Protective effects of fucoidan against 4-nitroquinolin-1-oxide provoked genetic damage in mouse bone marrow cells

Fucoidan is a unique bioactive and dietary polymer enriched mainly in the cell wall matrix of the brown seaweeds. This present study was intended to reveal the antigenotoxicity effect of fucoidan on 4-nitroquinolin-1-oxide (4-NQO) induced genetics damage and apoptosis in mice bone marrow cells. The 4-NQO caused genetic damages in the form of chromosome/chromatic breakage was estimated by micronuclei assay whereas apoptosis by annexin-V FITC kit and DNA damage by comet assay kit. In addition, oxidative damage in terms of plasma lipid peroxidation (LPO) and 8-OHdG was also estimated. In the experimental regime, six groups with each in five either sex of mice were used. Fucoidan constituted (50,100,200 mg/kg bwt) by orally for 5 days consequently and on 6th day, 4-NQO was administered (7.5 mg/kg bwt) by i.p. The results clearly show that negative control (H₂O) and fucoidan alone constituted mice were not exhibited significant effect on LPO, genetic damages whereas positive control group (4-NQO 7.5 mg/kg bwt, i.p.) showed significant effect on genetic damage by showing increased level of LPO (6.25 vs 1.3 μM MDA), 8-OHdG (12 vs 4%), micronuclei about six-fold, 5-fold of comet, and 4-fold of apoptosis when compared with negative control, 11.6 ± 2.07, 5.00 ± 1.58, and 4.14 ± 0.65 respectively. Fucoidan pretreatment significantly protected the 4-NQO-induced genetic damage by 77% decreased level of micronuclei and 96% comet at dose of 200 mg/kg bwt over the positive control whereas LPO, 8-OHdG, and apoptosis were restored as equal to negative control. This study found as fucoidan possessing significant antigenotoxicity property by protecting 4-NQO-induced genetic damage in mice bone marrow cells as dose dependent manner suggest as valuable food supplements and medicine for mankind from environmental toxicants.

Keywords: Antigenotoxicity; Fucoidan; LPO; 8-OHdG; Micronuclei; Comet; 4-NQO

Since ancient time, seaweeds have traditionally been utilized as supplement in functional foods, healthcare, and medicine from many parts of the East Asian country (Park et al. [32]; Hwang et al. [20]). Even though hydrocolloid polysaccharides such as agar, carageenan, and alginate enormously presented in most of the seaweeds, fucoidan is a unique bioactive and dietary polymer enriched mainly in the cell wall matrix of the brown seaweeds such as kombu, mozuku, mekabu, limumoui, bladderwrack, and wakame (Atashrazm et al. [8]; Kuznetsova et al. [24]), and some extend in marine invertebrates like sea urchin and sea cucumber (Kordjazi et al. [23]). In brown seaweeds, in addition to fucoidan, they are also included minerals, tannins, polyphenols, vitamins along with proteins, lipids, and carotene pigments (Eluvakkal et al. [15]; Moghadamtousi et al. [31]). Fucoidan is a sulfated polysaccharide predominantly constituted sulfated fucose along with minor components of glucuronic acid, xylose, and galactose (Ale et al. [2]; Arumugam et al. [6]) enfold with various biological activities such as anticoagulant, antiinflammatory, antiangiogenic, antiviral, antimetastatic, cytotoxicity, and antioxidants (Wang et al. [35]; Lee et al. [27]; Abdelkadder et al. [1]; Fitton et al. [16]). Consequently, its mode of action explored mainly through induction of cell cycle arrest, apoptosis, and immune system activation are primarily depend upon the rout of administration as well as the method of isolation from seaweeds (Kan et al. [21]; Kyung et al. [25]; Ale et al. [2]). There are studies reported antioxidant and antimutagenic properties in some edible brown seaweeds such as Laminaria digitata, Himanthalia elongata, Fucus serratus, Laminaria japonica, Hijikia fusiforme, Ascophyllum nodosum, Sargassum micracanthum, and Turbinaria conoides due to in the presence of fucoidan (Leite-Silva et al. [28]; Arumugam et al. [6]).

The center cores of genetic consequence such as DNA damage and apoptosis are mainly takes place and manifested by cytoplasmic shrinkage and chromatin condensation that facilitating the removal of cells without inflammation. Many research reports suggested that fucoidan-induced apoptosis through interacting with several components in the apoptotic pathway. Brown seaweed Undaria pinnatifida is most commonly used as algal seafood in Northeastern Asia and its fucoidan consumed up to 1000 mg/kg per day was safe in rodents that registered no toxicity in Sprague-Dawley rats observed by unaltered chromosomes structure/numbers when fed orally up to 2000 mg/kg per day (Chung et al. [12]). Similarly, other reports on mice model study clearly demonstrated the fucoidan as significantly preventing lipopolysaccharide (LPS) induced endotoxemia through suppressing LPS enhanced proinflammatory cytokines (TNFα and IL-6) and hypercoagulability (Kuznetsova et al. [24]). In another study, low molecular weight fucoidan of Laminaria japonica found as nontoxic observed in terms of chromosome aberration when fed up to 2000 mg/kg per day orally in mice and up to 5000 mg/mL constituted in Chinese hamster ovary cells as well, thus suggested for safe food supplement (Hwang et al. [20]). Fucoidan in okinawa and mozuku prepared from Cladosiphon okamuranus reported no significant toxicity in rats fed orally at a dose of 600 mg/kg per day (Gideon and Rengasamy [17]). Further, fucoidan of Fucus vesiculosus possessing various pharmacological properties exhibit significant hepatoprotective effect (200 mg/kg) by protecting tissues injury, hepatic cell death, and stellate cell death as well (Hong et al. [19]; Abdelkadde et al. [1]). In addition, presence of antigenotoxicity in F. vesiculosus aqueous extract was identified by proving activity against doxorubicin induced DNA damage in terms of chromosome aberrations and comet in human lymphocytes (Leite-Silva et al. [28]).

Concern about the carcinogenicity is a long standing issue that toxic potential was initially evaluated through genotoxicity test, most probably micronucleus assay and second most appropriate comet assay as well. However, most of works carried out on genotoxicity is based on in vitro assays without studying toxicant deposition, metabolism, and elimination (Chen et al. [11]). Therefore, in vivo testing methods considered as most indispensable to demonstrate the potential of genotoxin through genotoxicity test. Hence, micronuclei and comet assays are considered as most reliable methods of studying genotoxicity by either in vitro or in vivo by exposing various genotoxin (Tice et al. [34]; Silva Pereira et al. [33]). Thus, the genotoxic agents and their metabolites are able to bind with DNA and causes genetic damage in terms of chromosome breaks, micronuclei formation, and cell death. In addition, lipid peroxidation and oxidative DNA damage can also be observed due to genotoxic stress caused by free radicals generated either by genotoxin or its intermediates (Lemes et al. [29]). In fact, a few studies so far conducted both in vitro and in vivo in relation to antigenotoxicity characteristic of fucoidan, in this article, study made on antigenotoxicity effect of fucoidan against 4-NQO-induced genetic damage and apoptosis in mice bone marrow cells is presented.

Trevigen's comet assay kit (4250-050-K), annexin V-FITC assay kit (APOAF), fucoidan (F8315, ≥ 95), may-grunwald stain (205435), and 4-NQO (N8141, ≥ 98%) of Sigma-Aldrich, USA, were used for this study. Lipid peroxidation (MDA) assay kit (10009055), 8-hydroxy-2-deoxyguanosine (8-OHdG) EIA kit (589320), and phosphate buffered saline used were procured from Cayman Chemicals Company, USA, and Hi-media, Mumbai, India, respectively. All solvents used in this study were of analytical grade.

In the present study, protocol was accordingly tailored to the guidelines of University ethical committee. Animal care was pursued as per depiction of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. Both sexes of Swiss albino mice (20–25 g) used in this study reared under 12-h light/dark and 22 ± 2 °C. They were allowed free access to tap water and pelleted rodent diet orally.

In vivo micronucleus assay was carried out by the method described in Arumugam and Ramesh ([4]). Brown seaweed products of fucoidan on genotoxicity caused by 4-NQO was evaluated in mice of both sex by dividing into six groups with five each (Fig. 1). The group one was a negative control where mice fed only distilled water whereas in the second group animals were administered fucoidan 200 mg/kg animal weight by orally and third which was positive control received 7.5 mg 4-NQO /kg animal weight by i.p. Mice in other groups fed daily 50, 100, and 200 mg fucoidan/kg designated as fourth, fifth, and sixth group, respectively up to 5 days and 6th day was fed with 7.5 mg 4-NQO/kg animal weight. After 24 h, all mice slightly under anesthesia with diethylether and blood samples were collected from femoral vein by venipuncture into serum separation tubes. Subsequently, all the mice were sacrificed by cervical dislocation, and the bone marrow cells from the femur were obtained using a syringe (22G) having little amount of fetal calf serum (FCS) in to 1 mL FCS tube. Supernatant discarded after centrifugation and the cells were resuspended in a small volume of fresh FCS. A small drop of the suspension was transferred to a glass microscope slide and a smear was prepared. At least four smears was made from each animal and scored by staining with May-Grunwald. The antigenotoxicity of fucoidan was determined by counting for each animal (experimental/control) around 2500 polychromatic erythrocytes (with or without micronuclei) and a corresponding number of normal chromatic erythrocytes (NCEs) under a light microscope.

Graph: Fig. 1 Protective effect of fucoidan on 4-NQO-induced micronuclei in mice bone marrow cells (Micronucleated polychromatic erythrocytes [MnPCEs], body weight [bwt], 4-nitroquinoline-1-oxide [4-NQO] compared with control, fucoidan +4-NQO compared with 4-NQO, P ≤ 0.05)

Plasma separation tubes (EDTA) contain blood samples were centrifuged at 2500×g for 10 min at 4 °C. Then, the supernatant (200 μL) was transferred into fresh tubes and used it further for the estimation of lipid peroxidation (LPO) and oxidative DNA damage accordingly using assay kits (lipid peroxidation and 8-OHdG Assay Kit). For the lipid peroxidation, absorbance was recorded at 530 nm using a spectrophotometer whereas for 8-OHdG read at 420 nm by ELIS reader (Arumugam and Ramesh [5]).

In addition, 4-NQO-induced genetic damage was verified in terms of scoring comet by using Trevigen's comet assay kit with slight modification. The same micronuclei assay experimental mice were utilized one of their femur bones to extract bone marrow cells using syringe (22G) into phosphate buffer saline (PBS) for comet assay. Extracted bone marrow cells were washed twice with PBS by gentle aspiration. According to the protocol, molten LM agarose (at 37 °C) was mixed with bone marrow cells (1 × 105/mL) in 1:10 (v/v) ratio, and immediately 50 μL were pipetted onto comet slide precoated with 1% normal melting point agarose. Subsequently, the treated comet slide was kept at 4 °C for 10 min for better agarose adhesion. Then all the slides were transferred into ice-cold lysing solution for 60 min at 4 °C followed by immersing into freshly prepared alkaline unwinding solution (pH > 13: 200 mM NaOH, 1 mM EDTA in 50 mL of dH2O) for 1 h at 4 °C in the dark. Later on, the slides were placed in alkaline buffer (200 mM NaOH, 1 mM EDTA in 1 L of dH2O) in an electrophoresis chamber, and the gel was running with 21 V for 30 min and washed twice in dH2O followed by 70% ethanol. Slides were allowed to dry at room temperature and stored in desiccant. Silver staining was carried out and captured ~ 50 comet images per slide under light microscope that were analyzed in terms of head, tail, and tail moment (Anderson et al. [3]) using comet assay software.

Protective effect of fucoidan on 4-NQO induced apoptotic bone marrow cells were measured by according to the protocol of annexin-V FITC assay kit through flow cytometric (Arumugam and Ramesh [4]). Extracted bone marrow cells were washed twice with PBS and washed cell (1 × 106 cells/mL) were mixed with 500 μL of binding buffer (5 μL of annexin-V FITC and 5 μL of PI) and kept in the dark for 15 min at ambient temperature. Immediately, the stained cells were monitored under flow cytometer, and the values were recorded as a percentage of apoptotic cells by flowjo software.

The results of LPO, 8-OHdG, micronuclei, DNA damage, and apoptosis were presented as mean ± standard deviation for five mice of each group. Differences between groups were tested (P ≤ 0.05) by one-way ANOVA using SPSS Software version 16.0.

It is well known that genotoxicity test is conducted widely in mammalian system to examine the mutagenicity caused by various physical and chemical genotoxin. It is linked with human cancer as they cause the genetic damage in terms of DNA adducts/lesions which are significant step ahead in the process of mutagenesis/carcinogenesis (Basu [9]). Therefore, it is scientifically significant to develop drugs of plant origin which is safe and nontoxic to get rides of various diseases including cancer (De Flora and Ferguson [13]). The biological properties of fucoidan from brown seaweeds have been studied well (Lee et al. [27]; Fitton et al. [16]; Arumugam et al. [6]). However, antigenotoxic activity of fucoidan on 4-NQO induced micronucleated polychromatic erythrocytes (MnPCEs) in mice bone marrow cells was scanty. 4-NQO, a synthetic water-soluble carcinogen, is widely used as an oral carcinogen in murine models, and its toxicity depends upon the route of administration (Han et al. [18]). It enhanced the mutation frequency when given by the rout of intraperitoneally in mice. It produced genotoxicity in the form of micronuclei/chromosomal breakage though its metabolite, 4-hydroxyaminoquinoline 1-oxide (Ac-4-HAQO) and also induced apoptosis by forming DNA adducts at the N2, C8, and of N6 position (Kanojia and Vaidya [22]; Arumugam and Ramesh [4]). On the other side, lipid peroxidation and DNA strand breaks are takes place through oxidative stress caused 4-NQO by producing superoxide and hydroxyl radicals (Arumugam and Ramesh [4]; Downes et al. [14]). In the present study results reveal that 4-NQO-induced the frequency of MnPCEs (66.00 ± 5.70) six-fold higher than that of negative control, 11.60 ± 2.07 MnPCEs/2500 PCEs (Fig. 1). Fucoidan alone–treated group was exhibited no significant changes in the MnPCEs frequency (10.20 ± 1.92) whereas significantly reduced when different doses of fucoidan (50, 100, and 200 mg/kg) followed by 4-NQO (7.5 mg/kg) treatment although the MnPCEs frequency was significantly enhanced by 4-NQO received alone. Antigenotoxicity of fucoidan was found to be 20% at lower dose, 50 mg fucoidan/kg, and its MnPCEs frequency was 55.20 ± 1.92 over the negative control, 11.60 ± 2.07. But at higher dose, 200 mg fucoidan/kg, antigenotoxicity was 93%, and its MnPCEs frequency was 15.40 ± 1.14 over the negative control (11.60 ± 2.07). Whereas, moderate dose of 100 mg fucoidan/kg exhibited 66% antigenotoxicity, and its MnPCEs frequency was 30.0 ± 3.81 over the negative control, 11.60 ± 2.07. This observed results suggest that fucoidan exhibit antigenotoxicity, and the effect was increased with increasing dose (50 mg/kg to 200 mg/kg). Fucoidan effectively reduced the 4-NQO-induced MnPCEs frequency, and the reduction rat was 1.2, 2.2, and 4.3-fold high over the positive control (4-NQO 7.5 mg/kg, 66.00 ± 5.70) at dose of 50, 100, and 200 mg/kg, respectively (Fig. 1). This result further confirms that the antigenotoxic effect of fucoidan was dose dependent. To support, Chung et al. ([12]) reported by Ames test that fucoidan up to 500 μL/plate did not show mutagenicity beside 4-NQO-induced mutagenicity inhibition of 71% compared with control. Leite-Silva et al. ([28]) proved that aqueous extract of Fucus vesiculosus showing strong antigenotoxic effect against doxorubicin induced genetic damage, and the effect was mainly due to in the presence of sulfated-polysaccharide, fucoidan and it was further found that the extract treated animals did not exhibit any effect on chromosome aberrations/mitotic index.

4-NQO caused intracellular oxidative stress in terms of lipid peroxidation through generating over load of free radicals. Besides, it causes acute toxicity, mutagenicity, and tissue damage within 3 h of administration by stimulating prooxidant state in vivo. As the results, lipid peroxidation was ultimately reflected in form of MDA (Ashok Kumar et al. [7]). In fact, 4-NQO-induced oxidative stress was quantified in terms of elevated level of lipid peroxidation in mice. In this study result, group received 4-NQO alone significantly (P > 0.05) elevated the LPO level of 6.25 ± 0.49 μM MDA as ≥ 4.7-folds higher than that of control, but fucoidan alone–treated group was ≤ 1.33 ± 0.16 μM MDA (Fig. 2). Similarly, it was observed that 4-NQO significantly manifested the LPO level over the control that was quantified by LPO assay kit (Arumugam and Ramesh [5]). In contrast, fucoidan (50,100, and 200 mg/kg) followed by 4-NQO (7.5 mg/kg) constituted mice showed tremendous reduction of LPO level, 3.42 ± 0.15, 2.41 ± 0.12, and 1.45 ± 0.06 μM MDA respectively, when compare with 4-NQO alone–treated group, 6.25 ± 0.49 μM MDA. This results proved that fucoidan was effectively restored the 4-NQO-induced oxidative stress in terms of reducing the LPO level as well as the incidence of micronuclei up to about ≤ 6-folds over control. This observation is in conformity of exhibiting remarkable antioxidant potential of fucoidan assessed by decreasing of oxidative stress in terms of protecting genotoxin altered MDA and enzymatic antioxidants in rats (Abdelkadder et al. [1]). It is well-known fact that prolonged exposure of genotoxic/carcinogenic agents lead to the development of degenerative diseases such as cancer through the basis of reactive oxygen species, DNA damage, and mutation (Lima et al. [30]). It is already proved that 4-NQO causes oxidative DNA adduct through its intermediates, 4-HAQO, or by generating free radical on guanine (Kanojia and Vaidya [22]; Arumugam and Ramesh [5]) Therefore, in the present study, protective effect of fucoidan by 4-NQO-induced oxidative damage in mice was evaluated. Oxidative DNA damage was measured in the form of 8-hydroxydeoxyguanosine (8-OHdG) in mice plasma. The result shows that the level of 8-OHdG in control and fucoidan alone–treated mice exhibited ≤ 3.6% compared with positive control (4-NQO, 7.5 mg/kg, i.p.) which significantly (P > 0.05) enhanced the 8-OHdG level about 12% which was 3.4-fold higher than negative control (Fig. 3). Similarly, 4-NQO-induced 8-OHdG DNA adducts recorded was three fold higher than that of negative control, 5.13 ± 3.41 (Arumugam and Ramesh [5]). It clearly showed that different doses of fucoidan (50,100, and 200 mg/kg) followed by 4-NQO constituted mice explored the level of 8-OHdG ~ 11–3%. These results stated that high dose of fucoidan was significantly (P > 0.05) protected the 4-NQO-induced oxidative DNA damage as equal to negative control (3% 8-OHdG). Subsequently, following higher dose (100 mg/kg) of fucoidan was better cosseted against 4-NQO induced oxidative DNA damage than that of lower dose over the positive control (12% 8-OHdG; Fig. 3). To support, fucoidan of brown seaweeds was documented possessing rich antioxidant properties evaluated through scavenging DPPH, superoxide-hydroxyl radicals including chelating ability and reducing power (Kordjazi et al. [23]).

Graph: Fig. 2 Fucoidan pretreatment protected plasma lipid peroxidation elevated by 4-NQO in mice. (Lipid peroxidation [LPO], control [C], body weight [bwt], 4-nitroquinoline-1-oxide [4-NQO] compared with control; fucoidan + 4-NQO compared with 4-NQO, P ≤ 0.05)

Graph: Fig. 3 Fucoidan pretreatment protected plasma 8-OHdG elevated by 4-NQO in mice. (8-Hydroxy-2'-deoxyguanosine [8OHdG], control [C], body weight [bwt], 4-nitroquinoline-1-oxide [4-NQO] compared with control, fucoidan + 4-NQO compared with 4-NQO, P ≤ 0.05)

In addition, genetic damage in form of comet in mice bone marrow cells was recorded in terms of various factors such as % head, % tail, and % tail moment using comet assay software after single gel electrophoresis by comet assay kit. Comet assay is one of the typical techniques for detection of DNA damage and widely used in genotoxicity test, biomonitoring in human peripheral blood and DNA repair as well (Ladeira et al. [26]). This experimental results clearly shows that the negative control and fucoidan alone–treated groups were not exhibited significant effect on genetic damage/comet formation (Table 1; Fig. 4) but significant increase in genetic damage was found in the positive control (4-NQO 7.5 mg/kg, i.p.) by recording 18% head, 18% tail and 2.3% tail moment over the negative control, 95.00 ± 1.58, 5.00 ± 1.58, and 0.41 ± 0.13 respectively (Table 1). On the other hand, fucoidan significantly protected the 4-NQO-induced genetic damage of 40%, 79%, and 96 % at the dose of 50,100, and 200 mg/kg, respectively over the positive control, 2.71 ± 0.16. The effect of fucoidan was reflected through enhancing head factor about 5%, 11%, and 18 % over the positive control, 76.60 ± 5.46, and it was also similarly decreasing tail factor over the positive control, 23.40 ± 5.45 (Table 1). Thus revealed that fucoidan significantly (P > 0.05) protected the 4-NQO–induced genetic damage in mice bone marrow cells as dose dependent manner as like micronuclei. Similar to this present result, fucoidan of Fucus vesiculosus found effective against doxorubicin-induced DNA damage was reported (Leite-Silva et al. [28]). Since fucoidan prevent effectively LPS which is an endotoxin-induced DNA damage in mice, it could be used for the development of drug against negative effect of endotoxin (Kuznetsova et al. [24]). In addition, in this study, antiapoptotic effect of fucoidan was examined in mice bone marrow cells by flow cytometer using annexin-V FITC assay kit. Four different groups of cells measured and expressed as percentage of total apoptotic cells which was calculated by adding both early and late apoptotic cells (Fig. 5). Total apoptotic cells induced by 4-NQO alone–treated group were 15% over the negative control, 6%. During the genotoxic stress, cells undergo apoptosis if the DNA damage was beyond repair. Han et al. ([18]) reported that the 4-NQO induced an apoptosis through p53-dependent mitochondrial signaling pathway. In this observation, based on the micronuclei assay, maximum fucoidan dose (200 mg/kg) received group was adapted to analyze the antiapoptotic effect in mice bone marrow cells. Fucoidan treatment alone did not shown any effect on apoptotic cells whereas fucoidan pretreated 4-NQO induction group showed significant reduction of apoptotic cells, and the reduction observed was 5% over the positive control, 15% that was significant when compared negative control, 4% (Fig. 5). Hence, 4-NQO-induced apoptotic (15%) and genetic damage (18%) were well correlated and significantly protected by fucoidan (93%) (Fig. 5; Table 1). Byon et al. ([10]) observed the radioprotective effects of fucoidan on bone marrow cells (BMCs) by analyzing radiation exposed BMC cell viability as well as by immune responses. The study reported that fucoidan-treated BMCs inhibited the radiation-induced apoptosis and altered the production of immune-related cytokines from BMCs by increasing proliferation of allogeneic splenocytes (Byon et al. [10]).

Protective effect of fucoidan on 4-NQO induced genetic damage in mice bone marrow cells

The values are presented as mean ± SD [n = 5]. The percentage inhibition was made by following formula (tail moment of positive control — fucoidan + 4-NQO/positive control — control × 100)

Graph: Fig. 4 Image of comet recorded with light microscope after single gel electrophoresis by comet assay

Graph: Fig. 5 Protective effect of fucoidan on 4-NQO induced apoptosis in mice bone marrow cells (Body weight [bwt], 4-nitroquinoline-1-oxide [(4-NQO] compared with control, fucoidan +4-NQO compared with 4-NQO, P ≤ 0.05)

This study concludes as 4-NQO induced genetic damage in terms of LPO (6 μM MAD), micronuclei (65%), 8-OHdG (12%), comet (18%), and apoptosis (15%) either directly or by oxidative stress was well correlated and significantly (P > 0.05) elevate over the respective negative control. Indeed, fucoidan pretreatment significantly exhibit antigenotoxic effect (96%) with dose dependent order by effectively restoring the 4-NQO-induced genetic damages such as LPO, 8-OHdG, micronuclei, comet and apoptosis in mice. Therefore, this study strengthens further that fucoidan considered as valuable food supplements and medicine for mankind from environmental toxicants.

This contributed research work was supported by University Grant Commission (UGC), Government of India, New Delhi (TAM-8496).

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