other non-radical reactive derivatives (hydrogen peroxide (H2O2), ozone (O3), singlet oxygen (1O2), hypochlorous acid (HOCl), nitrous acid (HNO2), peroxynitrite (ONOO–), dinitrogen trioxide (N2O3), lipid peroxide (LOOH)) (Shao et al., 2007; Pham-Huy et al., 2008; Bielen et al., 2013; Phaniendra et al., 2015). Vitamins and trace metals co-factors have an essential role in the non-enzymatic antioxidant mechanisms (Singh et al., 2011; Bielen et al., 2013; Phaniendra et al., 2015; Su et al., 2017). Artificial induction of non-enzymatic antioxidants leads to the counteraction of free radicals by direct and/or indirect ways (Bielen et al., 2013; Hamid et al., 2016; Mohammad Hosseini et al., 2019; Sahiti et al., 2020). Antioxidants can block the action of free radicals on the cell surface and in the blood. Tocopherol locates on the biological membrane of cells reduces the risk of free radicals entering the cell. Also, when combined with other antioxidants, vitamin E promotes faster cell "cleansing" and protection. They are also able to neutralize free radicals by transferring them positively charged atoms. Based on this, in our study, we used vitamin E for treatment because it is considered as the most powerful exogenous antioxidant and free-radical scavenger (Pham- Huy et al., 2008; Al-Attar, 2011; Sahiti et al., 2020). It is also known that stabilization of one antioxidant can lead to exhibiting cooperative behavior and enhance other's antioxidant mechanisms (Al-Attar, 2011; Bielen et al., 2013; Sahiti et al., 2020). For example, vitamin C has a regenerative effect on vitamin E from α-tocopherol radicals damage to membranes, zeaxanthin synthesis in the xanthophyll cycle, and inhibits activation of the caspase cascade and DNA damage, etc. (Shao et al., 2007; Al- Attar, 2011; Bielen et al., 2013;Sahiti et al., 2020). The results of our study indicated a pernicious toxic effect of HMs on the rat uterus. Histopathological studies on uteri of different exposure groups in the present study revealed its dose-dependent deleterious effects in all structural elements. Thus, the heterogeneity of uterine transformation was represented mainly by degenerative © 2023 Jordan Journal of Biological Sciences. All rights reserved - Volume 16, Number 3 462 and atrophic changes (degeneration and decrease in the height of luminal and glandular epithelium, decrease in the number of glands, their size and lumen), interstitial edema, inflammatory cells infiltration, microcirculatory disorder, connective tissue disorganization, and uterus wall thinning. Simultaneously, there was a decrease of the uterus thickness on the 37.99 % (p < 0,0001) vs 26.03 % (p < 0,0001), HM vs HM+E groups compared to the control. The reduction of the uterine wall (37.99%, p<0.0001) was caused by thinning of mucous and muscular membranes, respectively. It resulted in an increased intrauterine lumen and a decreased number of endometrial folds. This can complicate the movement of sperm and oocyte fixation (Höfer et al., 2009; Lukacinova et al., 2012; Nakade et al., 2015; Hamid et al., 2016; Cedars et al., 2017; Hanson et al., 2017; Chen et al., 2019; Doncova et al., 2019; Mohammad Hosseini et al., 2019). It seems that this reaction of the uterus was caused by an increased concentration of Zn, Cu, Fe, Mn, Pb, and Cr in the organ tissue. It was also confirmed by the correlation between spectrophotometric and morphometric values imbalance. Similar morphological and morphometric changes in the uterus have been described in other studies (Höfer et al., 2009; Lukacinova et al., 2012; Nakade et al., 2015; Nasiadek et al., 2018; Doncova et al., 2019). However, different HMs combinations (mono- and/or polyelemental), their concentrations, and exposure time were used. In contrast, it was reported about the opposite effect of pollutants on the uterus, which was manifested by hyperplasia and dystrophy of the uterine mucosa (Höfer et al., 2009; Nasiadek et al., 2018). Authors indicate that HMs accumulation in the body can both stimulate and inhibit the activity of sex hormones and their effect on receptors in the uterus (Höfer et al., 2009; Chatterjee and Chatterji, 2010; Katz et al., 2016; Hanson et al., 2017). Moreover, we have previously found an HMs effect on developing dystrophic/atrophic and/or oncological changes in other organs (the bladder, bone marrow, breast, and others) (Romaniuk et al., 2015; Romaniuk et al., 2017; Romaniuk et al., 2018). Our results point out that in contrast to HMs exposure of rats in HM group, the less significant histopathological lesions were identified in the rats' uterus after vitamin E treatment (HM+E group). Thus, after vitamin E treatment, moderate atrophy (decrease in height of columnar cells and fibrosis) and inflammation in the uterus were observed. Moreover, treatment in HM+E group caused the less pronounced reduction of rat uterine thickness (less on 16.17%, p<0.01) against the background of suppression of the accumulation of the metal in the uterus tissue, compared to HM group. Based on this, a vitamin E supplement may be beneficial in slowing progressive uterus damage. Such morphological results coincide with the data on the effectiveness and importance of the natural or artificial compounds with detoxifying and antioxidant properties (Pham-Huy et al., 2008; Bielen et al., 2013; Yadav et al., 2016; Romaniuk et al., 2019; Romaniuk et al., 2018; Sahiti et al., 2020). Unfortunately, a definitive defense mechanism against the impact of pollutants on the organism has not been identified or reported. HMs exposure (both short- and long-term) leads to their accumulation in the organs (Hamid et al., 2016; Romaniuk et al., 2017; Su et al., 2017; Nwosu et al., 2018; Ali et al., 2019; Mohammad Hosseini et al., 2019). However, the imbalance of their concentrations in the uterus and other organs differed among reports (Höfer et al., 2009; Lukacinova et al., 2012; Rzymski et al., 2016; Nasiadek et al., 2018). Our study showed an increase of HMs accumulation (p<0.001) in rats' uterus. Moreover, the concentration and intensity of their accumulation differed in each case. Thus, the lowest relative bioaccumulation in both groups (HM and HM+E groups) belonged to Zn and the maximum to Pb (Pb>Fe>Cr>Mn>Cu>Zn for HM group and Pb>Fe>Cr>Cu>Mn>Zn for the HM+E group in descending order). It should be noted that corrector treatment caused the change in Mn and Cu accumulation order. In the HM+E group, the HMs levels were higher than control levels but were significantly lower (for Zn, Cu, Fe, Mn, Pb, and Cr; p<0.0001) compared to the HM group. This difference may be due to several factors, such as the properties of each individual metal, competition bonds (synergistic and antagonistic) both between metals and between metals with vitamin E, accumulative characteristics of HMs and bioaccumulative characteristics of the uterus, and others (Jaishankar et al., 2014; Nakade et al., 2015; Hamid et al., 2016; Rzymski et al., 2016; Romaniuk et al., 2018; Mohammad Hosseini et al., 2019; Sahiti et al., 2020; Wang et al., 2020). In addition, we demonstrated the relationship between the excess concentration of HMs in uterine tissue and the variability of morphometric values of the uterine wall. Strong negative correlations were found between organ thickness and HMs accumulation in the uterine tissues in both groups (HM group (r=−0.96, p<0.0001) and HM+E group (r=−0.91, p<0.0001)). In this case, the greatest influence on uterine wall thickness had Pb and Cr. On the other hand, the lowest influence had Zn and Cu. The essential HMs are prone to lower accumulation in the uterus on the background of detoxification by vitamin E. At the same moment, toxic or potentially dangerous metals have bigger accumulative properties. The lower HMs accumulation and reduced morphological changes in uterine tissue (HM+E group compared to HM group) validated the feasibility of the use of natural supplementation with antioxidant and detoxifying properties. Based on our results and analysis of the literature, as a general concept, it has become clear that rats' uterus changes depending on the HMs influence. The long-term intake of low HMs doses and their accumulation in the uterus (as in other organs) led to the gradual imbalance of intracellular homeostasis, atrophy, inflammation, suppression of cellular transduction mechanisms, disruption of compensatory defense mechanisms, redox imbalance, and oxidative stress (decrease of the cellular antioxidants and increased oxidative DNA damage, lipid peroxidation, and reactive oxygen species) (Lodovici and Bigagli, 2011; Jaishankar et al., 2014; Hamid et al., 2016; Diantin et al., 2018). The inflammation, provoked by the HMs action on the background of already existing pathological changes, also increased the free radical generation. Under the influence of chronic stress, adaptive mechanisms were exhausted and led to the development of atrophic changes of uterus cells. The aggravated cellular hypoxia leads to the progression of periglandular and perivascular fibrosis. Most likely, oxidative stress appeared to be one of the main mechanisms of the HMs effect on the body, which led to morphological transformations in the uterus. It was confirmed by reports © 2023 Jordan Journal of Biological Sciences. All rights reserved - Volume 16, Number 3 463 on the long-term adverse effects of free radicals under oxidative stress on the background of antioxidant capacity depletion (Shao et al., 2007; Pham-Huy et al., 2008; Phaniendra et al., 2015; Yadav et al., 2016; Romaniuk et al., 2017; Romaniuk et al., 2019). Vitamin E allows free radicals to abstract a hydrogen atom from the antioxidant molecule rather than from polyunsaturated fatty acids, thus breaking the chain of free radical reactions, the resulting antioxidant radicals being a relatively unreactive species. At the same time, the prolongation of the experimental conditions caused activation of adaptive mechanisms and the subsequent start of the recovery processes (Al-Attar, 2011; Yadav et al., 2016; Romaniuk et al., 2018; Mohammad Hosseini et al., 2019; Albishtue et al., 2020; Sahiti et al., 2020; Tahtamouni et al., 2020). Such histopathological transformations of the uterine wall can disrupt the estrous cycle, cause hormonal imbalance, and reduce fertility (Höfer et al., 2009; Lukacinova et al., 2011; Doncova et al., 2019). Moreover, it has been shown that low doses of various metals (lead, mercury, and cadmium) are associated with metal-specific reproductive system lesions (Doncova et al., 2019). However, long-term HMs exposure activates epigenetic and adaptive mechanisms as evidenced by an increase in the total number of litters and neonates (i.e., vulnerable groups showed increased reproductive activity). HMs can impair reproductive function by affecting other organs in both female and male rats. Moreover, the HMs accumulation in different organs is much higher than that in the uterus (Höfer et al., 2009; Lukacinova et al., 2012; Sahiti et al., 2020; Wang et al., 2020; Shraideh et al., 2021). In addition, the proven reprotoxic properties of HMs have been manifested as deteriorations of physical and reproductive health parameters in subsequent generations (Lukacinova et al., 2011; Doncova et al., 2019; Mohammad Hosseini et al., 2019;). Also, from the results of this study, there was group-dependent body weight loss. The most pronounced weight loss was observed in the HM group. Such results can be explained by chronic HM intoxication, which is accompanied by endocrine disorders of the thyroid gland, atrophic changes in internal organs, alteration of electrolyte balance and lipid metabolism, injury of hepatic function and the induction of neurobehavioral function (Su et al., 2017; Fiati Kenston et al., 2018). Increased environmental pollution is reflected in increased risks of deterioration of plants, animals, and humans. This has been confirmed by links between HMs excesses in the organs (including the uterus) of wild animals (from potentially contaminated areas) and human population density, age, season, and extent of territory contamination (Wirth et al., 2010; Jaishankar et al., 2014; Hamid et al., 2016; Ljungvall et al., 2017; Romaniuk et al., 2017; Avilova et al., 2018; Shah et al., 2020; Lytvynenko et al., 2021). Based on the variability of the consequences of pollutants, the prediction of the development of pathological changes is a complex process. This requires a consideration of HMs combinations and concentrations, their exposure time, the intake way, the features of local environmental pollution, the presence of concomitant pathologies and so on. 5. Conclusions Long-term exposure (within 90 days) to the HMs combination had a pernicious toxic effect on the rats' uterus. A strong negative correlation between the accumulation of HMs (zinc, copper, iron, manganese, lead, and chromium) in uterus tissue with morphological (degenerative and atrophic) and morphometric (reduced uterine-wall thickness) changes was detected. Uncontrolled exposure to HMs was found to lead to serious complications and adverse reproductive health risks. The HMs exposure combined with vitamin E treatment was accompanied by significantly lower accumulation of chemical elements in the uterine wall and restraint of morphological lesions in the rats' uterus. 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