Assessment of the Antigenotoxic Effects of Alginate and ZnO/Alginate–Nanocomposites Extracted from Brown Alga Fucus vesiculosus in Mice

Mitomycin C (MMC) is an alkylating chemotherapy drug that could induce DNA damage and genetic alteration. It has been used as a model mutagen for in vivo and in vitro studies. The current study aimed to evaluate the protective role of Zinc oxide alginate–nanocomposites (ZnO-Alg/NCMs) against MMC–induced genotoxicity in mice. Animals were treated as follows: the control group, the groups treated with Algin (400 mg/kg b.w), the groups treated with ZnO-Alg/NCMs (400 mg/kg b.w), the group treated with MMC, and the groups treated with MMC plus Algin or ZnO-Alg/NCMs. Pre-treatment with Algin and ZnO-Alg/NCMs was repeated for one or seven days. Zinc oxide alginate-nanocomposites (ZnO-Alg/NCMs) were synthesized with the aim of incorporating the intrinsic properties of their constituents as an antigenotoxic substance. In this study, alginate was extracted from the brown marine alga Fucus vesiculosus, Zinc oxide nanoparticles were synthesized by using water extract of the same alga, and loaded in alginate to synthesize ZnO-Alg/NCMs. ZnO-NPs and ZnO-Alg/NCMs were characterized by TEM, SEM, EDX, and Zeta potential. The obtained results confirmed that by TEM and SEM, ZnO-NPs are rod shaped which modified, when loaded in alginate matrix, into spherical shape. The physical stability of ZnO-Alg/NCMs was reported to be higher than ZnO-NPs due to the presence of more negative charges on ZnO-Alg/NCMs. The EDX analysis indicated that the amount of zinc was higher in ZnO-NPs than ZnO-Alg/NCMs. The in vivo results showed that treatment with MMC induced genotoxic disturbances. The combined treatment with Algin and ZnO-Alg/NCMs succeeded in inducing significant protection against MMC. It could be concluded that ZnO-Algin/NCMs is a promising candidate to protect against MMC–induced genotoxicity.


Introduction
Exposure to various endogenous and environmental agents such as metals, pesticides, and alkylating agents, along with therapeutic compounds including antitumor and antibiotics [1], could induce DNA damage, genetic alteration, endothelial dysfunction, and tissue injury. Mitomycin (MMC) is an alkylating DNA reactive antibiotic agent with anti-proliferative properties isolated from the Gram-positive actinobacteria Streptomyces caespitosus [2]. It has been used in the treatment of gastric, bladder, pancreatic, and colon cancer [3]. MMC was observed to induce genotoxic stress in human primary endothelial HCl for 1 h. Then, it was rinsed with 3% Na 2 CO 3 solution in relation to 1:30 (w/v) at 65 • C for 150 min, and then filtration was performed. Then, 10% HCl solution was amended in relation of 1:1 and allowed to form into white alginic acid to a pH of 2-3 within 30 min. Extreme pH was neutralized by the addition of dilute Na 2 CO 3 , with blending to ensure homogenization of the solution, until a pH of 7 was attained. After a pH of 7 was attained, Isopropyl alcohol (IPA) was added to obtain the Na alginate, and then dried at 60 • C in an electrical oven.

Alginate Yield
The Na-alginate yields that were obtained from the F. vesiculosus and P. pavonica extraction process were calculated, based on the weight of Na-alginate after drying, compared to the algal dry weight. The calculation of Na-alginate yield percentage was achieved using the following formula: D is the initial dry weight of algae used, A is the weight of alginate.

Alginates Characterisation
Fourier transforms infrared Spectroscopy (FTIR) is an important device that is used to identify the functional groups that relate to alginate. The previously extracted dry alginate was analyzed using FTIR spectroscopy and compared with standard alginic acid obtained from Sigma (Saudi Investment Group and Marketing) Ltd. Dry alginate samples were mixed with pellets of potassium bromide and the FTIR spectra were then investigated within the range of 400-4000 cm −1 using a "Termo Fisher Nicolete IS10, (Waltham, MA, USA) Spectrophotometer". 1 H NMR spectra were acquired in 0.1% w/v solutions of sodium alginate in DEMSO. Negative and positive-ion electrospray mass spectrometry (ESI-MS) was carried out on a Micromass Q-TOf and Q-TOf Ultima instruments (Waters, Manchester, UK). Nitrogen was used as the desolvation and nebulizer gas at a flow rate of 250 and 15 L/h, respectively. Source temperature was 80 • C and the desolvation temperature was 150 • C.

Green Synthesis of Zinc Oxide Nanoparticles
Zinc oxide nanoparticles were synthesized by using F. vesiculosus. Zinc acetate dehydrate (0.02 M) was supplemented to 40 mL of D water under constant stirring. Ten milliliters of algal aqueous extract (1 gm of alga added to 100 mL D water and boiled for 1 h) was added dropwise to this solution with stirring. After 10 min, NaOH (2.0 M) was added until reaching pH 12 and a pale white aqueous solution was obtained. The precipitate was filtered and washed twice with DD water followed by ethanol to obtain a solution free of contaminations. The ZnO-NPs were yielded after drying at 60 • C in a vacuum oven overnight [31,32].

Synthesis of ZnO/Alginates Nanocomposites (ZnO/Alg-NCMs)
A total of 2 gm of Sodium alginate were dissolved in 100 mL D water, at 40 • C, then 0.075 g ZnO-NPs was added, and the mixture was stirred for 2 h. After 2 h the solution was filtrated and dried at 60 • C for 2 days.

Characterizations of Zinc Oxide Nanoparticles and ZnO/Alg-NCMs
The characterization of ZnO/Alg-NCMs was performed by Fourier transform infrared spectroscopy (FTIR) using a (JASCO Europe S.r.l., Cremella, Italy), FT-IR 5300 spectrophotometer. Morphology and particle size of the nanoparticles were characterized via scanning electron microscopy JEOL JSM-6510/v, Tokyo, Japan (SEM), and transmission electron microscopy (JEOL JSM-6510/v, Tokyo, Japan), (TEM). The chemical contents of the nanoparticles were scanned by energy dispersive X-ray spectroscopy (EDS) using a Zeta potential analyser (Malvern Zeta size Nano-Zs90) Malvern United States.

Chromosome Abnormalities in Bone Marrow Cells
An investigation of chromosomal aberration was conducted in the bone marrow of mice injected intraperitoneally with colchicine, two hours before euthanasia [33]. Chromosome extractions from bone marrow cells were taken according to the method of Yosida and Amano [34]. One hundred well spread metaphases were analyzed per mouse and the metaphases with gaps, chromosome or chromatid breakage, and fragments were documented. Metaphases with gaps, chromosome or chromatid breakage, and fragments were recorded.

Chromosome Abnormalities in Spermatocytes
The diakinase-metaphase I cells collected from the spermatocytes were made following the air-drying technique of Evans et al. [35]. Slides were stained with 7% Giemsa stain in phosphate buffer (pH 6.8). A total of 500 well spread metaphases (5 animals/group) were scored for chromosome aberrations in germ cells. The types of aberrations in spermatocytes including XY univalent, autosomal univalent, and fragments were scored.

Sperm Morphology Assay
Sperm morphology assays were performed according to Rasgele [36]. Both of the cauda epididymides from each animal were dissected, excised, and minced in isotonic sodium citrate solution (2.2%). Smears were prepared and sperms were stained with Eosin Y [36]. At least 1000 sperms per animal (5 animals/group) were assessed for morphological abnormalities of the sperm shape. Sperm head and tail abnormalities were determined as having either normal or abnormal morphology.

Statistical Analysis
The genotoxicity analysis, the significance of each treatment, was assessed by t-test. All statements of significance were established on a probability of p ≤ 0.05.

Alginate Yields
Results in Figure 1 summarize that F. vesiculosus denoted larger amounts of alginate yields than P. pavonica. Both algae were collected from the beach at Jeddah, in the Red Sea in the same season. Alginate yields and quantity depended on various influences, for instance season of their collecting, type of algal age and brown algal species [37][38][39], age, harvesting methods [40], and extraction methods [41]. The alginates extracted from F. vesiculosus are more susceptible to high temperatures than alginates from any other species [10].

Fourier-Transform Infrared Spectroscopy (FTIR) Analysis
FTIR spectroscopy of the alginic acid (slandered) and FTIR analysis of alginate extracted from brown alga F. vesiculosus were investigated, Figure 2

Fourier-Transform Infrared Spectroscopy (FTIR) Analysis
FTIR spectroscopy of the alginic acid (slandered) and FTIR analysis of alginate extracted from brown alga F. vesiculosus were investigated, Figure 2  , and 528 cm −1 . The broad peak present at 3448 in alginic acid is shifted to 3449 cm −1 in alginate extracted from F. vesiculosus, and very narrow peaks at 2923 and 2855 present in alginic acid are shifted to 2922 and 2854 cm − 1, respectively, in alginate extracted from F. vesiculosus. The peak present at 1620 cm −1 in alginic acid shifted to 1625 cm −1 in alginate extracted from F. vesiculosus, however two others new peaks, 1653 and 1519 cm −1 , are present in the same regions in alginate extracted from F. vesiculosus. The wave number 3448 cm −1 indicates OH bonds in alginic acid [42], the peak present in wave number 2923 cm −1 denotes C-H stretching bands [43]. The wave number (2855) reveals C-H functional group [44]. The wave number 1620 cm −1 relates to the peak of carbonyl stretching [45]. The wave number 1418 cm −1 indicates deformation of C-H [42]. The band found at wave number 1126 cm −1 relates to n(C-O) [46]. The wave number present at 1095 cm −1 indicates PO2 symmetric (Phosphate II) [47]. The wave number 1031 cm −1 indicates C-O stretching [48]. The bands present in wave number 892 cm −1 relates to C-C and C-O [42]. The bands found at wave number 781 cm −1 relate to Out-of-plane bending vibrations [49], the wave number 725 cm −1 relates to Out-of-plane bending vibrations [49], and the peak present at wave number 675 cm −1 relates to CH out-of-plane bending vibrations [50].

Electrospray Mass Spectrometer
Alginate standard was used in ESI-MS analysis which indicated the great similarity to sample that isolated from brown alga.    These results are agreement with [56]. The 1 H NMR profile exposed the incidence of guluronic acid H-5 (GG-5G) that was determined at 4.282 ppm (signal III), as reported by Usoltseva et al. [57].   [56]. The 1 H NMR profile exposed the incidence of guluronic acid H-5 (GG-5G) that was determined at 4.282 ppm (signal III), as reported by Usoltseva et al. [57]. denote the relation of β-anomeric protons in the alginate sample in addition to the entrance of protons signals within 4.5 to 5.7 ppm. Signals recognized to the anomeric hydrogen of guluronic acid (G) at 5.66-5.70 pm and the H-5 of mannuronic acid at 4.56-5.03. These results are agreement with [56]. The 1 H NMR profile exposed the incidence of guluronic acid H-5 (GG-5G) that was determined at 4.282 ppm (signal III), as reported by Usoltseva et al. [57].

SEM and TEM Image
The shape and distribution of biosynthesis ZnO-NPs and ZnO/Alg-NCMs that were biosynthesized by brown alga F. vesiculosus, and alginates extracted from the same alga investigated by Scanning Electron Microscope (SEM). The rod shape was observed with ZnO-NPs and ZnO/Alg-NCMs Figure 5. The surface of ZnO/Alg-NCMs is observed on fibrous nanocomposite surfaces from sodium alginate and ZnO-NPs indicating that ZnO nano has been effectively modified on the surface of sodium alginate [58]. The results proved by the TEM image in Figure 5 demonstrated that ZnO-NPs are rod shaped, one diameter size ranges from 20 to 38 nm, meanwhile ZnO/Alg-NCMs are a spherical shape with size ranges from 7.75 to 17.75 and a high distribution. The results shown in Figure 5 demonstrate that ZnO-NPs was modified when embedded with alginate due to ZnO-NPs being rod shaped and ZnO/Alg-NCMs being a spherical shape. Trandafilović et al. [59] demonstrated micrographs of ZnO-dispersed NPS showing a huge number of spherical particles well spread in the alginate matrix. Nano-ZnO in the sodium alginate is well distributed [60]. Hamouda et al. [31,32] observed the green-synthesized ZnO-NPs by green alga Ulva fasciata had a rod shape.

Zeta Potential
The constancy of the attained nanomaterials was investigated by the zeta potential measurement [61]. Figure 6 reported the Zeta potential of the apparent surface charge of zinc oxide nanoparticles, and ZnO/Alg-NCMs bio-fabricated by brown alga F. vesiculosus. The results show the Zeta potential value was −1.91 and −3.78 of biosynthesis zinc oxide nanoparticles and ZnO/Alg-NCMs, respectively. The negative charge indicated that the highest physical stability of ZnO/Alg-NCMs was more than ZnO-NPs bio-synthesised by F. vesiculosus. Haider and Mehdi [62] reported the value of the negative charge of the zeta potential shows the efficacy of the capping compounds in stabilizing AgNPs by denoting more negative charges that reserve all the particles away from each other. The negative values explain the repulsion between the particles and, thereby, realization of more stability of the AgNPs structure eluding agglomeration in aqueous solutions [63]. The zeta potential value of nanocomposites is greater than the zeta potential of ZnO NPs synthesized intracellular with Lactobacillus plantarum [61]. with size ranges from 7.75 to 17.75 and a high distribution. The results shown in Figure 5 demonstrate that ZnO-NPs was modified when embedded with alginate due to ZnO-NPs being rod shaped and ZnO/Alg-NCMs being a spherical shape. Trandafilović et al. [59] demonstrated micrographs of ZnO-dispersed NPS showing a huge number of spherical particles well spread in the alginate matrix. Nano-ZnO in the sodium alginate is well distributed [60]. Hamouda et al. [31,32] observed the green-synthesized ZnO-NPs by green alga Ulva fasciata had a rod shape.

Zeta Potential
The constancy of the attained nanomaterials was investigated by the zeta potential measurement [61]. Figure 6 reported the Zeta potential of the apparent surface charge of

Energy Dispersive X-ray Measurements
The results in Figure 7 represent peaks of metals content of the zinc oxide nanoparticles and ZnO/Alg-NCMs bio-fabricated by the brown alga F. vesiculosus that were assessed by Energy dispersive X ray spectrophotometry. The five metals are C, O, Ca, Cu, and Zn with weight percentage 31.12, 28.34, 0.33, 2.22, and 37.98, respectively. Meanwhile seven metals are presented in case of ZnO/Alg-NCMs bio-fabricated by brown alga F. vesiculosus: C, O, Na, Ca, Cu, Zn, and Sb with weight percentage 28.78, 41.61, 4.44, 13.46, 2.78, 3.28, and 5.65, respectively. The results suggested that some metals, such as Ca and Na, present in alginates, were not present in ZnO-NPS, and also the weight percentage of carbon and oxygen are higher in ZnO/Alg-NCMs than those present in ZnO-NPS, due to the fact that carbon and oxygen are the main contents of alginate polymers. The existence of a sodium (Na) peak proves the presence of sodium alginate [64]. potential shows the efficacy of the capping compounds in stabilizing AgNPs by denoting more negative charges that reserve all the particles away from each other. The negative values explain the repulsion between the particles and, thereby, realization of more stability of the AgNPs structure eluding agglomeration in aqueous solutions [63]. The zeta potential value of nanocomposites is greater than the zeta potential of ZnO NPs synthesized intracellular with Lactobacillus plantarum [61].

Energy Dispersive X-ray Measurements
The results in Figure 7 represent peaks of metals content of the zinc oxide nanoparticles and ZnO/Alg-NCMs bio-fabricated by the brown alga F. vesiculosus that were assessed by Energy dispersive X ray spectrophotometry. The five metals are C, O, Ca, Cu, and Zn with weight percentage 31.12, 28.34, 0.33, 2.22, and 37.98, respectively. Meanwhile seven metals are presented in case of ZnO/Alg-NCMs bio-fabricated by brown alga F. vesiculosus: C, O, Na, Ca, Cu, Zn, and Sb with weight percentage 28.78, 41.61, 4.44, 13.46, 2.78, 3.28, and 5.65, respectively. The results suggested that some metals, such as Ca and Na, present in alginates, were not present in ZnO-NPS, and also the weight percentage of carbon and oxygen are higher in ZnO/Alg-NCMs than those present in ZnO-NPS, due to the     Table 2 shows the number and percentage of the chromosomal aberrations induced in control and treated animals. The mean percentage of metaphases with chromosomal aberrations reached 24.6% (p < 0.01) 24 h after intraperitoneal injection with Mitomycin C. The percentages of aberrant cells in animals treated with alginate (Algin) and ZnO/Alg-NCMs were statistically not significant compared to the control group. Treatment with alginate and ZnO/Alg-NCMs for 7 days caused a highly significant (p < 0.01) reduction in the percentage of chromosomal abnormalities induced by MMC. The percentage of reduction reached 33.3 and 56.9% after treatment with alginate and ZnO/Alg-NCMs, respectively, Table 2. Additionally, Table 2 illustrates the protective effect of alginate and ZnO/Alg-NCMs in reducing the different types of aberrations, Figure 8 demonstrates different types of chromosomal aberrations observed in bone marrow cells.

Chromosome Abnormalities in Spermatocytes
A highly significant (p < 0.01) elevation in spermatocytes' chromosomal aberration was observed after MMC treatment which reached 17.6 ± 0.44%. Treatment with Alginate and ZnO/Alg-NCMs for 7 days ameliorated these genotoxicities and reduced the aberrations statistically in a highly significant (p < 0.001) manner (Table 3). XY-univalents and autosomal univalents were the main aberrations observed in diakinase metaphase I cells Figure 9.

Sperm Shape Abnormalities
As shown in Table 4, MMC induced a statistically highly significant (p < 0.01) percentage of sperm abnormalities in male mice which reached 14.2%. The dominant abnormalities found were amorphous, triangular, and without hook heads or coiled tail. Simultaneous treatment of mice with Alginate and ZnO/Alg-NCMs reduced the percentage of sperm abnormalities. It reached 8.64 and 7.16% (p < 0.01), respectively. The percentage of reduction reached 39.15 and 49.57% after treatment of mice with Alginate and ZnO/Alg-NCMs, respectively, Figure 10. The total number of scored metaphases is 500 (5 animals/group); Frag. = fragment, Rt. = Robertsonian translocation. a : significant at 0.01 level (t-test) compared to control (non-treated). b : significant at 0.01 level (t-test) compared to treatment. The total number of scored metaphases is 500 (5 animals/group); a : significant at 0.01 level (t-test) compared to control (non-treated); b : significant at 0.01 level (t-test) compared to treatment.

Sperm Shape Abnormalities
As shown in Table 4, MMC induced a statistically highly significant (p < 0.01) percentage of s 14.2%. The dominant abnormalities found were amorphous, triangular, and without hook head with Alginate and ZnO/Alg-NCMs reduced the percentage of sperm abnormalities. It reached 8.6 age of reduction reached 39.15 and 49.57% after treatment of mice with Alginate and ZnO/Alg-N Chromosome abnormalities in bone marrow cells were employed as a cytogenetic end point in genetic risk assessment. One of the most significant bioassays for assessing the genotoxicity of various substances is in vivo chromosomal abnormalities [65,66]. In the present study, MMC induced a high and statistically significant percentage of chromosome aberrations in mouse bone marrow cells. These finding are in agreement with those observed by Khalil et al. [67] who documented that MMC induced chromosomal aberrations in Swiss albino mice. MMC has been shown to cause mitotic delay [68] which could affect the yield of chemically induced abnormalities [69]. MMC is an alkylating agent that induces DNA damage leading to an arrest of replication and transcription as well as apoptosis [70]. The activated metabolite of MMC mitosene reacts via N-alkylation with 7-N-guanine nucleotide residues in DNA at the location of 5 -CpG-3 sequence that causes DNA crosslinking and, thus, inhibits DNA synthesis [71].   MMC induced a high and statistically significant percentage of chromosome abnormalities in diakinesis metaphase I mouse spermatocytes. The most common type of abnormality seen in mouse spermatocytes after treatment with MMC was the univalents. There are reports that the X and Y chromosomes occasionally separate to form univalents in mouse primary spermatocytes [72]. It is termed non-pairing of XY, or X-Y dissociation. Leonard and Linden [73] concluded that autosomal univalents resulting from asynapsis or desynapsis are rare in control animals, since chiasma formed at the diploten stage maintain pairing association until the end of metaphase. X and Y chromosomes do not have homologous segments and so, X-Y univalents are the most common aberration. In the first meiotic prophase, homologous chromosomes undergo pairing, chiasma formation and crossing over [74]. In bivalents, when the process of pairing fail to progress in a normal manner, the two homologs would persist as unassociated univalents which could undergo random separation at anaphase I, leading to aneuploidy in the following metaphase.
The present study demonstrated that the mean percentages of sperm shape abnormalities were significantly increased after MMC treatment. These results support those obtained by Kumari et al. [75]. Sperm shape abnormalities reflect changes in the DNA content [37]. Also, sperm head abnormalities are frequently taken as a typical test for monitoring the mutagenic potential for many chemicals [76]. Tail deformities were conveyed to decrease fertility in human and animals [77].
The results indicated that pre-treatment with alginate for 7 days significantly reduced chromosomal aberrations in somatic and germ cells. Alginate oligosaccharide was observed to decrease H 2 O 2 -induced oxidative stress [11]. Guo et al. [13] documented that pretreatment of mice with alginate oligosaccharide inhibited the oxidative stress process and were well protected against acute Doxorubicin cardiotoxicity. Oligosaccharides identified from brown algae were found to exhibit an anti-proliferative activity via induction of cell cycle arrest and apoptosis on human prostate cancer cells both in vitro and in vivo [17].
The present results showed that pre-treatment with ZnO-Alg/NCMs for 7 days significantly reduced chromosomal aberrations in somatic and spermatocytes and reduced the percentage of morphological sperm abnormalities. Bionanocomposites are a combination of biopolymers and inorganic materials, mainly from metal oxides like silver nitrate, titanium oxide, silicon oxide, and zinc oxide in nano dimension size. The incorporation of zinc oxide nanoparticles into sodium alginate possesses an antibacterial effect which inhibits the growth of various Gram-positive pathogens [25]. Nystrom and Bisrat, [78] showed that such a relationship between the specific surface dissolution rate and the solubility of equilibrium increased with a decrease in particle size. The hyperbolic relationship between particle size and surface-specific dissolution rate increases solubility [79] due to the larger surface area, which promotes dissolution [80]. Moreover, a decline in the particle size into nanoparticles enhances the solubility and bioavailability of the active ingredients [81].

Conclusions
The current study revealed F. vesiculosus denoted more alginate yields than P. pavonica. FT-IR, ESI-MS and 1 H NMR proved that the compound that was extracted from F. vesiculosus is alginate. TEM, SEM, EDX, and Zeta potential proved that ZnO/Alg-NCMs is spherical in shape, more negatively charged than ZnO-NPs, and the amount of zinc in ZnO/Alg-NCMs was lower than in the case of ZnO-NPs. The current study also revealed that MMC increased chromosomal abnormalities in bone marrow and spermatocytes and significantly elevated the percentage of sperm shape deformities. Oral administration of ZnO-Alig/NCMs was found to be safe and may be a potential candidate to ameliorate the detrimental effects of different chemical-induced oxidative damage and cytogenetic alterations.

Data Availability Statement:
The datasets spent and/or analyzed during this study are available from the corresponding author on reasonable request.