INTRODUCTION:

Aluminium (Al) is a ubiquitous element with known toxicity in the human body, mainly in the central nervous system, may act as fetal teratogens.^1,2 Al is a silver white powdered substance with a melting point of 660.37°C. It is readily inflammable and on new Al surfaces in air or water, a hard transparent few molecules thick oxide layer forms due to its high affinity for oxygen. Additionally, Al is a light metal with a 2.7 g/cm³ of density.³ Furthermore, Al is also found in foods naturally as well as the use of Al containing food additives. Potatoes, spinach, tea, etc. are natural foods that contain a high amount of Al.

The Al concentrator in food can also increase through the use of Al cookware, utensils, and wrappings.⁴ In addition to this, Al is also present in many pharmaceutical products like vaccines, antacids (aluminum hydroxide), buffered aspirins and also in consumer products like cosmetics. In multiple daily doses for an extended period, antacids and buffered aspirins are taken containing 5-562 mg/kg of Al.⁵ Everyday people are being exposed to Al through several sources as it is present almost everywhere around us with a large number of applications. It is most extensively found in the environment and about 8% of the earth crust contains Al.⁶ Air, water, and dietary sources are cited as primary sources of Al. It is used for the purpose of packaging of almost 95% beverage cans.⁷Other possible sources are housing materials (such as cookware utensils, serving utensils, etc.), cookware, Al foils, cosmetics, etc.In the 70s of last century, the medical fraternity considered the toxicity of, so called ‘biologically inert’ metal, Al. A large volume of research, since then, is conducted to get insight and fight against the Al toxicity. While the awareness about the toxic effects is bringing down the daily and avoidable uses of the Al in developed countries, being the cheap, Al wares are the commonly used cooking utensils and containers in India. Al toxicity causes Alzheimer disease (AD), dialysis dementia, parkinsonism, and amyotrophic lateral sclerosis. It also affects skeletal system brain tissue, bone, blood cells, liver and kidney.⁸ It has been shown that Al has severe toxic effects on the mouse embryo/fetus, which leads to significant increase in resorption rate, decreased body weight, and major anomalies fetal death, and skeletal anomalies.⁹ Transplacental passage of Al from pregnant mice to fetus organs is noted after maternal transcutaneous exposure.¹⁰ It has been reported that oral Al exposure increases the incidence of fetal abnormalities in rats and mice.¹¹Exposure to Al is inevitable because of its abundance in the earth’s crust, use in cookware, foods and drinks, medications, etc.² Nervous system is the most sensitive to Al toxicity and it’s inducing cognitive deficiency and dementia in brain. Accumulation of excessive amounts of Al may leads to gonadal dysfunction in both human and animals. Alcohol consumption harm fetus during pregnancy. It also may toxic effects in the reproductive process through miscarriage, aneuploidy, anomaly, disordered fetal growth, developmental delay, perinatal death.¹² The effects of alcohol on the reproductive system become very interesting for researcher. The most important endocrine consequences of long-term alcohol use are its effects on the gonads. It is also noted that moderate alcohol consumption increase the HDL level and reduce the cholesterol level in the bloodand reduce the risk of stroke and stress, anxiety and tension, AD.¹³ Moderate alcohol consumption and risk of coronary heart disease among women with type 2 diabetes mellitus.¹⁴ It is also noted that moderate alcohol consumption lowers the risk of type 2 diabetes.¹⁵ Consumes large amounts of alcohol can result in acute and delayed impairments in cognitive and executive functions, spatial learning and memory impairment. These impairments lead to medical and social problems including dementia, violence and decreased work productivity.¹⁶ However, the Al load caused by Al exposure, may be influenced by ethanol coexposure.^17,18 Consumption of alcohol is suggested to increase susceptibility of rats to certain effects of Al but it is also noted that consumption of beer may be affording a protective for the toxic effect of Al.^19,20,21 Brain is the primary organ affected by Al toxicity. Only a few studies have described its effect on the structure of the ovary. Furthermore, the use of ethanol against Al toxicity needs to be investigated. Many studies on the effects of alcohol on female reproductive system but combined effects of Al and ethanol has not been documented yet. The aim of the study was to identify the effects of Al administration on the microscopic structure of ovary and to clarify any possible protection conferred by the concomitant administration of ethanol.

MATERIALS AND METHODS:

Sixteen wistar female rats of an average weight of 200 gm and an average age of 120 days were used in this study. Animals were kept individually in plastic cages in noise free, airconditioned animal house with temperature maintained at 75°F and on a light dark cycle of 12: 12 hours. Humidity was maintained with a minimum of 50%. Rats were fed on diet pellets, tap water ad libitum and treated with utmost humane care. The experimental protocol was approved by the Institute Animal Ethics Committee and the procedures were performed according to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, India). After one week of acclimatization, rats were randomly divided (with the help of Random Allocation Software Version 1.0, May 2004) into 4 groups namely-

Group I (Control group, 4 animals) received the normal saline water.

Group II (Experimental group, 4 animals) received aluminium chloride (4.2mg/kg body weight) dissolved in drinking water containing sodium chloride (0.9%).

Group III (Experimental group, 4 animals) received ethanol (1gm/kg body weight) dissolved in drinking water containing sodium chloride (0.9%).

Group IV (Experimental group, 4 animals) received aluminium chloride (4.2mg/kg body weight) and also ethanol (1 gm/ kg body weight).

The treatments were carried out through oral feeding gavage once daily for periods of 90 days. Their weights were recorded daily. After 3 months, the animals were anaesthetized with pentobarbitone (i.p) and an intracardiac perfusion of normal saline followed by 10% formol saline was performed. The ovary of all groups of animals were dissected out and blotted. The ovary was separated from the fallopian tube and weighed. The ovary of all animals was processed for routine paraffin embedding.

Histological study:

The ovary was fixed in 10% formaldehyde solution, passed through ascending series of ethanol baths, cleared in xylene and embedded in paraffin. Tissues were sectioned at 5 µm and stained with Haematoxylin and Eosin (H&E) according to John D Bancroft Theory and Practice of Histological Techniques to observe the structure. The stained slides were labeled properly and placed under light microscope obtained with a digital camera attached to the microscope for observation.

RESULTS:

Examination of H & E-stained sections of ovaries obtained from the control rats revealed a normal ovarian histological structure with an outer simple cubical germinal epithelium, an ovarian cortex characterized by multiple follicles in various stages of development, corpus luteum and a central medulla made of the vascularized ovarian stroma (Fig.1a). High magnification showing primordial follicle starts to mature with transformation of flattened follicular cells at the periphery to cuboidal morphology. The morphological changes in follicular cells indicate the onset of follicular maturation. This follicle is bigger than primordial follicle and consists of single or double layer of follicular granulosa cells at the periphery, more abundant follicular fluid and an enlarging oocyte. The zona pellucida of the oocyte is distinct (Fig.1b). H&E-stained sections of ovaries obtained from aluminium treated rats revealed degenerated atretic follicles with degenerated ova and vacuolation of cells (Fig.2a). High magnification showed degenerated zona pellucida (Fig.2b). Ethanol treated group showing absence of growing follicles, increased large corpora lutea. Dilated and congested vessels were observed in the growing follicle in high magnification (Fig3a. & 3b). H&E stained sections of ovaries from combined treated aluminium and ethanol rats revealed absence of growing follicles, degeneration of growing follicle with desquamation of pyknotic granulosa cells and degenerated oocyte. Multiple vacuoles of degenerated granulosa cells with dilated congested vessels and edema seen. Hyaline material seen inside the degenerating follicles. (Fig.4a & 4b).

Fig.1a. Control group I ovarian sections showing multiple growing follicles (GF) in various stages of development, newly formed corpus luteum (CL) with small spindle-shaped basophilic cells. Haematoxylin & Eosin stain, 100X.

Fig.1b. Control group I ovarian sections showing primordial follicle starts to mature with transformation of flattened follicular cells at the periphery to cuboidal morphology (black arrow). The morphological changes in follicular cells indicate the onset of follicular maturation and consists of single or double layer of follicular granulosa cells (green arrow) at the periphery, more abundant follicular fluid and an enlarging oocyte. The zona pellucid of the oocyte is evident (red arrow). Haematoxylin & Eosin stain, 400X.

Fig.2a. Photomicrograph of ovary of the aluminium treated group II showing atretic Follicles (T) with degenerated ova and vacuolation of cells in some of them. Haematoxylin & Eosin stain, 100X

Fig.2b. Photomicrograph of ovary of the aluminium treated group II showing degenerated zona pellucidum (arrow). Haematoxylin & Eosin stain, 400X.

Fig.3a. Photomicrograph of ovary of the ethanol treated group III showing absence of growing follicles, increased large corpora lutea (CL) and increased atretic follicles. Haematoxylin & Eosin stain, 100X

Fig.3b. Photomicrograph of ovary of the ethanol treated group III showing Dilated and congested vessels (B). Haematoxylin & Eosin stain, 400X.

Fig.4a. Photomicrograph of ovary of the aluminium and ethanol treated group IV showing degeneration of atretic follicle (T) with desquamation of pyknotic granulosa cells and degenerated oocyte. Multiple vacuoles (v) of degenerated granulosa cells with dilated congested vessels and edema seen. Hyaline material (H) seen inside the degenerating follicles. Haematoxylin & Eosin stain, 100X.

Fig.4b. Photomicrograph of ovary of the aluminium and ethanol treated group IV showing degeneration of atretic follicle with multiple vacuoles and dilated congested vessels and edema seen. Haematoxylin & Eosin stain, 400X.

DISCUSSION:

Al has no known biological role in living organisms and may be classified as a toxic metal.²² Soil contamination resulting in substantial concentrations of different pollutants, including metals, has been observed in plants²³ which then may be ingested by herbivores. In polluted sites, a decline in the density of rodent populations has been widely observed.^24,25,26 In vertebrates, this element may be deposited in different tissues, including the central nervous system, becoming a neurotoxin.^27,28 Disorders of steroidogenesis may arise through the deposition of Al in the hypothalamus and pituitary gland. However, the mechanisms of Al toxicity are not fully understood.^29,30 In present study rats treated with Al showing atretic follicles with degenerated ova and vacuolation of cells in ovary. Degenerated zona pellucidum seen in growing follicle. Knowledge of the effects of Al on the female reproductive system is limited. In female mice, Mohammed and collaborators showed histopathological changes in the ovaries and decreased fertility, as measured by the number of pregnant females and the number of absorbed fetuses, after 12 weeks of Al chloride administration (dose range 1000–1400 mg/kg).³¹ Fu and collaborators noted a disruption of the rat ovary structure after 64, 128, and 256 mg/kg Al intake, while Trif and collaborators reported significant lengthening of the sexual cycle in female rats after 0.2, 0.4, and 1 mg/kg aluminum sulfate administration in the uterus.^32,33 There are few data on the impact of aluminum doses, equivalent to the environmental levels of the metal, on the female reproductive system. Research on adult rat females employing low Al doses showed no effect of Al on body and uterus weights.^34,35 Much higher Al concentrations (1000– 1400 mg/kg) reduced female body weight, reduced absolute uterus weight, and caused histological changes in ovarian sections.³¹ Few studies suggest that Al may also modify sexual behavior. Indeed, Abu-Taweel and collaborators found a significant decrease of social contacts and sexual behavior after Al application, but they used higher doses (300 and 600 mg/kg) than those in our experiment.³⁶ Heavy metals may insert their deleterious effects on fish reproduction and gamete development via disruption of the endocrine system and the inhibition of hormone production, such as disruption of hypothalamic-pituitary system.³⁷ The lesions confirmed that exposure to heavy metals can induce histological and pathological changes, as already mentioned in other studies.^38,39,40,41

An increasing literature suggests that chronic alcohol abuse may induce failure of female sexual function.^42,43,44 Association of alcoholism with menstrual abnormalities, problems with reproduction, and changes in secondary sex characteristics has been made. The deleterious effects of alcohol on male sexual function are well documented.^45,46 The relation between chronic alcohol abuse and male impotence, sterility, and feminized bodily habitus is widely recognized.^47,48,49 The mechanisms of alcohol-induced male sexual dysfunction have been explored, and have been shown to be related both to primary testicular failure and to suppression of hypothalamic - pituitary responsiveness.^46,50,51 An increasing literature suggests that chronic alcohol abuse may induce failure of female sexual function. Association of alcoholism with menstrual abnormalities, problems with reproduction, and changes in secondary sex characteristics has been documented. Although systematic endocrinological studies have yet to be performed, evidence is now beginning to accumulate that alcohol may adversely affect sexual function in females as well.^52,53,54,55 It has been suggested that the metabolic endocrinological functioning of the fetal placental unit max' be deranged and that fetal growth and development may be diminished by excessive ingestion of alcohol during pregnancy.^{56,57,58,59,60,61} The absence of recognizable corpora hemorrhagica and corpora lutea in the ovaries of premenopausal age female alcoholics has been reported. In the present study ethanol treated ovary showing absence of growing follicles, increased large corpora lutea, and increased atretic follicles. Dilated and congested vessels were observed in the growing follicle. The mechanism of alcohol-induced ovarian failure remains in question. The ovary contains alcohol dehydrogenase, and ovarian metabolism of alcohol with the local accumulation of toxic metabolites comparable to phenomena believed to occur in the testes is thus possible.⁶² Several hypotheses for specific mechanisms of direct alcohol induced ovarian toxicity might be proposed. Ovarian conversion of ethanol to acetaldehyde, with resultant mitochondrial dysfunction, might occur. Several steps in estrogen genesis, including the conversion of pregnenolone to progesterone by 3 beta-hydroxy steroid dehydrogenase and delta 5-4 isomerase, are NAD dependent.⁶³ The depletion of ovarian stores of NAD by conversion to NADH as the result of ovarian ethanol metabolism might diminish estradiol synthesis. It had been suggested from previous studies that the presence of corpora lutea in ethanol tissues would be greatly reduced.^64,65 In a study performed by Oakberg, et al., it was noted that formation of an antrum within a follicle with less than 7 to 8 layers marked degeneration of that follicle.⁶⁶ According to literature, the premature presence of an antrum produces pressure on the immature oocyte. This pressure can lead to rupture of the zona pellucida, which can harm the ooplasm. The zona pellucida is a layer that surrounds the oocyte. The harming of the ooplasm results in fragmentation of intracellular structures and eventual cell death.⁶⁷ Since the corpus luteum is responsible for producing progesterone, and there are known low levels of progesterone in ethanol fed mice from previous studies.⁶⁸

Most previous studies focused on the individual toxic effects of a single chemical; however, there is a possibility that humans and animals can be exposed to a mixture of toxic agents.⁶⁹ In this animal study, we have for the first time investigated the effects of two commonly used Al and ethanol on the rat’s ovary. We have examined the underlying ovarian histopathological changes due to aluminium chloride toxicity in presence of ethanol coexposure. Fluoride or Al treatment alone or in combination revealed a decline in the activities of both 3β- and 17β-HSDs in ovary suggesting a block in the steroidogenic pathway. Experimental studies have shown that the dietary factors such as calcium, amino acids and vitamins C, E, and D can mitigate the toxic effects of individual treatments of aluminium and fluoride.^70,71,72 However, when the animals were co-exposed to both ethanol and aluminium showing degeneration of growing follicle with desquamation of pyknotic granulosa cells and degenerated oocyte. Multiple vacuoles of degenerated granulosa cells with dilated congested vessels seen. Hyaline like material inside the degenerating maturing follicles and increase in the edema fluid of the ovary noted. The present study explored the histopathological changes in ovary by Al itself and in presence of pro-oxidants ethanol.

CONCLUSSION:

From the foregoing data, it is evident that the administration of aluminium or ethanol alone induced reproductive toxicity in female rats. This toxicity was enhanced by their combined treatment in affecting steroidogenesis in ovary.

ACKNOWLEDGEMENTS:

The authors would like to thank Assistant Director, I/C Central Lab Animal House and Sr. Lab Technician, Department of Anatomy, Dr RP Govt. Medical College, Kangra India to carry out this project in the Institute. Authors wish to thankfully acknowledge the support received from Department of Pharmacology and Department of Pathology Government Medical College, Amritsar, India to carry out the work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 09.06.2020 Modified on 08.08.2020

Research J. Pharm. and Tech. 2021; 14(7):3809-3815.

DOI: 10.52711/0974-360X.2021.00660