According to global statistics, cancer is the second major cause of death worldwide [1] representing a global public health concern with an increasing tendency, mostly due to population growth and a fast-aging population, particularly in developed countries [2]. The most common malignancies diagnosed in male patients comprise prostate, colorectal, lung, and bladder cancer; while breast, colorectal, uterine, and lung cancer are the most common in female patients [1]. Since cancer patients are met with numerous physical, emotional, and socio-economic challenges that have a major impact on their quality of life, major efforts have been invested in the development of faster and more efficient screening, diagnostic, and treatment strategies [3]. Despite remarkable progress in the understanding of cancer development that is accompanied by major advances in cancer therapy over the past decade, the fact that malignancies are generally aggressive, heterogenous, and dynamic pathologies [4] represents a persistent challenge for clinical oncology even in the new millennium [4].

The World Health Organization (WHO) defines cancer therapy as a treatment aimed to considerably prolong the life of oncology patients and to ensure a proper quality of life for cancer survivors [5]. While the leading cancer therapies comprise conventional options such as surgery, radiation therapy, chemotherapy, and proton therapy [2], complementary therapies have received more prominence and popularity as adjuvant remedies to improve the efficiency of conservative cancer treatment as well as a strategy to increase the quality of life of patients [6]. Although complementary remedies are by and large not included in standard patient care, particularly in developed countries as opposed to most African or Asian countries [7, 8, 9], their use is on the rise, most notably to enhance immunity, relieve pain, or ameliorate side-effects stemming either from the ailment itself or from conservative treatment [8]. Complementary therapies encompass a rather large and diverse group of approaches, that may be divided into three major categories, specifically mind–body therapies (such as meditation, hypnosis, acupuncture, relaxation techniques, yoga, tai chi, or massage therapy), traditional medicine (Chinese medicine, Ayurveda, or naturopathic medicine), and biologically based practices or natural products such as vitamins, botanicals, dietary supplements, herbs, spices, special foods, or diets [9]. Among these, apricot kernels, and their active component amygdalin, have received particular attention from the public as well as the scientific community.

Amygdalin (D-mandelonitrile-b-D-gentiobioside; AMG) is a cyanogenic glycoside composed of two molecules of glucose: one molecule of benzaldehyde, which has analgesic effects; and one molecule of hydrocyanic acid, which is regarded as an antineoplastic agent [10]. The compound may be found in kernels of plants belonging to the Rosaceae family such as almonds, apricots, peaches, or apples [11]. AMG per se is non-toxic until hydrogen cyanide (HCN) is released primarily through enzymatic hydrolysis catalyzed by $\beta$-glucosidases. HCN will then inhibit the mitochondrial cytochrome-c oxidase, causing alterations to the oxidative metabolism as well as oxidative phosphorylation, leading to cell death caused by energy depletion and oxygen uptake [12]. While the absorption, distribution, metabolism, and excretion of cyanides have been studied extensively due to their importance as industrial chemicals and environmental toxins, a thorough understanding of AMG toxicokinetics is still missing [13].

AMG has become a popular alternative treatment in patients suffering from diabetes mellitus, asthma, bronchitis, and a complementary cancer remedy [10]. The use of AMG or its synthetic derivate Laetrile in cancer therapy has been widely supported by in vitro studies on colon [12], cervical [14], bladder [15], or prostate cancer cells [16]. The rationale for using this molecule rests on the fact that as opposed to cancer cells, non-malignant cells present with higher levels of rhodanese—a liver mitochondrial enzyme that catalyzes the detoxification of HCN by sulphuration [17]. Nevertheless, the effects of AMG observed in in vivo studies are inconsistent; besides, peroral ingestion of AMG comes along with the risk of cyanide poisoning. Consequently, AMG has not yet been granted Food and Drug Administration (FDA) approval for its use as a therapeutic agent in the United States due to insufficient clinical verification of its therapeutic efficiency [18]. As such, particular attention must be paid to any effects AMG may exhibit on healthy individuals, since the current knowledge on the behavior of AMG on the general morphology and physiology is limited.

This paper is part of a larger investigation striving to determine the effects of AMG within a larger systemic context of health, physiology, and reproduction [19, 20, 21, 22, 23]. As opposed to most studies on AMG, taking advantage of rodents, we chose rabbits as our laboratory model. Whilst undoubtedly the use of mice or rats comes along reduced maintenance costs, small size, and ease of breeding as well as a wide array of available commercial reagents, and genetically modified models [24], the attractiveness of rabbits lies in their intermediate size between rodents and larger animal models, a longer life span, and higher success rate in the translation of findings to humans. From a practical point of view, the size of rabbits permits greater access to tissues and cells from one single animal, and a repeated sampling of blood. What is more, semen collection is more convenient, and as opposed to rodents, ejaculated spermatozoa may be readily obtained when needed [24, 25].

Possible beneficial or adverse effects of AMG on male reproduction have been previously studied with regard to the endocrine balance as well as the quality of ejaculated spermatozoa. Kolesár [26] reported no significant changes to the levels of pituitary hormones following a 28-day administration of pure AMG or apricot kernels. Similarly, the levels of 17$\beta$-estradiol remained by and large unaffected, significantly decreased levels of progesterone were observed in all experimental groups when compared to the Control. At the same time, a notable although non-significant decrease in testosterone was observed, particularly following intramuscular injection of AMG at a dose of 0.6 and 3.0 mg/kg b.w. A follow-up study [21] revealed that short-term administration of pure AMG significantly decreased rabbit sperm motility characteristics in a dose-dependent manner. On the other hand, the consumption of apricot seeds was accompanied by a slight improvement in sperm motion behavior. It must be, however, noted that a better interpretation of the available evidence requires taking the complexity of the male reproductive milieu into consideration. Since on one hand, the male reproductive system acts as a sensitive barometer to external or internal stimuli, while on the other hand, the sperm vitality and fertilizing ability rely exclusively on the proper function of male reproductive structures, possible effects of AMG on the male reproductive system need to be assessed and elucidated. Therefore, this study aims to reveal whether a short-term intramuscular AMG application or a peroral supplementation of apricot kernels could cause changes to the histological, oxidative, and inflammatory profile of the testicular tissue in rabbits.

Over the past years, several in vivo studies have emerged suggesting that AMG does not cause significant changes to the overall health status [22], bone [23], or renal structure [19] of the experimental animals. Nevertheless, it has been reported that intramuscular AMG administration may be associated with time-dependent changes to the balance of male reproductive hormones [26] and a decrease in sperm motion and kinematic characteristics [21]. In this sense, it must be remembered that tumorigenesis and spermatogenesis share some common characteristics, as both processes are characterized by rapidly dividing and differentiating cells, high energy requirements, and an exceptional metabolic profile. On the other hand, the structural and functional uniqueness renders the testes to respond in a very sensitive manner to any imbalance of the internal milieu or surrounding exterior. Since the male fertilization ability by and large depends on the structural integrity and functional activity of male gonads, this paper strived to elucidate the in vivo response of testicular tissue towards pure amygdalin administered intramuscularly, or to crushed apricot kernels supplemented orally.

Our data reveal that pure AMG, as opposed to apricot kernels, may affect the testicular dynamics, particularly when higher (“therapeutic”) doses are used. These effects manifested themselves primarily through a compromised activity of antioxidant enzymes representing the first line of defense against oxidative stress, accompanied by an increase of oxidized proteins, and inflammatory and apoptotic processes; however, these alterations were not translated into notable changes to the testicular architecture.

While the toxicity and efficacy of AMG in in vivo studies needs to be further understood, it has been previously postulated that orally administered AMG releases a high amount of cyanide [34], either through the activity of $\beta$-D-glucosidase found in foods, through the activity of the microflora present in the small intestine and colon [18], or through indirect hydrolysis by water into glucose, benzaldehyde, and hydrocyanic acid [35]. A relatively high risk for the toxicity of AMG was one of the reasons why we chose to apply pure AMG intramuscularly, and opted for an oral administration of apricot kernels, which is considered a safer option for consumption since the kernel comprises other potential bioactive components and nutrients including carbohydrates, carotenoids, phenols, vitamins, terpenoids, esters, and volatile compounds [36]. Hence, we may assume that AMG consumed in the form of a heterogeneous mixture of components present in the kernel may act in symbiosis with other biomolecules with beneficial health effects. Nevertheless, it is important to pay close attention to the pharmacodynamics and interactions of AMG with other biomolecules present in foodstuffs that can be consumed and ingested. Our results also agree with previous animal studies employing higher doses and oral or intravenous AMG administration, according to which a direct lethal effect of the molecule was not observed; however, minor changes to the structure and/or function of organ systems were detected [23, 37, 38]. According to Kovacikova et al. [22], short-term administration of pure AMG, or apricot kernels had a negligible effect on the overall health status of rabbits and the pre-established doses did not pose any risk to their hematological or biochemical blood parameters. On the other hand, intramuscular administration of 3.0 mg/kg b.w. AMG led to a slight enlargement of renal glomeruli accompanied by a thickening of the glomerular basement membrane as observed by Kolesarova et al. [19]. Changes to the renal structure manifested themselves in a significantly decreased diameter of renal corpuscles, glomeruli, and tubules, as well as a decreased height of epithelial tubules. Similarly to our observations, oral administration of apricot kernels had no significant impact on the renal structure. In the meantime, Kovacova et al. [23] reported that neither pure AMG nor apricot kernels affected the femoral weight or length while maintaining the compact and trabecular bone tissue intact. Nevertheless, the study revealed that rabbits administered with 3.0 mg/kg b.w. AMG i.m. presented histological alterations to the bone microstructure consistent with a decreased density and size of secondary osteons as well as the presence of plexiform bone tissue. As such, summarizing currently available evidence we may speculate that pure AMG may present with a certain degree of deteriorating effects independently on the route of administration.

Cyanide as a resulting product of AMG metabolism acts as a potent neurotoxin, which may cause alterations to vital organs including the heart, kidney, brain, or liver, although, Shivanoor and Muniswamy [39] observed no changes in the body or reproductive organ weights of rats perorally treated with (0.64 and 1.2 mg/kg) sodium cyanide for 90 days. As discussed earlier, the primary toxicity of HCN lies in its ability to bind to the cytochrome oxidase terminal in the mitochondrial respiratory chain causing an obstruction in the cells’ ability to use oxygen [40], ROS overgeneration that is accompanied by lipid peroxidation, and cell death [41, 42]. Furthermore, oxidative overload may develop if the effects of ROS are not properly counteracted with antioxidants [43].

While lipid peroxidation is considered the central mechanism of oxidative damage, only slight changes in the levels of MDA were observed in this study. Inversely, a significant increase in the levels of protein carbonyls was observed following intramuscular treatment with 3.0 mg/kg b.w. AMG. This observation agrees with Albogami et al. [37] who suggest that AMG decomposition is associated with the overproduction of benzaldehyde, which will then interact with hydrogen peroxide to form carboxylate and benzenediol [44]. Hence, we may hypothesize that protein carbonylation may be the primary oxidative mechanism of AMG through which ROS accumulation and excessive levels of benzaldehyde may trigger protein oxidation even before the lipids are compromised by peroxidation.

Intramuscular treatment particularly with 3.0 mg/kg b.w. AMG significantly modulated the activity of all antioxidant enzymes and decreased the levels of glutathione as a prominent testicular non-enzymatic antioxidant. Changes in the patterns of antioxidant molecules indicate a shift in the oxidative milieu driven by ROS overproduction and a depletion of the inherent antioxidant capacity, which corroborates previous studies on AMG toxicity [22, 37, 43]. A compromised capacity of SOD to dismutate superoxide to hydrogen peroxide (H${}_2$O${}_2$), accompanied by a decreased ability of CAT or GPx to break H${}_2$O${}_2$ down to water and oxygen, may lead to the accumulation of toxic reactive intermediates with a subsequent loss of structures critical for a proper sperm production [45]. As such, we may speculate that enzymatic antioxidants are particularly prone to deterioration driven by high doses of AMG and/or HCN, leading to the buildup of oxidized biomolecules within testicular structures and hence an increased risk for reproductive failure.

Previous reports have revealed a close association between testicular damage and increased antioxidant depletion accompanied by oxidative damage to the proteins and lipids [46]; nevertheless, our histology and morphometry data indicate only slight changes to the testicular architecture following treatment with AMG or apricot kernels. Inversely, Albogami et al. [37] have observed pathological alterations in the testicular tissue, such as fewer spermatogonia, cellular necrosis, epithelial disarray, and degeneration of Leydig and Sertoli cells. This discrepancy may be explained by the fact that such changes were observed only in rats administered with extremely high AMG doses (200 mg/kg b.w.), whereas animals exposed to lower AMG doses (50 and 100 mg/kg b.w.), presented with normal cell arrangement in the lumen of the seminiferous tubules with no histopathological alterations and active spermatogenesis. Hence, we may postulate that carefully selected low AMG doses may not have a detrimental impact on the structural integrity of male reproductive organs.

It is apparent, however, that induced cell death could be a paramount mechanism of AMG toxicity even in the reproductive tissues. Our data collected from the assessment of caspase activity agree with Erkkilä et al. [47] who observed germ cell death with a concomitant decrease of adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) in human seminiferous tubules cultured in the presence of cyanide. The cessation of energy production may stem from proton (H${}^{+}$) gradient collapse caused by disruptions in the mitochondrial permeability transition pores and maintenance of the mitochondrial membrane potential [48]. The apoptotic machinery will then lead to alterations in the mitochondrial ATP/ADP exchange and a decreased rate of oxidative phosphorylation [49]. Other targets of HCN may include the rate of cytochrome c release or changes in the redox states of pyridine nucleotides such as nicotinamide adenine dinucleotide phosphate (NADPH), all of which will affect the mitochondrial permeability transition and ATP levels [47]. Since the exact site(s) of the apoptotic action of AMG and/or HCN remains to be clarified, further studies will be needed to understand the molecular machinery of cell death that is catalyzed by both molecules.

Cell disintegration and subsequent death may be defined as a cluster of molecular processes that may trigger the secretion of cytokines [50]. As unraveled by earlier reports, cyanide toxicity is associated with the activation of the transcription factor nuclear factor-kappa B (NF-$\kappa$B) pathway [51], which is a major pro-inflammatory molecular pathway promoting the secretion of cytokines, out of which TNF-$\alpha$, chemokines, or interleukins have been implicated in the etiology of male reproductive dysfunction [52]. In this paper, we studied four major cytokines that have been involved in testicular dysregulation, specifically IL-1, IL-6, INF-$\gamma$, and TNF-$\alpha$. Higher levels of all target molecules were found to be increased particularly following intramuscular administration of 3.0 mg/kg b.w. AMG. Accordingly, their elevated production has been observed during a local or systemic inflammation, with a direct negative impact on proper testicular steroidogenesis and spermatogenesis [53]. Besides their direct involvement in a disturbed communication amongst the germ, Leydig and Sertoli cells needed for adequate sperm production [54], it has been proposed that these cytokines could play a role in the regulation of testicular cell proliferation and apoptosis [52], which supports our observations of the testicular caspase activity. Finally, it has also been suggested that high cytokine levels could directly inhibit spermatogenesis through an enhanced oxygen metabolism, and thus lead to a higher incidence of oxidative insults to the testicular structures [52, 53]. Nevertheless, the exact mechanism by which AMG and its metabolic products are involved in the inflammatory process is unknown and deserves further understanding at a local as well as systemic level.

The results of this study revealed that neither of the selected doses of apricot kernels caused any structural alterations to the testicular tissues. Moreover, oral treatment with apricot kernels had slight antioxidant and anti-inflammatory effects, which agrees with the findings of Albogami et al. [37]. It seems that like other bioactive molecules, amygdalin presents a peculiar dichotomy—whilst high AMG doses may have toxic to lethal effects, carefully selected low doses present a biological activity that exhibits beneficial effects on the living system. Pure AMG or foodstuffs containing this molecule have been shown to act as ameliorating agents in studies of induced toxicity, particularly through chemotherapeutic agents [55, 56]. It seems that AMG or apricot kernels could become an interesting complementary option to support conventional therapy of a variety of ailments; this future prospect is, however, subject to larger cohort and/or case-control studies to clarify the existing discrepancies.

Despite notable changes observed in the Control and experimental groups, sample size is the most significant limitation of this study. A relatively low number of animals representing each pre-established group may have impacted the power and complexity of this study. At the same time, an undersized population of animals used in our experimental design may not be fully representative of the population, hence affecting the precision and validity of the collected data. As such, follow-up and/or complementary in vivo studies on a larger population of animal models (rodents or rabbits) may add more veracity to the data obtained in this report. Another limiting factor lies in the aim to only investigate subacute doses within a shorter timeframe. Hence, to generalize the results, the treatment period should have taken longer, whilst considering higher doses of AMG as well as apricot kernels. At the same time, knowledge of the specific signaling pathways that are affected or regulated by AMG on a genetic, epigenetic, or molecular level is more than necessary to conclusively determine the suitability of pure AMG or apricot kernels as relevant therapeutic agents.

The present study suggests that short-term intramuscular administration of amygdalin exerted negative effects on rabbit testicular tissue through oxidative, inflammatory, and pro-apoptotic mechanisms, which may lead to a decreased reproductive potential of the animals. On the other hand, oral supplementation of apricot kernels did not present with changes to the testicular milieu. The specific conditions leading to the toxicity of amygdalin and its food sources should be further studied to determine optimal doses, timeframes, and routes of amygdalin administration to ensure its safety and effectiveness as a potential complementary cancer treatment option in the future.

The datasets generated during and/or analyzed in this study are available from the corresponding author upon reasonable request.

AK and ET designed the study. ET, MĎ, MH, JP and EK performed the research. ĽC and ĽO oversaw the animal care and treatment regime. MH and RJ performed the animal sacrifice and sample collection. ET and MĎ analyzed the data. ET and AK wrote the manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work. All authors contributed to editorial changes in the manuscript, read and approved the final manuscript.

Institutional and national guidelines for the care and use of animals were followed appropriately, and all experimental procedures were approved by the State Veterinary and Food Institute of Slovak Republic, no. 3398/11–221/3 and Ethic Committee.

We wish to thank the CeRA Team of Excellence for their financial and promotional support.

This research was funded by the Slovak Agency for Research and Development, grant numbers APVV-18-0312 and APVV-21-0095; and by the Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic and the Slovak Academy of Sciences, grant number VEGA 1/0266/20.

Given her role as Guest Editor, Eva Tvrdá had no involvement in the peer-review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Amedeo Amedei. The authors declare no conflict of interest.