Contemporary Comprehensive Review on Arsenic-Induced Male Reproductive Toxicity and Mechanisms of Phytonutrient Intervention

Arsenic (As) is a poisonous metalloid that is toxic to both humans and animals. Drinking water contamination has been linked to the development of cancer (skin, lung, urinary bladder, and liver), as well as other disorders such as diabetes and cardiovascular, gastrointestinal, neurological, and developmental damage. According to epidemiological studies, As contributes to male infertility, sexual dysfunction, poor sperm quality, and developmental consequences such as low birth weight, spontaneous abortion, and small for gestational age (SGA). Arsenic exposure negatively affected male reproductive systems by lowering testicular and accessory organ weights, and sperm counts, increasing sperm abnormalities and causing apoptotic cell death in Leydig and Sertoli cells, which resulted in decreased testosterone synthesis. Furthermore, during male reproductive toxicity, several molecular signalling pathways, such as apoptosis, inflammation, and autophagy are involved. Phytonutrient intervention in arsenic-induced male reproductive toxicity in various species has received a lot of attention over the years. The current review provides an in-depth summary of the available literature on arsenic-induced male toxicity, as well as therapeutic approaches and future directions.


Introduction
Arsenic (As) is a naturally occurring toxic metalloid with odourless, colourless, and tasteless properties. The most commonly available forms of arsenic include inorganic As, organic As, and arsine gas [1]. Arsenic is ranked as the 20th most abundant element in the terrestrial, 14th in the marine, and 12th in the human ecosystem. It possesses significant health concerns due to its ubiquitous existence, discharged into the environment from volcanic and industrial activities [2,3]. Charles Dickens used arsenic as a tonic, called Fowler's solution (potassium arsenate in water). It was also employed to treat leukaemia, psoriasis, and chronic bronchial asthma [4,5]. As is a well-known poison whose use is restricted to the production of pesticides, herbicides, cotton desiccants, and exfoliants in agriculture, applied as doping material in the semiconductor industry, bronzing, pyrotechnics, as well as the manufacturing of special kinds of glasses and preservation of wood [6,7]. As is found naturally in both trivalent and pentavalent forms, but the pentavalent form is much more common and is the best form for eliminating arsenic from biological systems [8,9]. The commonly available pentavalent forms of arsenic are arsenic pentoxide, arsenic acid,

Metabolism of Arsenic
In many organisms, along with humans, the inorganic form of As gets rapidly reduced from arsenate (pentavalent) to arsenite (trivalent) in the blood, facilitated by glutathione (GSH) [86]. The latter is 2-10 folds more toxic and rapidly taken up by the cells than the former [10]. Once absorbed, arsenite binds to globin of Hb-and -SH-containing proteins such as Glutathione and Cysteine and is distributed to the skin, hair, and mucosa owing to thiol-cystine amino acids. Skin, hair, epithelium of gastrointestinal tract (GIT), and epididymis had the highest As retention [87][88][89][90]. Aquaglyceroporins (AQPs) are involved in transporting As into cells [91], whereas efflux is carried out by major facilitator superfamily (MFS) transporters and ATP-binding cassette (ABC) transporters [92]. As shown in Figure 1, initially, biotransformation of arsenic involves oxidative methylation of arsenite to arsenate, mediated by the enzyme arsenite methyltransferase (As3MT), a primary methyl donor, followed by oxidation, arsenite to a pentavalent metabolite of arsenate, monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) [93,94]. MMA and DMA were taken up less effectively by organs and tissue than arsenite and rapidly excreted into urine [95]. In contrast, evidence suggests that metabolites of As tend to induce chromosomal mutations [96] and show genotoxicity [97]. Methylated forms of As were also found in human urine, natural water, and bird eggshells [98].

Metabolism of Arsenic
In many organisms, along with humans, the inorganic form of As gets rapidly reduced from arsenate (pentavalent) to arsenite (trivalent) in the blood, facilitated by glutathione (GSH) [86]. The latter is 2-10 folds more toxic and rapidly taken up by the cells than the former [10]. Once absorbed, arsenite binds to globin of Hb-and -SH-containing proteins such as Glutathione and Cysteine and is distributed to the skin, hair, and mucosa owing to thiol-cystine amino acids. Skin, hair, epithelium of gastrointestinal tract (GIT), and epididymis had the highest As retention [87][88][89][90]. Aquaglyceroporins (AQPs) are involved in transporting As into cells [91], whereas efflux is carried out by major facilitator superfamily (MFS) transporters and ATP-binding cassette (ABC) transporters [92]. As shown in Figure 1, initially, biotransformation of arsenic involves oxidative methylation of arsenite to arsenate, mediated by the enzyme arsenite methyltransferase (As3MT), a primary methyl donor, followed by oxidation, arsenite to a pentavalent metabolite of arsenate, monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) [93,94]. MMA and DMA were taken up less effectively by organs and tissue than arsenite and rapidly excreted into urine [95]. In contrast, evidence suggests that metabolites of As tend to induce chromosomal mutations [96] and show genotoxicity [97]. Methylated forms of As were also found in human urine, natural water, and bird eggshells [98].  Figure 1. Inorganic arsenic conversion pathways into mono-, di-, and trimethylated products when the toxic form of As(III) is taken up by a cell, it is methylated to MMA (monomethylarsonic acid) and DMA (dimethylarsinic acid) with the help of the enzyme arsenic methyl transferase (AsMT) and can then be eliminated from the body through urine. However, in a pathological state, SAM depletion causes As(III) aggregation, affecting cellular homeostasis in a variety of ways, including oxidative balance, inflammation, and genetic and epigenetic processes. This disruption in homeostasis causes cellular damage and, eventually, cell death. As(V): inorganic pentavalent arsenic; As(III): inorganic trivalent arsenic; As3MT: arsenite methyltransferase, MMA(V): methyl arsonate; MMA(III): monomethylarsonous acid; DMA(V): dimethyl arsenate; DMA(III): dimethylarsinous acid; GSH: glutathione; GSTO: Glutathione S-transferase omega, GSSG: Glutathione disulfide As-GSH: Arsenic glutathione; AsS: Arsenic sulphide; AsB: Arsenobetaine; ABC transporter: ATP-binding cassette transporters; AS3MT: arsenite methyltransferase.

Arsenic-Induced Oxidative Stress and DNA Damage
At a biochemical level, inorganic arsenic in the pentavalent state replaces the phosphate in several reactions, the trivalent form of inorganic and organic (methylated) arsenic reacts with critical thiols and inhibits their activity [93]. As induces the formation of reactive oxygen species (ROS), enhances free radicals, and diminishes the activity of thiol group-rich antioxidants such as glutathione (GSH) [99]. Production of ROS will take place during the generation of intermediate arsine species [100]. Methylated arsenicals cause the release of iron from ferritin and initiate the production of hydroxy radicals and progress ferroptosis [101]. Arsenic shows mitochondrial toxicity by inhibiting succinic dehydrogenase activity and uncoupling oxidative phosphorylation with the production of O 2, which gives rise to other forms of ROS [102]. Oxidative stress leading to the activation of ERK/AKT/NFkβ pathway is one of the primary molecular mechanisms involved in arsenic-induced male reproductive toxicity [10]. Mitogen-activated protein kinases (MAPKs) are proteins that regulate the signalling of cells in response to environmental stimuli. The ERK signalling pathway is involved in a variety of male reproductive functions, such as spermatogenesis and Sertoli cell function. Activation of ERK1/2 inhibits the function of Sertoli cells and increases apoptosis in the testes. Protein kinase B (AKT) regulates oxidative stress in conjunction with the immune system by regulating cell growth, survival, proliferation, and inflammation. MAPK and AKT both directly phosphorylate nuclear factor kappa B (NF-B), which increases NF-kB binding to target DNAs and the expression/activity of NF-kB-controlled genes. Spermatogenesis and the function of Sertoli cells in the testes are governed by the transcription factor NF-kB. Activation of NF-kB inhibits spermatogenesis in both humans and mice. Arsenic activated the ERK/AKT/NF-B signalling pathways in numerous cell types. The expression of ERK1/2, IKK, PI3K, AKT, and NF-B, as well as the phosphorylation of ERK/AKT, increased in rats exposed to sodium arsenite (1, 5, or 25 mg/L) for 6 months. This causes reproductive toxicity via ERK/AKT/NF-kB signalling [68][69][70][103][104][105][106][107]. Figure 2 depicts As-induced oxidative stress mechanisms.

Effects of Arsenic on the Male Reproductive System in Animals
Epidemiological studies have suggested that arsenic is one of the most hazardous reproductive toxicants present in the environment, which is significantly accumulated in the reproductive tissues like the testes, epididymis, seminal vesicle, and prostate gland [108,109]. The inhibition of testosterone biosynthesis by arsenic is depicted in Figure 3. Arsenic exposure exerted cellular and molecular perturbations such as oxidative stress, inflammation, induction of autophagy, and apoptosis, which obstructed male gonadal de-

Effects of Arsenic on the Male Reproductive System in Animals
Epidemiological studies have suggested that arsenic is one of the most hazardous reproductive toxicants present in the environment, which is significantly accumulated in the reproductive tissues like the testes, epididymis, seminal vesicle, and prostate gland [108,109]. The inhibition of testosterone biosynthesis by arsenic is depicted in Figure 3. Arsenic exposure exerted cellular and molecular perturbations such as oxidative stress, inflammation, induction of autophagy, and apoptosis, which obstructed male gonadal development and led to reproductive dysfunction in humans and animals [110,111]. Table 3 Shows studies on arsenic-induced reproductive toxicity in animals. Arsenic has been shown to damage the histology of various tissues, such as the liver, brain, and kidney [112,113]. Notably, arsenic exposure during development significantly altered tight junctions' proteins, leading to an increase in blood-brain barrier permeability [114]. As a result, the age-dependent inhibition of the PI3K/Akt/mTOR signalling pathway may contribute to the induction of autophagy and the facilitation of arsenic transfer through the cerebellum's cerebral cortex and hippocampus leaky blood-brain barrier [115]. Recent studies from the Ommati research group also shed some light on hypothalamic-pituitarygonadal (HPG) axis disruption and subsequent toxicity in mice and its continued impact on offspring as well (refer to Figure 3) [37,41,43,116,117]. Similarly, the blood-testis barrier (BTB) is one of the most impermeable blood-tissue barriers in mammals. It divides the seminiferous epithelium into basal (intraluminal) and apical (intraluminal) compartments. Meiosis I and II, spermiogenesis, and spermiation all occur in a specialised microenvironment behind the BTB in the apical compartment, whereas spermatogonial renewal and differentiation, as well as cell cycle progression up to the preleptotene spermatocyte stage, occurs outside the BTB in the basal compartment of the epithelium. However, the BTB is not a static ultrastructure. Instead, it undergoes extensive remodelling during stage VIII of the seminiferous epithelial cycle of spermatogenesis to allow preleptotene spermatocytes to pass through the BTB. However, the BTB's immunological barrier cannot be compromised, even temporarily, during the epithelial cycle in order to prevent the production of antibodies against meiotic and postmeiotic germ cells. Adhesion protein complexes (e.g., occludin-ZO-1, N-cadherin-catenin, claudin-5-ZO-1), steroids (e.g., testosterone, estradiol-17), nonreceptor protein kinases (e.g., focal adhesion kinase, c-Src, c-Yes), polarity proteins induce testicular damage via their initial actions at the BTB, resulting in germ-cell loss, reduced sperm count, and male infertility or subfertility. Metallothioneins [cysteine-rich low molecular weight metal-binding proteins localised to the membrane of the Golgi apparatus that protect cells from cytotoxicity of essential heavy metals (such as zinc, selenium, and copper) and non-essential heavy metals (such as arsenic, mercury, silver, and cadmium) by binding to these metals via the thiol groups] are largely responsible for heavy metal accumulation in the body. As a result, significant and harmful amounts of heavy metal accumulation can accumulate in a person over time, exceeding the capacity of metallothioneins in the process [118][119][120][121][122][123].
Testicular histopathological investigations showed that sodium arsenite decreased seminiferous tubule diameter in Wistar rats [16] and outbred Institute of Cancer Research (ICR) mice led to a significant decrease in the lumen in the arsenic-treated group compared to the control group [124]. Vacuolisation, acidophilic cells, and epithelial degeneration were associated with increased inflammatory cytokines in male rats exposed to sodium arsenite through drinking water [125]. Moreover, sodium arsenite exhibits severe damage to the testicular structure and elevated cleaved caspase 3 (CC3) which is an apoptotic marker in the cluster differentiation 1 (CD1) mouse testes cell line [126]. Increased expression of CC3 also indicates increased apoptosis. Together, sodium arsenite and arsenate at a concentration of 0.01 and 10 mg/L in drinking water showed a decrease in catalase activity and vacuolisation of seminiferous tubules in rats [127]. Arsenic exposure inhibited the spermatogenesis process and decreased the mobility and viability of sperm [128]. An increase in apoptotic spermatozoa has been observed with arsenic exposure, the central mechanism involved in arsenic-induced decreased sperm count [129]. Sodium arsenite exposure at a concentration of 10 mg/L through drinking water for eight weeks exhibited a decrease in sperm counts and enhanced sperm head abnormalities, which led to an increase in the infertility risk and pre-implantation loss in Wistar rats [130]. Moreover, in mice, arsenic trioxide at doses of 0.3 and 3 mg/kg s.c. for 35 days reduced the number of spermatozoa, increased epithelial aberration and exfoliation of germ cells in the tubule lumen, and altered the nucleus/cytoplasm ratio of Leydig cells [131]. In mouse testes, arsenic trioxide at concentrations of 0.2, 2, and 20 ppm in drinking water for six months impaired sperm motility and affected the ultra-structure of the acrosome structure and sperm tail by downregulating the protein expression of DPY19L2, AKAP3, CFAP44, and SPAG16 [132]. Similarly, arsenic trioxide inhibited the expression of the DDX25 and CRM1 mRNAs, as well as the downstream proteins HMG2, PGK2, and H4 necessary for spermatogenesis in mice [133]. Oral exposure to arsenic trioxide at doses of 0.3 and 30 µg/kg for 15 days resulted in a dose-dependent frequency of sperm production in mice with aberrant head morphology [134]. Diabetic rats exposed to sodium arsenite at a concentration of 10 mg/L in drinking water for 40 days caused a drop in serum testosterone, sperm counts, motility, morphology, and acrosomal and plasma membrane structure [135]. Decreases in sperm count and viability with increased arsenic accumulation, lipid peroxidation, and protein carbonylation in the testes have been observed by administration of sodium arsenate at a concentration of 10, 25, 50, 100, and 200 ppm for 40 days through drinking water in mice [136]. Rats exposed to sodium arsenite at doses of 1, 5, and 25 mg/L through drinking water for 6 months showed compromised sperm counts and motility, and testosterone and altered 19 proteins related to reproduction such as Vdac3, Prkaca, Hspa41, Spaca1, Ma1b, Gpx4, Safb1, Trim28, Rbp1, Hsd11b1, Mapk3, Gpd2, Ace, Hspa11, Dnaja1, Ybx3, Smcp, Nasp, and Cabs1 were altered [104].

Pre-Natal Arsenic Exposure Induced Male Reproductive Toxicity in Animals
The growing evidence suggests that parental and/or pre-natal arsenic exposure to animals resulted in post-natal developmental toxicity. In-utero exposure to sodium arsenite from embryonic day-10 to day-18 at a concentration of 10 ppb induced an increase in leptin levels, and at 42.5 ppm reduced the litter size compared to control mice [137]. Chronic As trioxide exposure to parental male at a dose of 1 mg/L showed genotoxic damage in F0-F3, altered methylation patterns, changes in reproductive parameters, morphological damage in the ovaries (F0 and F1) and testicles (F1-F3), and compromised sperm quality (F0-F3, except F2) [138]. Exposure of sodium arsenite at the dose of 10 mg/L in drinking water to pregnant females from GD1-21 affected body weight and initial sexual development in male pups and relative anogenital distance also showed changes in the expression of SOD1, SOD2, CAT, and GSTK1 gene in male pup rat [139]. Administration of sodium arsenite at a concentration of 10 mg/L showed lower sperm production, sperm count, motility, and quality in the epididymis of rats [140]. Daily sperm production and the number of spermatids in the rat epididymis were reduced after oral treatment of sodium arsenite at doses of 0.01 and 10 mg/L for 56 days [141]. Lead at the dose of 819 mg/L was exposed to pregnant rats through drinking water until weaning, followed by the exposure of arsenic to male offspring at a dose of 2.3 mg/L, which showed a decrease in daily sperm production, relative weights of testes, epididymis, seminiferous tubules, and prostate, and decreased the activity of 3β-HSD and 17β-HSD in male offspring [142]. Sodium fluoride and sodium arsenite at 100 mg/L and 50 mg/L, respectively, via drinking water during the pre-pregnancy period, decreased the testicular weights, serum FSH, LH, and testosterone levels, and increased Beclin1 and LC3 expressions in Sprague Dawley (SD) rats' testes [143].

Pre-Natal Arsenic Exposure Induced Male Reproductive Toxicity in Animals
The growing evidence suggests that parental and/or pre-natal arsenic exposure to animals resulted in post-natal developmental toxicity. In-utero exposure to sodium arsenite from embryonic day-10 to day-18 at a concentration of 10 ppb induced an increase in leptin levels, and at 42.5 ppm reduced the litter size compared to control mice [137]. Chronic As trioxide exposure to parental male at a dose of 1 mg/L showed genotoxic damage in F0-F3, altered methylation patterns, changes in reproductive parameters, morphological damage in the ovaries (F0 and F1) and testicles (F1-F3), and compromised sperm quality (F0-F3, except F2) [138]. Exposure of sodium arsenite at the dose of 10 mg/L in drinking water to pregnant females from GD1-21 affected body weight and initial sexual development in male pups and relative anogenital distance also showed changes in the expression of SOD1, SOD2, CAT, and GSTK1 gene in male pup rat [139]. Administration of sodium arsenite at a concentration of 10 mg/L showed lower sperm production, sperm count, motility, and quality in the epididymis of rats [140]. Daily sperm production and the number of spermatids in the rat epididymis were reduced after oral treatment of sodium arsenite at doses of 0.01 and 10 mg/L for 56 days [141]. Lead at the dose of 819 mg/L was exposed to pregnant rats through drinking water until weaning, followed by the exposure of arsenic to male offspring at a dose of 2.3 mg/L, which showed a decrease in daily sperm production, relative weights of testes, epididymis, seminiferous tubules, and

Mechanisms of Arsenic-Induced Male Reproductive Toxicity in Animals
The major toxicity mechanisms include inflammatory response, oxidative stress, autophagy, and apoptosis.

Arsenic-Induced Oxidative Stress
Being a metalloid, arsenic showed molecular toxicity by oxidative stress [155,156]. Arsenic induces the generation of free radicals and diminishes the activity of the antioxidant system in the body. In the Leydig and Sertoli cells of mice testes, exposure to sodium arsenite at concentrations of 50 and 1000 ppb for 24, 48, and 72 h caused cytotoxicity and impaired antioxidant systems [144]. In vitro cell cultures of rodent testes and epididymis treated with sodium arsenite at concentrations of 1, 10, 50, and 100 µM for 2 and 24 h increased ROS, TBARS, and sperm DNA damage, decreased catalase, peroxidase, and superoxide dismutase, and decreased serum testosterone [157]. Sodium arsenite at a dose of 10 mg/L through drinking water was shown to induce overexpression of SOD1, CAT, GSTK1, and MT1 in the testes and epididymis of rats [148].

Arsenic-Induced Apoptosis
Reduced sperm counts, increased caspase-3 activity, increased TUNEL-positive cells, changes in mRNA levels of Bax and Bcl-2 decreased serum testosterone levels, and downregulated expression of steroidogenic genes (LHR, StAR, and ABP) were observed in mice exposed to sodium arsenite and sulphur dioxide at doses of 5 mg/L and 5 mg/m 3 , respectively, through double distilled water for 60 days oral administration [158]. In mice, arsenic trioxide and antimony at doses of 4 and 15 mg/kg through intragastric administration for two months resulted in lower serum testosterone levels, fewer spermatogonia and sperm counts, and lower T-AOC, SOD, and MsrB 1 levels, as well as increased Beclin-1, Atg-5, LC3B/LC3A, caspase-8, Cytc, cleaved caspase-3, and p53 [159]. Figure 4 shows arsenic-induced apoptosis in the testis.

Arsenic-Induced Inflammation
Chickens exposed to copper sulphate and arsenic trioxide at the doses of 300 and 30 mg/kg through the feed for 4, 8, and 12 weeks showed an increase in mRNA levels of proinflammatory cytokines and inflammatory factors. They showed an increase in mRNA

Arsenic-Induced Autophagy
Autophagy is a major cellular mechanism that allows cells to break down and reuse old cell parts, allowing them to operate more efficiently. Several studies have shown that arsenic-induced disruption in autophagy mechanisms causes a variety of diseases such as cancer, metabolic disorders, and reproductive toxicity [160,161]. Sodium arsenite exposure at concentrations of 3, 6, and 9 µM for 24 h resulted in the accumulation of autophagosomes with the upregulation of LC3β, Atg7, Beclin-1, and Vps34 autophagic markers expression in mouse testes Leydig tumour cell lines (MLTC-1) [152]. In the GC-1 spermatogonial (spg) cell line, arsenic trioxide at concentrations of 10 and 20 µM resulted in a drop in GSH and an increase in Malondialdehyde (MDA) levels, as well as elevation of ATG3, p62, LC3I, and LC3II mRNA expression, indicating mitochondrial dysfunction [154]. From five weeks before mating of parental mice and post-natal days up to adulthood, As trioxide exposure at concentrations of 0.2, 2, and 20 ppm in distilled water resulted in an increase in PI3K, Atg5, Atg12 gene expression and Beclin-1, LC-3I, II, and P62 protein expression in HPG axis tissues in F1 males [41]. In the testes of chickens, As trioxide at doses of 0.625, 1.25, and 2.5 mg/kg body weight for 30, 60, and 90 days elevated oxidative stress, exacerbated LC3-II, dynein, Beclin-1, ATG5, and ATG4B mediated autophagy, and triggered apoptosis [151]. Exposure of sodium arsenite to MLTC-1 cell lines at concentrations of 1, 2, and 4 mg/L for 48 h resulted in downregulation of Star, P450scc, P45-c17, and 17β-HSD genes and in contrast, increased mRNA and protein expression of 3β-HSD was observed [153]. Upregulation of H3K9me3 methyltransferase, Suv39h1, and downregulation of Jmjd2a demethylase were observed by sodium arsenite exposure at a dose of 1, 5, and 25 mg/L through drinking water for 6 months which led to steroidogenic gene repressions such as Lhr, StAR, P450scc, 3β-HSD, 17β-HSD, Cyp17a1, and Arom [147]. Additionally, sodium arsenite at the concentration of 5 and 50 ppm for 6 months reduced LHR, StAR, 3β-HSD, and 17β-HSD expressions and downregulated StAR, 17β-HSD, and Ddx3y mRNA levels in mice testes [145].

Arsenic-Induced Inflammation
Chickens exposed to copper sulphate and arsenic trioxide at the doses of 300 and 30 mg/kg through the feed for 4, 8, and 12 weeks showed an increase in mRNA levels of proinflammatory cytokines and inflammatory factors. They showed an increase in mRNA and protein levels of Hsp60, Hsp70, and Hsp90 as a protective effect from inflammatory damage [150]. NF-kβ, TNF-α, i-NOS, COX-2, and PTGEs mRNA overexpression were observed in chickens at dietary doses of 7.5, 15, and 30 mg/kg for 30, 60, and 90 days, whereas Hsp70 and Hsp90 mRNA expressions were also raised as a cell defence mechanism against As exposure [149]. Ferroptosis has been reported as one of the toxicity pathways of arsenic [162]. Sodium arsenite exposure through drinking water induced ferroptosis signalling in the testis of mice and GC-2 spg cell lines at concentrations of 0.5, 5, and 50 ppm for six months [163].

Effects of Prenatal Exposure to Arsenic in Humans
A study on 1390 pregnant women in Wuhan, China who were exposed to As revealed a reduction in birth weight, birth length, and risk of SGA in newborns [164]. A case study conducted with Unexplained Recurrent Spontaneous Abortion (URSA) patients at Beijing Maternal and Child Health Care Hospital found an increased level of arsenic in the blood, which suggests that blood arsenic may increase the risk of URSA in women of childbearing age [165]. A cohort study of 205 pregnant women in Hanam province, Vietnam revealed that prenatal exposure to drinking water containing high levels of arsenic showed increased cord arsenic levels, 8-OHdG, 8-nitroguanine, DNA strand break, and MN frequency, illustrating the genotoxic effects of arsenic [157]. Low levels of arsenic exposure during pregnancy showed maternal and neonatal thyrotoxicity [166]. In-utero exposure to arsenic in 706 pregnant women has shown an increase in birth length, decreased head circumference, and reduced adiposity in infants. A cohort study in Mexico City revealed transplacental arsenic exposure had shown an increased risk of SGA and large-forgestational-age (LGA) and enhanced maternal arsenic blood levels [167]. Systematic review and metanalysis of maternal arsenic exposure showed a decrease in the birth weight, head circumference, and birth length in which gestational exposure increased hypomethylated cytosines in active retrotransposons long interspersed nuclear elements (LINEs) and long terminal repeat (LTRs) [168].

Effects of Arsenic on the Male Reproductive System in Humans
A case-control study reported a positive correlation between urinary arsenic species with Unexplained Male Infertility (UMI) and a decrease in methylation of arsenic in 101 patients. Exposure to arsenic increased very low birth weight (VLBW) and preterm birth (PTB) in the people of Ohio, USA [169]. Prenatal exposure to sodium arsenite at a concentration of 85 ppm from 8 to 18 days showed transgenerational inheritance of impaired spermatogenesis phenotyping involving a decrease in the methylation status of Igf2, DMR2, and H19 DMR with a relative increase in mRNA and abnormal expression of Igf2 and H19 [170]. Arsenic exposure in 452 males was associated with increased urinary hormone excretion [171]. A cohort study of 127 male subjects in the hospitals of Nanjing Medical University, China showed the presence of urinary arsenic levels of inorganic arsenic (iAs), MMA, DMA, and arsenobetaine (AsB) which were correlated with male infertility risk [172]. Combined heavy metal exposure of arsenic and lead and cadmium has shown increased 8-OHdG, 8-isoPGF2α, and HNE-MA, which illustrated that higher urinary As, cadmium, and lead levels were associated with increased oxidative stress leading to alteration in semen quality [173]. A cohort study of 96 subjects in China revealed elevated levels of creatinine, arsenobetaine, DMA, MMA, arsenite, and arsenate were associated with poor semen quality [17]. In NJMU hospitals, a positive correlation was found between environmental arsenic exposure and male sexual dysfunction [171,172]. In conclusion, these findings suggest that As plays a critical role as a toxicant in the dysfunctions of male reproductive toxicity. The overall effect of arsenic on male reproductive cells is depicted in Figure 5.

Ameliorating Agents for Arsenic Toxicity
Phytonutrients are plant-produced natural substances or compounds. They have health-promoting bioactive effects via enhancing immunity and exerting antioxidant characteristics (shown in Table 4). The major molecular mechanisms involved in arsenic toxicity were oxidative stress, apoptosis, autophagy, and inflammation. Many phytonutrients showed protective benefits against arsenic-induced reproductive toxicity. A study using Chlorophytum borivilianum showed a reduction in As-induced lipid peroxidation, acid and alkaline phosphatase, and cholesterol in mouse Leydig and Sertoli cells [174]. Further, α-lipoic acid (LA) at a dose of 70 mg/kg was effective against arsenic-induced testicular toxicity, and results showed that LA decreased the mRNA expression of caspase-3 [175]. A total of 30 days of treatment with the formulated high-protein diet (FHPD), containing 15% casein and 7% pea protein in arsenic-exposed rats, inhibited lipid peroxidation, and the findings inferred that FHPD may have chelation properties and improve urinary arsenic excretion by increasing methylation [176]. The oral administration of Pulsatilla nigricans at a dose of 35 mg/kg for 90 days increased sperm maturation by increasing sorbitol dehydrogenase levels, as well as increasing GSH, SOD, and CAT levels and decreasing LDH and γ-GT levels in the testis [177]. The activation of NF-kβ and expression of i-NOS, COX-2, TNF-α, and IL after As exposure was inhibited by giving ellagic acid at doses of 10 and 30 mg/kg orally for 14 days in rat testis [178]. The expressions of Nfe2l2, StAR, and Ppargc1a, sperm morphology, and antioxidant levels in mice were restored after 40 days of oral administration of ellagic acid and ferulic acid at doses of 50 mg/kg each [179]. Lutein treatment at a dose of 40 mg/kg given orally for five weeks increased mRNA expression of Nrf-2 downstream genes (HO-1, GST, and NQO1) in response to As exposure [180]. Proanthocyanidin, grape seed extract, given orally at doses of 100 and 200 mg/kg for 5 weeks, boosted T-AOC, Nrf-2 expression, GSH, and SOD activity, while decreasing MDA and 8-OHdG levels [181]. Green tea component, epigallocatechin-3-gallate (EGCG) at a dose of 20 mg/kg intraperitoneally for 40 days restored

Ameliorating Agents for Arsenic Toxicity
Phytonutrients are plant-produced natural substances or compounds. They have health-promoting bioactive effects via enhancing immunity and exerting antioxidant characteristics (shown in Table 4). The major molecular mechanisms involved in arsenic toxicity were oxidative stress, apoptosis, autophagy, and inflammation. Many phytonutrients showed protective benefits against arsenic-induced reproductive toxicity. A study using Chlorophytum borivilianum showed a reduction in As-induced lipid peroxidation, acid and alkaline phosphatase, and cholesterol in mouse Leydig and Sertoli cells [174]. Further, α-lipoic acid (LA) at a dose of 70 mg/kg was effective against arsenic-induced testicular toxicity, and results showed that LA decreased the mRNA expression of caspase-3 [175]. A total of 30 days of treatment with the formulated high-protein diet (FHPD), containing 15% casein and 7% pea protein in arsenic-exposed rats, inhibited lipid peroxidation, and the findings inferred that FHPD may have chelation properties and improve urinary arsenic excretion by increasing methylation [176]. The oral administration of Pulsatilla nigricans at a dose of 35 mg/kg for 90 days increased sperm maturation by increasing sorbitol dehydrogenase levels, as well as increasing GSH, SOD, and CAT levels and decreasing LDH and γ-GT levels in the testis [177]. The activation of NF-kβ and expression of i-NOS, COX-2, TNF-α, and IL after As exposure was inhibited by giving ellagic acid at doses of 10 and 30 mg/kg orally for 14 days in rat testis [178]. The expressions of Nfe2l2, StAR, and Ppargc1a, sperm morphology, and antioxidant levels in mice were restored after 40 days of oral administration of ellagic acid and ferulic acid at doses of 50 mg/kg each [179]. Lutein treatment at a dose of 40 mg/kg given orally for five weeks increased mRNA expression of Nrf-2 downstream genes (HO-1, GST, and NQO1) in response to As exposure [180]. Proanthocyanidin, grape seed extract, given orally at doses of 100 and 200 mg/kg for 5 weeks, boosted T-AOC, Nrf-2 expression, GSH, and SOD activity, while decreasing MDA and 8-OHdG levels [181]. Green tea component, epigallocatechin-3-gallate (EGCG) at a dose of 20 mg/kg intraperitoneally for 40 days restored sperm kinetic characteristics, structural membrane integrity (SMI), and functional membrane integrity (FMI). It potentiated the activity of the Nrf-2 pathway and the production of different antioxidants [182]. Treatment with N-Acetyl Cysteine (NAC) at a concentration of 40 ppm for five weeks improved sperm motility, morphology, and weight of seminal vesicles. It boosted the GSH concentration and activity by acting as a precursor for GSH [183]. Further, NAC given intraperitoneally at a dose of 75 mg/kg for 40 days enhanced sperm parameters, 3β-HSD, 17β-HSD, and SOD and Catalase activities against As exposure by chelating As [184]. Polydatin treatment at doses of 50, 100, and 200 mg/kg for 60 days with oral administration restored As-induced sperm destruction by raising SOD and CAT levels in the testicular tissue of rats [185]. Melatonin at a dose of 25 mg/kg for 30 days repaired As-induced damage to the mean seminiferous tubular diameter (MSTD), mean testicular biopsy scores (MTBS), and proliferating cell nuclear antigen (PCNA) in rats [182]. Pistia stratiotes treatment at the dose of 100 mg/kg for 14 days of oral administration had shown protective action against As-induced sperm damage by restoring sperm motility, viability, count, and semen volume in rats [186]. Oral administration of chlorogenic acid at doses of 100 and 200 mg/kg for four weeks in the testicular tissue of mice exhibited antioxidant, anti-inflammatory, anti-apoptotic, and Nrf-2 activation against As toxic effects [185]. Oral therapy with selenium and diphenyl diselenide (DPDS) at a dose of 2.5 mg/kg for 45 days reduced inflammation, myeloperoxidase, NO, TNF-α, and IL-1 activity in the testes and epididymis of As-exposed rats [187]. Sodium arsenite-induced reproductive damage in hamsters was reversed by αtocopherol succinate (α-TOS) and sodium selenite (SS) at a dose of 6 mg/kg and 0.025 mg/kg from the 1st day of gestation till delivery showed decreased teratogenic effects. In contrast, SS was shown to increase the methylation process, whereas α-TOS enhanced antioxidant activity [57].  Decreased teratogenic effects. SS increased methylation process and α-TOS enhanced antioxidant activity. [198]

Other Phytonutrients That Promote Male Fertility
Coenzyme-Q10 is a naturally occurring hydrophobic molecule with potent antioxidant properties that have also been found to inhibit the generation of TNF-α, NO, and NF-Kβ as well as the activation of apoptosis in rats when given at a dose of 10 mg/kg intraperitoneally for five days [189,199]. Oral administration of quercetin at a dose of 50 mg/kg for 15 days improved serum testosterone levels, restored testicular architecture, and reduced TUNEL-positive cells [191], whereas exposure for 49 days showed powerful chelating and antioxidant properties to defend against lipid peroxidation, which recovered daily sperm production, sperm count, and reversed sperm DNA damage in the epididymis and testis of rats [192,193]. Oral administration of Withania sominifera at a dose of 100 mg/kg improved male libido by restoring spermatogenesis, sperm morphology, and testicular architecture, as well as boosting blood LH and testosterone levels [194]. Combined oral treatment of zinc chloride and vitamin C at doses of 20 mg/kg and 100 mg/kg restored sperm morphology, count, and seminiferous tubule diameter. Zinc works as a cofactor for SOD and can chelate As, demonstrating its antioxidant properties in rats, whereas Vitamin C at a dose of 200 mg/kg increased testosterone, FSH, and LH and improved histopathological lesions in Teddy goat bucks [195]. Oral administration of Alchornea cordifolia at 100 µg/kg for 30 days raised testosterone, FSH, spermatozoa count, and motility, as well as the expression of androgen receptor binding protein, and anti-apoptotic B-cell lymphoma-2 in the testis of rats [196]. Treatment with 200 mg/kg Vitamin E for 84 days increased spermatogenesis and restored blood LH, FSH, and testosterone levels [167], and improved semen quality in Teddy goat buck testicular tissue [200]. Oral administration of D-ribose-L-Cysteine at doses of 10 and 30 mg/kg over 28 days restored sperm count, motility, viability, LH, FSH, testosterone, and CAT, SOD, and GSH activities in rat testes [197]. In a recent study, nano vitamin C (NVC) was found to ameliorate arsenic-induced changes in testicular and sperm parameters. Significant increases in GPx, SOD, and CAT, as well as elevated serum levels of LH, FSH, and testosterone, were found in arsenic-treated male rats receiving 200 mg/kg of NVC [201]. Similarly, broccoli extract (300 mg/kg) treatment resulted in a significant reduction in oxidative stress and a rise in SOD, GPx, and total antioxidant capacity (TAC); nevertheless, a decrease in malondialdehyde (MDA) content was seen in the group compared to the As group. These results suggest that broccoli extracts are highly effective at reducing liver and kidney damage as well as improving haematological and biochemical variables in arsenic poisoning circumstances [202]. These findings clearly suggest that phytonutrients play a key role in combating arsenic-induced toxicity. However, further research needs to be conducted to explore the untapped potential of phytonutrients in ameliorating arsenic chronic toxicity.

Conclusions and Future Directions
Arsenic is a widespread metalloid that exerts a detrimental effect on male reproductive health in both animals and humans. Major sources of arsenic contamination include drinking water, food, and industrial waste. Arsenic impaired sperm quality, decreased sperm count, sperm viability, induced spermatozoa apoptosis, and damaged testicular and epididymal tissues and sperm DNA. Arsenic exposure also affects testosterone levels by impairing FSH and LH levels, affecting spermatogenesis, and producing male sterility in both animals and humans. Additionally, arsenic has shown transplacental developmental toxicity, which increases the chance of SGA and causes fetal abnormalities such as lower birth weight and preterm birth. However, the reproductive toxicity of arsenic is poorly understood, and the molecular mechanisms of arsenic-induced male reproductive toxicity remain unclear. Inflammatory response, oxidative stress, autophagy, and apoptosis are some of the possible arsenic-mediated toxicity pathways. On the other hand, phytonutrients have an essential protective function against arsenic-induced male reproductive toxicity. Phytonutrients, plant-based bioactive components, improve male fertility by boosting immunity and exerting antioxidant properties that diminish the oxidative and inflammatory stress generated by arsenic in reproductive cells. However, future research is needed to identify more phytoconstituents and understand their molecular mechanisms to completely mitigate and/or reverse the deleterious effects of arsenic poisoning. Furthermore, governments must take stringent measures to reduce arsenic levels in drinking and groundwater, food, and industrial effluents, thereby lowering human and animal exposure levels.

Conflicts of Interest:
The authors declare no conflict of interest.